Journal of food Science 1974 Volume.39 No.1

JOURNAL of FO O D SCIENCEBASIC SCIENCE

1 E f f e c t o f t u n a f i s h a n d s e l e n i u m o n t h e t o x i c i t y o f m e t h y l - m e r c u r y : A P r o g r e s s R e p o r t —H.E. Ganther and M.L. Sunde

i C a t h e p s i n a c t i v i t y o f P e n a e u s s e t i f e r u s - m u s c l e — R.R. Eiten- mi/ler

' i A s t u d y o f s o m e k i n e t i c p r o p e r t i e s o f p a r t i a l l y p u r i f i e d P e n a e u s s e t i f e r u s a r y l a m i d a s e — B.A. Bauer and R.R. EitenmMer

» A n e v a l u a t i o n o f a m e t h o d f o r m e a s u r i n g s h e a r f o r c e f o r a n i n d i v i d u a l m u s c l e f i b e r — R.L. Henrickson, J.L. Marsden and R.D. Morrison

R a t e o l h e a l i n g a s i t a f f e c t s t h e s o l u b i l i z a t i o n o f b e e f m u s c l e c o l l a g e n - S . £ . McCrae and P.C. Paul

■ O x y g e n u p t a k e in p o s t r i g o r b o v i n e m u s c l e — D.P. DeVore and M. So/berg

7- b o s s o f c a l c i u m a c c u m u l a t i n g a b i l i t y in t h e s a r c o p l a s m i c r e t i c u l u m f o l l o w i n g d e g r a d a t i o n b y c a t h e p s i n s — R.L. West, P.W. Moeller, B.A. Link and W.A. Land man n

j J I t r a s t r u c t u r a l p o s t m o r t e m c h a n g e s in n o r m a l a n d lo w -q u a l i t y p o r c i n e m u s c l e f i b e r s — T.R. Dutson, A.M. Pearson, R.A. Merkel and G.C. Spink

) e t e r m i n a t i o n o f b e n / o ( a ) p y r e n e in s m o k e c o n d e n s a t e s b y h i g h p r e s s u r e r a p i d l i q u i d - l i q u i d c h r o m a t o g r a p h y —/ . / ? . O'Hara, M.S. Chin, B. Dainius and /.LI. Kilbuck

. f f e c t o f h e a t a n d o t h e r f a c t o r s u p o n f o a m i n g p r o p e r t i e s o f w h e y p r o t e i n c o n c e n t r a t e s —S.H. Richert, C.V. Morr andC.M. Cooney

‘ • D i t e r m i n a t i o n o f S H - a n d S S - g r o u p s in s o m e f o o d p r o t e i n s u s i n g E l l m a n ’s r e a g e n t — T. Beveridge, S.J. Toma and S. 'Sakai 57 * * *

5 7 t- —a c t i o n o f d i e l e c t r i c p r o p e r t i e s in n o n f a t n jil-k a t f,u e n c i e s a n d t e m p e r a t u r e s o f i n t e r e s t in . .m i c r o w a v e

m o c e s s i n g — R.E. Mudgett, A.C. Smith, D.I.C. Wang'and\ , 4 ¡oldblith

5 5 I n t e n s i t y - t i m e c u r v e s f o r f l a v o r e d o i l - i n - w a t e r e m u l s i o n s — P.B. McNulty and H.R. M oskowitz

5 8 F l a v o r a n d s t o r a g e s t a b i l i t y o f e x p l o s i o n - p u f f e d p o t a t o e s . N o n e n z y m a t i c b r o w n i n g —/.F. Sullivan, R.P. Konstance, M.J. Calhoun, F.B. Talley, / . Cording /r. and O. Panasiuk

6 1 D a t a A n a l y s i s : A v a r i a b l e s e q u e n t i a l t e s t f o r s e l e c t i o n o f s e n s o r y p a n e l s — A 1 C . Gacula jr., L-A. Parker, /.J. Kubala

* and / . Reaume

6 4 S e n s o r y a n d g a s c h r o m a t o g r a p h i c p r o f i l e s o f c o f f e e b e v e r a g e h e a d s p a c e v o l a t i l e s e n t r a i n e d o n p o r o u s p o l y m e r s -C .G . Tassan and G.F. Russell

6 9 S o y b e a n p h o s p h a t i d y l c h o l i n e d e v e l o p s b i t t e r t a s t e o n a u - t o x i d a t i o n — D.j. Sessa, K. Warner and D.H. Honig

7 3 S o m e a r o m a c o m p o n e n t s o f r o a s t e d s e s a m e s e e d ( S e s m u m i n d i c u m L.)-C .H . Manley, P.P. Val/ong and R.E. Erickson

7 7 U s e o f a r a d i o m e t r i c t e c h n i q u e f o r t h e r a p i d d e t e c t i o n o f g r o w t h o f c l o s t i d i a l s p e c i e s — G.M. Evancho, D.LI. Ashton and A.A. Zwarun

8 0 E f f e c t o f v a r i o u s p e p t o n e s in t h e g r o w t h m e d i u m o n t h e a g g l u t i n a t i o n o f t e n S a l m o n e l l a s p e c i e s w i t h p o o l e d S p i c e r — E d w a r d s a n t i s e r a — W.J. Stamper and G.J. Banwart

8 3 C a r b o n y l p r o d u c t i o n f r o m l i p o l y z e d m i l k f a t b y t h e c o n

t i n u o u s m y c e l i a l c u l t u r e o f P e n ic i 11 iu m r o q u e f o r t i — B.K. Dwivedi and J.E. Kinsel/a

88 A c t i v i t y a n d s t a b i l i t y o f 0 - g a l a c t o s i d a s e i m m o b i l i z e d o n p o r o u s g l a s s — £ . 5 . Okos and W.j. Harper

9 4 B e n z y l i s o t h i o c y a n a t c a s a n a t u r a l l y o c c u r r i n g p a p a i n i n h i b

i t o r — C - S Tang9 7 . Z o n e e l e c t r o p h o r e s i s o f f o o d s t a b i l i z e r s in m a l o n a t e b u f f e r

—/ . C. Chang, M. IV. Renoll and P. M. T. Hansen

- C O N T E N T S C O N T I N U E D ( o n t h e i n s i d e o f t h e f r o n t c o v e r ) . . .

A P L ? t I C A T I O N O F T H E I N S T I T U T E O F F O O D T E C H N O L O G IS T S

— CONTENTS (CONTINUED) —

1 0 3 H e a t - i n d u c e d c h a n g e s in t h e z o n e e l e c t r o p h o r e t i c p a t t e r n s o f c a r r a g e e n a n s t a b i l i z e r s — P.M. T. Hansen and M.W. Renoll

1 0 8 M e c h a n i s m o f e m u l s i f i e r a c t i o n in a n i c e c r e a m s y s t e m —P. Lin and J.G. Leeder

1 1 2 D e c r e a s e o f l i n o l e a t e o x i d a t i o n r a t e d u e t o w a t e r a t i n t e r m e d i a t e w a t e r a c t i v i t y — T.P. Labuza and H.E. Chou

1 1 4 B o u n d w a t e r c a p a c i t y o f c o r n s t a r c h a n d i t s d e r i v a t i v e s b y N M R - / . Mousserl, M.P. Steinberg , A.I. Nelson and L.S. Wei

1 1 7 M i c r o s t r u c t u r e o f m o d i f i e d t a p i o c a s t a r c h - m i l k g e l s —L.F. Hood, A S . Seifried and R. Meyer

1 2 1 E f f e c t o f f r o z e n s t o r a g e o n t h e m i c r o s t r u c t u r e a n d s y n e r e s i s o f m o d i f i e d t a p i o c a s t a r c h - m i l k g e l s — L.F. Hood and A S . Sei fried

APPLIED SCIENCE and ENGINEERING

1 2 5 R a p i d t e c h n i q u e s f o r s a l t - c u r i n g f i s h . A R e v i e w —J.M. Medelsohn

1 2 8 E v a l u a t i o n o f b c t a l a i n p i g m e n t s a s s u b s t i t u t e s a u s a g e c o l o r a n t s — ].H. von Elbe, J.T. Klement, C.H. Amundson,R.G. Cassens and R.C. Lindsay

1 3 3 E f f e c t s o f d i e t a r y f a t a n d d l - a - t o c o p h c r y l o n s t a b i l i t y c h a r a c t e r i s t i c s o f p r e c o o k e d f r o z e n b r o i l e r p a r t s —].E. Webb,C.C. Brunson and / .D. Yates

1 3 7 S o y a a d d i t i v e s in b e e f p a t t i e s —M.D. lodge, C.G. Haugh,G.L. Zachariah, C.E. Par me lee and R.L. Pyle

1 4 0 H o t b o n i n g a n d v a c u u m p a c k a g i n g o f e i g h t m a j o r b o v i n e m u s c l e s — G.R. Schmidt and S. Keman

1 4 3 E f f e c t o f v a c u u m p a c k a g i n g o n w e i g h t l o s s , m i c r o b i a l g r o w t h a n d p a l a t a b i l i t y o f f r e s h b e e f w h o l e s a l e c u t s —¡.H. Hodges, V.R. Cahill and H.W. Ocher man

1 4 7 A n a n a l y s i s o f a t m o s p h e r i c f r e e z e d r y i n g — D.R. Heldman and G.A. Hohner

1 5 6 D e t e r m i n a t i o n o f f r e s h p a p a y a ’s t e x t u r e b y p e n e t r a t i o n t e s t s —A7. Peleg

1 6 0 F l o w p r o p e r t i e s o f t r o p i c a l f r u i t p u r e e s —M.A. Rao, L.N. O toya Palomino and L. W. Bernhardt 162 *

1 6 2 P i l o t a p p l i c a t i o n o f f r e e z e - h e a t p e e l i n g o f t o m a t o e s —S .Leonard and F. Winter

1 6 6 H i g h - l y s i n e c o r n f r a c t i o n s a n d t h e i r c h a r a c t e r i s t i c s — G .A '. Bookwalter, K. Warner, O.L. Breeke and E.L. Griffin Jr.

1 7 1 A m i n o a c i d c o m p o s i t i o n a n d b i o l o g i c a l q u a l i t y o f l i m a b e a n p r o t e i n —S . Maneepun, B.S. Luh and R.B. Rucker

1 7 5 A c o m p a r i s o n o f t h e e m u l s i f i c a t i o n c a p a c i t i e s o f s o m e p r o t e i n c o n c e n t r a t e s — D.D. Crenwelge, C.W. Dill, P.T. Tybor and W.A. Landmann

1 7 8 C o m p o s i t i o n a n d c h a r a c t e r i s t i c s o f g l a n d l e s s a n d l i q u i d c y c l o n e p r o c e s s d e g l a n d e d c o t t o n s e e d w h e y s —S.H.C. Lin, J.T. Lawhon, C.M. Cater and K. F. Mat til

1 8 3 U t i l i z a t i o n o f c o t t o n s e e d w h e y p r o t e i n c o n c e n t r a t e s p r o d u c e d b y u l t r a f i l t r a t i o n —/ . / ' . Lawhon, S.H.C. Lin, L.W. Rooney, C.M. Cater and K.F. Mat til

1 8 8 V i s c o s i t y a n d w a t e r a b s o r p t i o n c h a r a c t e r i s t i c s o f s l u r r i e s o f s u n f l o w e r a n d s o y b e a n f l o u r s , c o n c e n t r a t e s a n d i s o l a t e s — S.E. Fleming, F.W. Sosulski, A. Kilara and E.S. Humbert

1 9 2 S o l u b l e c o f f e e : S h e l f l i f e s t u d i e s —M E . Harris, S.J. Bishov, A.R. Rahman, M.M. Robertson and A.F. Mabrouk

1 9 6 D e h y d r a t e d c o c o n u t s k i m m i l k a s a f o o d p r o d u c t : C o m p o s i t i o n a n d f u n c t i o n a l i t y — /? . Hagenmaier, K.F. Mattil andC.M. Cater

2 0 0 S e n s o r y r a t i o s c a l e s r e l a t i n g h a r d n e s s a n d c r u n c h i n e s s t o m e c h a n i c a l p r o p e r t i e s o f s p a c e c u b e s —H.R. M oskowitz, R.A. Segars, J.G. Kapsalis and R.A. Kluter

2 0 3 P r e d i c t i o n o f h e d o n i c r a t i n g s o f r i c e b y s e n s o r y a n a l y s i s — H. G. Schütz and J. D. Damrell

RESEARCH NOTES2 0 7 D e g r a d a t i o n o f t h i a m i n e a n d r i b o f l a v i n d u r i n g e x t r u s i o n

p r o c e s s i n g — G . Beetner, T. Tsao, A. Frey and J. Harper

2 0 9 D a t a a n a l y s i s : R e g r e s s i o n a n a l y s i s w i t h r e p e t i t i o n s o f t h e i n d e p e n d e n t v a r i a b l e —/ . / . Kubala, M.C. Gacula Jr. and M .f Moran

2 1 1 E f f e c t o f m i c r o w a v e h e a t i n g o f p r e c o o k e d c h i c k e n o n C l o s t r i d i u m p e r f r i g e n s — S.E. Craven and H.S. Lillard

2 1 3 M y c o t o x i n s p r o d u c e d b y f u n g i i s o l a t e d f r o m i n s h e l l p e c a n s — A .F. Schindler, A.N. Abadie, J.S. Gecan, P.B. Mislivec and P.M. Brie key

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January-February 1974 Volume 39 : Volume 1

JOURNAL of FO O D SCIENCEDirector of Publications

John B. Klis

Managing EditorBernard Schukraft

Advertising ManagerEdward H. Hoffman

PublisherCalvert L. Willey

Scientific EditorBernard J. Liska

Asst, to Scientific E d ito rAnna May Schenck

Board of EditorsE. Briskey (74)L. Dugan (74)F. Francis (74)J. Liston (74)R. Pangborn (74) J. Powers (74)W. Stahl (74)

R. Cassens (75) A. Denton (75) D. Goll (75)H. Hultin (75) T. Labuza (75) R. Maxcy (75) P. Nelson (75)

W. Clark (76)R. Eiserle (76)G. Giddings (76) D. Heldman (76) P. Hopper (76)Y. Pomeranz (76) M. Solberg (76)

• M A N U S C R IP T S (3 c o p ie s ) s h o u ld b e s u b m i t t e d to :

D r . B e r n a r d J . L ik sa

I F T S c ie n t i f i c E d i to rP .O . B o x 3 0 6 7 L a f a y e t t e , IN 4 7 9 0 6 U S A

N O R E S P O N S IB IL I T Y is a s s u m e d b y th e I n s t i t u t e o f F o o d T e c h n o lo g is t s f o r s t a t e m e n t s a n d o p in io n s e x p re s s e d b y th e c o n t r i b u t o r s t o i t s p u b l ic a t io n s .

M A N U S C R IP T S s h o u ld c o n f o r m to t h e s ty le u s e d in Journal of Food Science. A u t h o r s s h o u ld r e q u e s t f r o m th e D i r e c to r o f P u b l i c a t i o n s t h e re v is e d l e a f le t “ S ty l e G u id e f o r R e s e a r c h P a p e r s .” Journal o f Food Science r e s e rv e s t h e p r iv i le g e o f e d i t in g m a n u s c r i p t s t o m a k e th e m c o n f o r m w ith th e a d o p t e d s ty le o f th e j o u r n a l o r r e tu r n i n g th e m to a u th o r s f o r re v is io n . E d i t in g c h a n g e s m a y b e r e v ie w e d b y a u t h o r s b e f o r e p u b l i c a t i o n .

P A G E C H A R G E S f o r P u b l i c a t io n s . T h e I F T E x e c u t iv e C o m m i t t e e h a s e s ta b l i s h e d a p a g e c h a rg e o f $ 5 0 p e r p r i n t e d p a g e

f o r a l l p a p e r s p u b l i s h e d in Journal of Food Science. T h e p a g e c h a rg e s h a ll n o t c o n s t i t u t e a b a r t o a c c e p ta n c e o f r e s e a r c h m a n u s c r i p t s b e c a u s e t h e a u t h o r is u n a b le t o p a y th e c h a r g e .

• S U B S C R IP T IO N S : A ll c o m m u n ic a t io n s r e la te d t o h a n d l in g o f s u b s c r ip t i o n s , in c lu d in g lo s s c la im s , c h a n g e o f a d d re s s , o r d e r s f o r b a c k is su e s , a n d 1 0 0 o r m o r e r e p r in t s s h o u ld b e s e n t to :

S u b s c r ip t i o n D e p a r tm e n tI n s t i t u t e o f F o o d T e c h n o l o g i s t s - S u i t e 2 1 2 02 2 1 N . L a S a lle S t r e e tC h ic a g o , IL 6 0 6 0 1 U S A

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f r o m S u b s c r ip t i o n D e p a r tm e n t .

© C o p y r i g h t 1 9 7 4 b y I n s t i t u t e o f F o o d T e c h n o lo g is t s . A ll r ig h t s r e s e rv e d . J O U R N A L O F F O O D S C IE N C E ( f o r m e r ly Food Research) is p u b l i s h e d s ix t im e s a y e a r ( b i m o n t h l y ) b y I n s t i t u t e o f F o o d T e c h n o lo g is t s , S u i t e 2 1 2 0 , 2 2 1 N . L a S a lle S t r e e t , C h ic a g o , I l l in o is 6 0 6 0 1 U S A . P r in te d in U S A . S e c o n d c la s s p o s ta g e p a id a t C h ic a g o , 111. a n d a t a d d i t i o n a l m a ilin g

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ABSTRACTS :I N T H I S I S S U E

E F F E C T O F T U N A F I S H A N D S E L E N I U M O N T H E T O X IC IT Y O F M E T H Y L M E R C U R Y : A P R O G R E S S R E P O R T . H .E . G A N T H E R & M .L . S U N D E . J. Food Sci. 3 9 , 1 - 5 ( 1 9 7 4 ) - J a p a n e s e q u a i l w e r e f e d m e r c u ry in t h e f o r m o f l y o p h i l i z e d c a n n e d t u n a , 1 7 % o f t u n a p r o v id in g u p to 0 .5 p p m H g in t h e t o t a l d i e t . H ig h e r le v e ls o f H g w e re o b t a i n e d b y a d d in g m e t h y l m e r c u r y h y d r o x i d e . N o o b v io u s to x i c e f f e c t s w e r e n o t e d in q u a i l f e d t u n a u p t o 4 7 w k , n o r w a s r e p r o d u c t iv e p e r f o r m a n c e im p a i r e d . M e th y l m e r c u r y a d d e d t o a t u n a - c o r n - s o y a d i e t w a s le s s t o x i c t h a n a n e q u iv a l e n t a m o u n t a d d e d t o a c o r n - s o y a d i e t . S e le n iu m a d d e d t o d ie ts ( 0 .5 p p m ) o f q u a i l o r r a t s d e c r e a s e d m e th y lm e r c u r y to x i c i t y a n d is a p p a r e n t l y t h e p r o t e c t iv e f a c to r in tu n a .

C A T H E P S IN A C T IV IT Y O F P E N A E U S S E T I F E R U S M U S C L E . R .R . E I T E N M I L L E R . J. Food Sci. 3 9 , 6 - 9 ( 1 9 7 4 ) - T h e m u s c le e x t r a c t o f w h i t e s h r im p (Penaeus setiferus) c o n ta in s c a th e p s in D a c t iv i ty b u t n o t c a t h e p s in A , B , o r C a c t iv i t ie s . T h e c a th e p s in D e n z y m e is o p t im a l ly a c t iv e a g a in s t a c id d e n a t u r e d h e m o g lo b in a n d e n d o g e n o u s p r o t e i n a t 4 2 ° C a n d p H 3 .0 . I t is o p t im a l ly a c t iv e a t p H 3 .5 w i th a c id - u r e a d e n a t u r e d h e m o g lo b in . T h e m u s c l e e x t r a c t is n o t a c t iv e a g a in s t c a s e in o r s e r u m a lb u m in . I n h i b i t o r s tu d ie s h a v e s h o w n t h a t E D T A , C a + + , Z n + + , C o + + , H g + + a n d C N a r e i n h i b i t o r y ; w h e re a s , c y s te in e s l ig h t ly a c t iv a te s t h e e n z y m e . N - e th y l- m a le im id e a n d p - c h lo r o m e r c u r ib e n z o a te d o n o t a l t e r t h e a c t i v i t y . S tu d i e s d i f f e r e n t i a t i n g f r e e a n d b o u n d a c t iv i t i e s in d ic a te d th a t t h e c a th e p s in D a c t iv i ty e x i s t s p r im a r i ly in a b o u n d s t a t e in th e m u s c le .

A S T U D Y O F S O M E K IN E T IC P R O P E R T I E S O F P A R T I A L L Y P U R I F I E D P e n a e u s s e t i f e r u s A R Y L A M I D A S E . B .A . B A U E R & R .R . E I T E N M I L L E R . J. Food Sci. 3 9 , 1 0 - 1 4 ( 1 9 7 4 ) - A p r o c e d u r e f o r th e p a r t i a l p u r i f i c a t i o n o f Penaeus setiferus ( w h i t e s h r im p ) m u s c le a r y la m i- d a s e is g iv e n . T h e e n z y m e s h o w e d h ig h e s t a c t iv i ty a t p H 6 .5 w i th ly s y l - |3 -n a p h th y la m id e a s s u b s t r a t e . P u r o m y c i n w a s a c o m p e t i t i v e in h ib i t o r o f t h e e n z y m e , a f a c t o r d i f f e r e n t i a t i n g th e a r y la m id a s e a c t iv i ty f r o m c la s s ic a l le u c in e a m i n o p e p t id a s e a c t iv i ty . M e ta ls a re n o t r e q u i r e d f o r a c t i v i t y , b u t a n in c r e a s e in h y d r o ly s i s is o b s e r v e d u p o n th e a d d i t i o n o f d iv a le n t c o b a l t o r m a n g a n e s e . A c r y la m id e g e l e l e c t r o p h o r e s i s w i th a n d w i t h o u t u r e a t r e a t m e n t in d i c a t e d a s u b u n i t s t r u c t u r e .

A N E V A L U A T IO N O F A M E T H O D F O R M E A S U R IN G S H E A R F O R C E F O R A N I N D I V I D U A L M U S C L E F I B E R . R .L . H E N R IC K - S O N , J .L . M A R S D E N & R .D . M O R R IS O N . J. Food Sci. 3 9 , 1 5 - 1 7 ( 1 9 7 4 ) - A m ic r o s e n s i t iv e s h e a r i n s t r u m e n t f o r m e a s u r in g th e s h e a r f o r c e o f a n in d iv id u a l m u s c le f ib e r w a s e v a lu a te d , f o r m a l d e h y d e f ix e d f ib e r s f r o m th e s a r to r iu s m u s c le w e re u s e d to e v a lu a te t h e p r e c i s io n o f th e i n s t r u m e n t . S a r to r iu s m u s c le f ib e r s f r o m th r e e ( 2 , 5 a n d 8 -h r ) p o s t m o r te m h o ld in g p e r io d s w e re a ls o s tu d i e d . E v a lu a t io n s m a d e o f f ib e r d i a m e t e r , k in k in e s s , s h e a r f o r c e a n d s h e a r s t r e s s i n d i c a t e d t h a t m u s c le r e s t r a i n t o n th e c a r c a s s f o r 5 - 8 h r p o s t m o r t e m , b e f o r e m u s c le e x c i s io n , r e d u c e d t h e r i g o r e f f e c t o n th e m u s c l e a n d i t s f ib e r s .

R A T E O F H E A T IN G A S IT A F F E C T S T H E S O L U B I L I Z A T I O N O F B E E F M U S C L E C O L L A G E N . S .E . M c C R A E & P .C . P A U L . J. Food Sci. 3 9 , 1 8 - 2 1 ( 1 9 7 4 ) - C o m p a r i s o n w a s m a d e o f e f f e c t s o f f o u r h e a t in g r a te s p r o d u c e d b y m ic r o w a v e h e a t in g , o v e n b r o i l in g , b r a i s in g a n d r o a s t i n g t o 7 0 ° C , a n d in c r e a s e d e x p o s u r e t o h e a t b y b r a i s in g t o 9 8 ° C p lu s 1 /2 h r h o ld in g a t t h a t t e m p e r a t u r e , o n c o o k in g lo s s e s , c o m p o s i t i o n , c o l la g e n e x t r a c t a b i l i t y , s h e a r f o r c e , p e n e t r a t i o n a n d m ic r o s c o p ic a p p e a r a n c e o f c u t s o f b e e f s e m i te n d in o s u s . M ic ro w a v e e n e r g y a p p e a r e d t o b e m o r e e f f e c t iv e in s o lu b i l iz in g c o l la g e n th a n a n y o f t h e c o n v e n t io n a l m e t h o d s a t 7 0 ° C . H o w e v e r , t h e lo n g e r h ig h t e m p e r a t u r e e x p o s u r e w i t h b r a i s in g t o 9 8 ° C p lu s 1 /2 h r g r e a t ly in c r e a s e d c o l la g e n e x t r a c t a b i l i t y o v e r a n y o f t h e o t h e r m e th o d s . M ic ro w a v e a n d o v e n - b r o i l e d s a m p le s w e r e s o f t e s t , a n d b r a i s e d to 9 8 ° C p lu s 1 /2 h r h a r d e s t a s m e a s u r e d b y d e p t h o f p e n e t r a t i o n . F o r c e r e q u i r e d t o s h e a r d id n o t v a ry s ig n i f i c a n t ly a m o n g t h e v a r io u s t r e a t m e n t s . T h e c h a n g e s o b s e r v e d b y m ic r o s c o p ic e x a m i n a t i o n t e n d e d t o s u p p o r t t h e in d i c a t io n s t h a t r a p id h e a t in g w i th m ic ro w a v e e n e r g y i n f l u e n c e s b o t h c o n t r a c t i l e a n d c o n n e c t iv e t i s s u e s d i f f e r e n t l y t h a n d o e s t h e s lo w e r h e a t in g b y c o n v e n t io n a l m e th o d s .

O X Y G E N U P T A K E IN P O S T R I G O R B O V I N E M U S C L E . D .P . D e V O R E & M . S O L B E R G . J. Food Sci. 3 9 , 2 2 - 2 8 ( 1 9 7 4 ) - O x y g e n u p t a k e b y p o s t r i g o r b o v in e s e m i m e m b r a n o s u s m u s c le w a s m e a s u r e d m a n o m e t r i c a l l y in i n t a c t m u s c le s lic e s . A c o n s t a n t o x y g e n h e a d s p a c e p r e s s u r e o f o n e a tm o s p h e r e w a s m a in t a in e d . O x y g e n u p t a k e f r o m l ip id o x i d a t i o n s a n d b a c t e r ia l d e m a n d s w a s in s ig n i f i c a n t . O x y g e n u t i l i z a t i o n r e s u l t in g f r o m r e s p i r a t i o n a c c o u n t e d f o r a p p r o x i m a t e l y 5 0 % o f t h e t o t a l u p t a k e a f t e r 3 0 h r . R e s p i r a to r y o x y g e n r e q u i r e m e n t s w e re h ig h e s t in t h e f i r s t 1 0 h r a n d t h e n d e c r e a s e d r a p id ly t o a s lo w , s t e a d y s t a t e . T h e r e m a in in g u p t a k e , r e s u l t in g f r o m h e m e p ig m e n t o x y g e n a t io n a n d o x y g e n d i s s o lu t io n i n t o t i s s u e f lu id s , w a s c o n s t a n t d u r in g t h e 3 0 - h r e x p o s u r e p e r io d . T h e e n d o g e n o u s a c t iv i t y o f s u c c in a te /N A D H c y t o c h r o m e c r e d u c t a s e d e c r e a s e d d u r in g t h e t im e c o u r s e o f o x y g e n u p t a k e . R e s p i r a to r y o x y g e n c o n s u m p t io n a p p e a r e d t o b e l im i t e d b y s u b s t r a t e d e p le t i o n s o r d e t e r i o r a t i o n s o f r e s p i r a to r y e n z y m e s . A t t e m p t s t o m e a s u re t h e e f f e c t s o f t i s s u e m y o g lo b in c o n c e n t r a t i o n o n o x y g e n u p t a k e in c o n t r o l a n d r e s p i r a t i o n - i n h i b i t e d s a m p le s r e s u l t e d in c o n f l i c t i n g d a ta .

L O S S O F C A L C IU M A C C U M U L A T IN G A B IL I T Y IN T H E S A R C O P L A S M IC R E T IC U L U M F O L L O W IN G D E G R A D A T I O N B Y C A T H E P - S I N S . R .L . W E S T , P .W . M O E L L E R , B .A . L I N K & W .A . L A N D M A N N . J. Food Sci. 3 9 , 2 9 - 3 1 ( 1 9 7 4 ) - A s tu d y w a s c o n d u c t e d to d e t e r m i n e i f s p le e n c a t h e p s in s c o u ld c a u s e a r e d u c t i o n in t h e c a l c iu m - a c c u m u la t in g a b i l i t y o f t h e i s o la te d s a r c o p la s m ic r e t i c u lu m th u s s h o r t e n in g t h e t im e r e q u i r e d f o r t h e o n s e t o f r ig o r m o r t i s . A 4 0 - 7 0 % s a t u r a t e d a m m o n iu m s u l f a t e p r e c ip i t a t e ( c a th e p s in 4 0 —7 0 % f r a c t i o n ) f r o m b o v in e s p le e n w a s f r a c t i o n a t e d b y g e l f i l t r a t i o n c h r o m a to g r a p h y t o i s o la te a c a t h e p s in B1 p e a k . T h e t r e a t m e n t s o f t h e s a r c o p la s m ic r e t i c u lu m i n c lu d e d : ( 1 ) T r y p s in d ig e s t io n f o r 3 m in a t 2 5 ° C ; (2 ) C a th e p s in B1 d ig e s t io n f o r 2 h r a t 2 5 ° C ; (3 ) C a t h e p s in 4 0 - 7 0 % f r a c t io n d ig e s t io n f o r 2 h r a t 2 5 ° C ; (4 ) C a t h e p s in B1 d ig e s t io n f o r 1 8 h r a t 2 ° C ; a n d (5 ) C a th e p s in 4 0 - 7 0 % f r a c t i o n d ig e s t i o n f o r 1 8 h r a t 2 ° C . T h e c o m b in a t io n o f c a t h e p s in s in t h e 4 0 —7 0 % f r a c t io n w a s m o r e e f f e c t iv e in l o w e r in g th e c a l c iu m - a c c u m u la t in g a b i l i t y th a n p u r i f i e d c a t h e p s in B l . B o th t h e c r u d e 4 0 - 7 0 % c a t h e p s in f r a c t i o n r n d t h e i s o la te d c a t h e p s in B l w e re e f f e c t iv e in r e d u c in g t h e C a 2 + - i c c u m u la t in g a b i l i t y a t e i t h e r 2° o r 2 5 ° C .

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U L T R A S T R U C T U R A L P O S T M O R T E M C H A N G E S IN N O R M A L A N D L O W -Q U A L IT Y P O R C I N E M U S C L E F I B E R S . T .R . D U T S O N , A .M . P E A R S O N , R .A . M E R K E L & G .C . S P IN K . J. Food Sci. 39. 32-37 ( 1 9 7 4 ) - T h e u l t r a s t r u c t u r e o f r e d a n d w h i te f ib e r s f r o m p o r c in e lo n g is s i- m u s m u s c le w a s c h a r a c t e r i z e d a t 15 m in a n d 2 4 h r p o s t m o r t e m f o r 1 0 n o r m a l a n d 1 0 l o w - q u a l i t y p ig s . T h e w id th o f t h e Z l in e w a s a p p r o x i m a te ly 1 2 0 0 A f o r r e d f ib e r s a n d 6 2 5 A f o r w h i t e f ib e r s , w i th t h e Z l in e s o f r e d f ib e r s b e in g m o r e d e n s e a p p e a r in g . M i to c h o n d r i a f r o m r e d f ib e r s w e re m o r e n u m e r o u s , la rg e r , m o r e d e n s e in a p p e a r a n c e a n d s h o w e d m o re c lo s e ly p a c k e d c r i s ta e t h a n m i t o c h o n d r i a f r o m w h i t e f ib e r s . L o n g i tu d in a l tu b u le s o f t h e s a r c o p la s m ic r e t i c u lu m f r o m r e d f ib e r s a p p e a r e d m o r e n a r r o w a n d t u b u l a r t h a n t h o s e f r o m w h i te f ib e r s . A t 2 4 h r p o s t m o r t e m th e Z l in e s , m i t o c h o n d r i a a n d s a r c o p la s m ic r e t i c u l u m w e re m o r e d is r u p t e d in w h i te t h a n in r e d f ib e r s . P o s tm o r t e m d i s r u p t i o n w a s g r e a te r f o r lo w - q u a l i ty t h a n f o r n o r m a l m u s c l e , p a r t i c u l a r ly f o r w h i t e - ty p e f ib e r s . A lso , t h e r a t e o f p o s t m o r t e m d i s r u p t i o n w a s f a s t e r in lo w - q u a l i t y m u s c le , e s p e c ia l ly f o r w h i t e t y p e f ib e r s .

D E T E R M I N A T IO N O F B E N Z O ( a ) P Y R E N E IN S M O K E C O N D E N S A T E S B Y H IG H P R E S S U R E R A P I D L I Q U I D -L I Q U I D C H R O M A T O G R A P H Y . J .R . O ’H A R A , M .S . C H IN , B . D A IN IU S & J .H . K IL B U C K .J. Food Sci. 3 9 , 3 8 —4 1 ( 1 9 7 4 ) —P u b l i s h e d m e t h o d s f o r a n a ly s i s o f b e n z o ( a ) p y r e n e in s m o k e c o n d e n s a t e s in v o lv e a l e n g th y s a m p le p r e p a r a t i o n f o l lo w e d b y s p e c t r o p h o t o m e t r i c e s t i m a t i o n o f t h e c o n t e n t o f th e c a r c in o g e n . A m o r e e f f i c i e n t m e t h o d h a s b e e n d e v e lo p e d in w h ic h th e b e n z o ( a ) p y r e n e is e x t r a c t e d f r o m th e s m o k e c o n d e n s a t e , w a s h e d w i th a c id a n d a lk a l i s o lu t io n s , e l u t e d f r o m a F lo r is i l c o lu m n , i s o la te d b y p r e p a r a t iv e t h in - l a y e r c h r o m a t o g r a p h y , a n d q u a n t i t i z e d o n a L iq u id - L iq u id C h r o m a to g r a p h a t 2 5 4 m q . T h e r a n g e o f r e c o v e r y is 6 0 - 8 8 % .

E F F E C T O F H E A T A N D O T H E R F A C T O R S U P O N F O A M IN G P R O P E R T IE S O F W H E Y P R O T E I N C O N C E N T R A T E S . S .H . R I C H E R T , C .V . M O R R & C .M . C O O N E Y . J. Food Sci. 3 9 , 4 2 - 4 8 ( 1 9 7 4 ) - T h e e f f e c t s o f h e a t in g w h e y p r o t e i n c o n c e n t r a t e (W P C ) d i s p e r s io n s a t 5 0 - 8 5 ° C f o r 3 0 m in a t d i f f e r e n t p H , o f c a lc iu m c o n c e n t r a t i o n a n d o f r e d o x p o t e n t i a l a n d s o d iu m la u r y l s u l f a te c o n c e n t r a t i o n le v e ls u p o n w h e y p r o t e in d e n a t u r a t i o n / a g g r e g a t io n a n d f o a m in g p r o p e r t i e s w e re s tu d ie d . E x p e r i m e n t s w e re c o n d u c t e d a c c o r d in g to a f iv e f a c t o r c e n t r a l c o m p o s i te e x p e r im e n ta l d e s ig n t h a t p e r m i t t e d c o m p u t e r a n a ly s is t o d e t e r m in e r e g re s s io n c o e f f i c i e n t s , c o r r e l a t i o n s a n d s t a t i s t i c a l s ig n i f ic a n c e , b a s e d o n a q u a d r a t i c m a th e m a t i c a l m o d e l . I n g e n e r a l , t h e m o r e s e v e re h e a t t r e a t m e n t s a t 8 0 - 8 5 ° C im p a i r e d t h e f o a m in g p r o p e r t i e s o f W P C d is p e r s io n s . T h o s e d e p e n d e n t v a r ia b le s (Y ) m o s t s ig n i f i c a n t ly a f f e c t e d b y a l te r in g e a c h o f t h e i n d e p e n d e n t v a r ia b le s (X ) w e r e : w h ip p in g t im e ( Y , ) , m a x i m u m o v e r r u n ( Y 2 ) , % u n d e n a t u r e d p r o t e i n ( Y s ) , % s o lu b le p r o t e i n ( Y 6 ) a n d lo g , 0 h e a t e d v is c o s i ty ( Y , ) . R e g r e s s io n a n a ly s is in d i c a t e d t h a t th e o n ly tw o i n d e p e n d e n t v a r ia b le s w h ic h s ig n i f i c a n t ly a f f e c t e d m a x im u m o v e r r u n ( Y 2 ) w e re h e a t in g t e m p e r a t u r e ( X , ) a n d r e d o x p o t e n t i a l ( X 3 ) ,e .g . , h ig h e r h e a t in g t e m p e r a t u r e s a n d lo w e r r e d o x p o t e n t i a l a d v e r s e ly a f f e c te d W P C f o a m in g c h a r a c te r i s t i c s . H e a t in g W P C d is p e r s io n s a t 5 0 - 7 0 ° C g r e a t ly im p r o v e d th e i r f o a m in g p r o p e r t i e s ; h o w e v e r , th is b e n e f ic ia l e f f e c t w a s o n ly t e m p o r a r y . D a ta a re g iv e n f o r c o m p a r i s o n o f p r o t e i n c o m p o s i t i o n a n d d e n a t u r a t i o n a n d f o a m in g p r o p e r t i e s o f s e le c te d W P C s o u rc e s .

D E T E R M I N A T IO N O F S H - A N D S S -G R O U P S IN S O M E F O O D P R O T E IN S U S IN G E L L M A N ’S R E A G E N T . T . B E V E R ID G E , S .J . T O M A & S. N A K A I. J. Food Sci. 3 9 , 4 9 - 5 1 ( 1 9 7 4 ) - A m e t h o d f o r m e a s u r in g s u l f h y d r y l (S H ) a n d d i s u l f id e (S S ) c o n t e n t s o f f o o d s u t i l i z in g E l lm a n ’s r e a g e n t w a s d e v e lo p e d . F o r S H d e t e r m i n a t i o n , 6 .5 M u r e a ( o r 0 .5 % N a d o d e c y l s u l f a te ) f o r eg g w h i t e a n d s k im m ilk a n d a m ix tu r e o f 6 .5 M u re a a n d 5M g u a n id in e HC1 f o r f l o u r a n d g lu te n w e re u s e d as d is s o c ia t in g a g e n ts . SS g r o u p s w e re r e d u c e d w i th 1—2 % 2 - m e r c a p to e th a n o l in th e p re s e n c e o f t h e s a m e d is s o c ia t in g a g e n t s a s f o r S H d e t e r m i n a t i o n a n d th e p r o t e in s w e re p r e c i p i t a t e d b y a d d in g 8 - 1 1 % t r i c h l o r o a c e t i c a c id . T o t a l S H (S H + r e d u c e d S S ) w a s a n a l y z e d a f t e r d is s o lv in g th e p r e c ip i t a t e s in

8M u r e a o r 0 .5 % N a d o d e c y l s u l f a t e a t p H 8 .0 . V a lu e s o f S H a n d S S f o r /M a c to g lo b u l in , o v a lb u m in , /c -c a se in , a s , - c a s e i n , a s s -c a s e in , e g g w h i te , s k im m ilk , t h e i r d r ie d p r o d u c t s , f l o u r a n d g lu t e n w e re in g o o d a g r e e m e n t w i th l i t e r a t u r e v a lu e s . R e c o v e r ie s o f S H a n d S S r a n g e d f r o m 9 1 - 9 8 % a n d f r o m 8 9 - 1 0 2 % , r e s p e c t iv e ly .

P R E D I C T I O N O F D I E L E C T R I C P R O P E R T I E S IN N O N F A T M IL K A T F R E Q U E N C I E S A N D T E M P E R A T U R E S O F I N T E R E S T IN M IC R O - W A V E P R O C E S S IN G . R .E . M U D G E T T , A .C . S M IT H , D .I .C . W A N G &S .A . G O L D B L 1 T H . J. Food Sci. 3 9 , 5 2 - 5 4 ( 1 9 7 4 ) - S t u d i e s o f n o n f a t m i lk s h o w t h a t d i e l e c t r i c b e h a v io r is p r e d i c t e d f r o m 3 0 0 - 3 , 0 0 0 M H z , 2 5 - 5 5 ° C b y t h e H a s te d - D e b y e m o d e ls f o r a q u e o u s i o n i c s o lu t io n s , a n d is r e la te d p r im a r i ly t o m o i s tu r e a n d a s h c o n t e n t s . M ilk c o n d u c t i v i t y a n d c a t io n b in d in g le v e ls w e re f o u n d to b e e s s e n t ia l ly c o n s t a n t w i th t e m p e r a tu r e . A n e f f e c t iv e d is s o lv e d m ilk s a l t s c o n c e n t r a t i o n o f a p p r o x i m a t e l y 0 .1 M s o d iu m c h lo r id e e q u iv a le n t s w a s d e t e r m i n e d b y c o n d u c t i v i t y m e a s u r e m e n ts . A m o d e l c o r r e c t io n f o r d i e l e c t r i c c o n s t a n t p r e d i c t i o n s d u e to m in o r w a te r b in d in g e f f e c t s b y n o n i o n i c c o n s t i t u e n t s w a s d e t e r m in e d f r o m a n a lo g d e p r e s s io n m e a s u r e m e n ts . T h e s e s tu d ie s s u g g e s t t h a t l iq u id f o o d s y s te m s o f lo w c o l lo id a l c o n t e n t b e h a v e a s a q u e o u s io n i c s o lu t io n s w h o s e d i e le c t r i c p r o p e r t i e s m a y b e e s t i m a t e d b y th e H a s te d - D e b y e m o d e ls in c o n j u n c t i o n w i th s im p le c o n d u c t i v i t y m e a s u r e m e n ts .

IN T E N S IT Y -T I M E C U R V E S F O R F L A V O R E D O IL - IN -W A T E R E M U L S IO N S . P .B . M c N U L T Y & H .R . M O S K O W IT Z . j. Food Sci. 3 9 , 5 5 - 5 7 ( 1 9 7 4 ) - T h e t a s t e i n t e n s i t y o f a n e t h o l e w a s e v a l u a te d b y a p a n e l o f 14 in d iv id u a ls . F iv e a n e t h o l e le v e ls a n d th r e e o i l f r a c t i o n s w e re e m p lo y e d . P a n e l is ts d i p p e d t h e i r t o n g u e , w i t h o u t s t i r r i n g , i n t o t h e e m u ls io n s , a n d j u d g e d t h e t a s t e in t e n s i t i e s a t in te r v a l s o f 5 s e c f o r a t o t a l o f 6 0 se c . I n a ll c a se s p e r c e iv e d i n t e n s i t y in c r e a s e d o v e r t im e , a n d t h e d a t a w e re f i t t e d b y a l t e r n a t e m a th e m a t i c a l f u n c t io n s . A la rg e i n t e r a c t i o n w a s d e m o n s t r a t e d b o t h in v i t r o a n d b y s u b je c t iv e e s t i m a te s , b e tw e e n a n e t h o l e a n d th e h y d r o p h i l i c s u r f a c t a n t , T w e e n 6 0 . T h e in t e r a c t i o n g r e a t ly r e d u c e d th e a n t i c i p a t e d d i f f e r e n c e s in p e r c e iv e d t a s t e i n t e n s i t i e s a m o n g d i f f e r e n t a n e t h o le le v e ls .

F L A V O R A N D S T O R A G E S T A B I L IT Y O F E X P L O S IO N - P U F F E D P O T A T O E S . N o n e n z y m a t i c B ro w n in g . J .F . S U L L I V A N , R .P . K O N - S T A N C E , M .J . C A L H O U N , F .B . T A L L E Y , J . C O R D IN G J R . a n d O . P A N A S I U K . J. Food Sci. 3 9 , 5 8 —6 0 ( 1 9 7 4 ) - S t o r a g e s tu d ie s w e re c o n d u c t e d to e v a lu a te t h e s ta b i l i ty o f e x p lo s i o n - p u f f e d , d r ie d p o t a t o d ic e w i th r e g a rd to th e d e v e l o p m e n t o f o f f - f l a v o r s d u e in p a r t t o t h e f o r m a t i o n o f t h e S t r e c k e r a ld e h y d e s , 2 - m e th y lp r o p a n a l ( 2 M P ) a n d 2 - a n d3 - m e th y lb u ta n a l ( 2 + 3 M B ). D ic e w e re p r e p a r e d f r o m tw o i m p o r t a n t p ro c e s s in g v a r ie t i e s o f p o t a t o e s , a t h ig h a n d lo w s u g a r c o n t e n t . C h r o m a t o g r a p h ic a n a ly s e s a n d o r g a n o le p t i c e v a l u a t io n s w e re m a d e m o n t h l y t o q u a n t i t a t i v e l y e x a m in e th e e x t e n t o f d e t e r i o r a t i o n . T h e r e s u l t s o f th is s to r a g e t e s t s h e w t h a t a l l s a m p le s s to r e d a t 2 3 ° C o r l o w e r r e g a rd le s s o f t h e p a c k a g e a tm o s p h e r e r e m a in s ta b l e w i t h r e g a r d t o b r o w n in g t h r o u g h o u t t h e 1 -y r p e r io d a s i n d i c a t e d b y 2 + 3 M B le v e ls a n d b r o w n in g f la v o r . L o w s u g a r s a m p le s s to r e d a t 3 8 ° C e x h i b i t a lo n g e r s h e l f l ife t h a n th e i r h ig h s u g a r c o u n t e r p a r t s . A s e x p e c t e d , o x id a t iv e o f f - f l a v o r s w e re e n c o u n t e r e d in s a m p le s s to r e d in a i r a t 2 3 ° C a n d s tu d i e s t o c o n t r o l t h e i r f o r m a t i o n a r e b e in g m a d e .

D A T A A N A L Y S I S : A V A R I A B L E S E Q U E N T I A L T E S T F O R S E L E C T I O N O F S E N S O R Y P A N E L S . M .C . G A C U L A J R . , L -A . P A R K E R , J .J . K U B A L A & J . R E A U M E . J. Food Sci. 3 9 , 6 1 - 6 3 ( 1 9 7 4 ) - A n a p p l ic a t i o n o f W a ld ’s s e q u e n t i a l a n a ly s is t o p a n e l s e l e c t i o n is a p p l ie d t o a q u a n t i t a t iv e r e s p o n s e m e a s u r e m e n t , m e a t t e n d e r n e s s . I n a p a i r e d d e s ig n , t h e e q u a l i ty d j = A j - B j = 0 h o ld s in a p a i r o f i d e n t i c a l s a m p le s . I t is s h o w n h a t t h e r e l a t i o n s h ip s E d ; > 0 a n d E d j < 0 c a n b e r e p r e s e n te d b y re g re s -

iii


s io n l in e s t o f o r m b o u n d a r y l in e s f o r s e le c t io n . T h e s e le c t io n o f a p a n e l i s t d e p e n d s o n w h e t h e r h is p e r f o r m a n c e o n te s t s l ie s in s id e o r o u t s id e th e s e b o u n d a r y l in e s . A d e v ia t io n f r o m th e e x p e c t a t i o n E d ; = 0 r e f le c t s th e i n a b i l i ty o f th e c a n d i d a t e t o d i s c r im in a te b e tw e e n s a m p le s . S e q u e n t ia l t e s t s in v o lv in g q u a n t i t a t i v e r e s p o n s e s s c r e e n c a n d i d a t e s f o r c o r r e c t i d e n t i f i c a t i o n o f s a m p le a n d m a g n i tu d e o f d i f f e r e n c e .

S E N S O R Y A N D G A S C H R O M A T O G R A P H IC P R O F I L E S O F C O F F E E B E V E R A G E H E A D S P A C E V O L A T I L E S E N T R A I N E D O N P O R O U S P O L Y M E R S . C .G . T A S S A N & G . F . R U S S E L L . J. Food Sci. 3 9 , 6 4 - 6 8 ( 1 9 7 4 ) —A n e w m e t h o d o f c a p t u r in g a n d c o n c e n t r a t i n g th e h e a d s p a c e v o la t i le s a b o v e b r e w e d c o f f e e b e v e ra g e s w a s in v e s t ig a te d . T h e m e th o d in v o lv e d th e u s e o f a p o r o u s p o ly m e r t o c o n c e n t r a t e t h e v o la t i le s a m p le b y s e le c t iv e r e m o v a l o f t h e w a te r v a p o r p r e s e n t . T h e s a m p le s c o l le c te d p o s s e s s e d a c h a r a c t e r i s t i c c o f f e e a r o m a a n d , w h e n c h r o m a t o g r a p h e d o n p a c k e d g la ss c o lu m n s , p r o v id e d q u a n t i t a t i v e d a t a o n 4 4 p e a k s . E ig h t p e a k s h a d s ig n i f i c a n t ly d i f f e r e n t a re a s d e p e n d in g o n w h e th e r th e b r e w w a s p r e p a r e d w i th d is t i l le d , h a r d , o r s o f t w a te r . S e n s o ry e v a lu a t io n o f t h e a r o m a o f c o f f e e b r e w s m a d e w i th t h e t h r e e ty p e s o f w a te r s h o w e d n o s ig n i f i c a n t d i f f e r e n c e s . T h e a r o m a c o m p o n e n t s p r e s e n t in t h e b r e w i n d ic a t e d t h a t a c o m p o u n d , o r g ro u p o f c o m p o u n d s , h a d a c h a r a c t e r i s t i c c o f f e e c h a r a c t e r . N o n e o f t h e c h r o m a to g r a p h ic a l ly s ig n i f ic a n t p e a k s w e re d e s c r ib e d a s m a jo r c o n t r i b u t o r s t o th e c h a r a c t e r i s t i c c o f f e e a r o m a . D i f f e r e n c e s d id e x i s t b e tw e e n th e d e s c r ip t i o n s a s c r ib e d to th e w h o le c o f f e e b r e w s a n d th o s e a s c r ib e d to t h e in d iv id u a l c o m p o n e n t s c o n s t i t u t i n g t h a t a r o m a . T h e s e d i f f e r e n c e s m a y b e a t t r i b u t e d t o e i t h e r o f tw o p o s s ib le a c t io n s : th e b le n d in g a n d i n t e r a c t io n o f t h e v a r io u s c o m p o u n d s in th e a r o m a , s o m e o f w h ic h w e re s u b o r d in a t e d w h ile n e w a r o m a s w e re c r e a t e d ; o r , d i f f e r e n t i a l th r e s h o ld f o r t h e c o f f e e a r o m a c o m - p o u n d ( s ) a n d o t h e r c o m p o n e n t s .

S O Y B E A N P H O S P H A T ID Y L C H O L IN E D E V E L O P S B I T T E R T A S T E O N A U T O X ID A T I O N . D .J . S E S S A , K . W A R N E R & D .H . H O N IG . J. Food Sci. 3 9 , 6 9 - 7 2 ( 1 9 7 4 ) —S o y b e a n p h o s p h a t i d y l c h o l i n e (S P C ) a n d h y d r o g e n a te d S P C w e re i s o la te d b y c o lu m n c h r o m a to g r a p h y f r o m c o m m e rc ia l l e c i th in a n d h y d r o g e n a te d l e c i t h in , r e s p e c t iv e ly . A q u e o u s s u s p e n s io n s o f th e s e p r e p a r a t i o n s w i th a d d e d C u + + w e re s to r e d a t 2 5 ° C . A s e v e n -m e m b e r t a s t e p a n e l r a t e d d i lu t i o n s c o n ta in in g 0 .1 % p h o s p h o l ip id f o r i n t e n s i t y o f b i t t e r n e s s , b a s e d o n th e s c o r in g s y s te m : 0 = n o n e to 3 = s t r o n g . B o th S P C a n d h y d r o g e n a te d S P C in i t i a l ly r a t e d a s c o re o f 0 .8 . A s c o re o f 1 .6 w a s g iv e n w h e n a s u s p e n s io n o f S P C e x h ib i t e d m a x im u m a b s o r b a n c e d u e to d ie n e c o n ju g a t io n . T h e s c o re in c r e a s e d to 3 .0 a f t e r 4 w k o f s to r a g e . T h e d e v e l o p m e n t o f b i t t e r ta s t e a p p e a r e d to b e a s s o c ia t e d w i th e x t e n t o f o x i d a t i o n o f S P C a s d e t e r m in e d b y th i o b a r b i t u r i c a c id a s s a y . S in c e n o c h a n g e s in t a s t e o c c u r r e d w i th h y d r o g e n a te d S P C t r e a t e d s im i la r ly , b i t t e r n e s s d e v e l o p m e n t is a t t r i b u t e d to a u t o x i d a t i o n o f th e c o n s t i t u e n t u n s a t u r a t e d f a t t y a c id s .

S O M E A R O M A C O M P O N E N T S O F R O A S T E D S E S A M E S E E D ( S e s m u m in d ic u m L .) . C .H . M A N L E Y , P .P . V A L L O N Sc R .E . E R I C K S O N . J. Food Sci. 3 9 , 7 3 - 7 6 ( 1 9 7 4 ) - P r e l i m i n a r y s tu d ie s o f th e b a s ic a n d n e u t r a l a r o m a c o m p o n e n t s o f r o a s t e d s e s a m e s e e d re v e a l th e p r e s e n c e o f m a n y m a te r ia l s k n o w n to b e f o r m e d f r o m p r e c u r s o r s in s im ila r f o o d m a te r ia l s b y th e ro a s t in g p r o c e s s . G r o u p s o f a lk y l p y r a z in e s ,

c y c l o p e n ta p y r a z in e s , f u r y l p y r a z in e s , p h e n o ls , g u a ia c o ls a n d a lk e n y l a ld e h y d e s w e re i n d e n t i f i e d in t h e s te a m d i s t i l l a te s o f t h e s e s a m e s e e d . P r e c u r s o r m a te r ia l s f o r th e d e v e l o p m e n t o f th e s e c o m p o u n d s u p o n r o a s t in g a re a v a i la b le in t h e s e e d a n d a re d is c u s s e d a lo n g w i th t h e p o s s ib le m e c h a n is m s o f f o r m a t io n .

U S E O F A R A D I O M E T R IC T E C H N I Q U E F O R T H E R A P I D D E T E C T I O N O F G R O W T H O F C L O S T ID IA L S P E C IE S . G .M . E V A N C H O , D .H . A S H T O N & A .A . Z W A R U N . J. Food Sci. 3 9 , 7 7 - 7 9 ( 1 9 7 4 ) - A r a d io - m e t r ic t e c h n iq u e ( B a c t e c ) w a s e v a lu a te d a s a p o s s ib le r a p id m e t h o d f o r d e t e c t in g g r o w th o f c lo s t r id ia l s p e c ie s . T ire J L I A n a e r o b ic C u l tu r e M e d iu m 7 A f a i le d to s u p p o r t t h e g r o w th o f s e v e n o f 2 0 c u l tu r e s t e s t e d . S tu d ie s w i th m e d iu m c o n s t i t u e n t s s h o w e d h e m in to b e i n h i b i t o r y . T h e t y p e o f p e p t o n e in f l u e n c e d re c o v e r y t im e , T h i o t o n e p e p t o n e h a v in g th e m o s t f a v o r a b le e f f e c t . A g i t a t i o n d u r in g in c u b a t i o n r e d u c e d r e c o v e r y t im e s b y a s m u c h a s 3 0 % . U s in g th e B a c te c 2 2 5 a n d th e r e c o v e r y m e d iu m d e v e lo p e d , g r o w th f r o m h e a t - s t r e s s e d s p e r e s c o u ld b e d e t e c t e d in a s l i t t l e a s 14 h r . A t im e a d v a n ta g e o f f r o m 2 0 - 1 5 2 h r w a s o b t a i n e d o v e r s t a t i c i n c u b a t i o n in t u b e s o f C o o k e d M e a t M e d iu m . T h e B a c te c m e t h o d o f f e r s a s ig n i f i c a n t t im e a d v a n ta g e o v e r c o n v e n t io n a l c u l tu r a l t e c h n iq u e s w h e n e v e r r a p id d e t e c t i o n o f h e a t - s t r e s s e d o rg a n is m s is e s s e n t ia l a s in s te r i l i ty t e s t in g p r o c e s s e d f o o d s b e f o r e i n c u b a t io n .

E F F E C T O F V A R I O U S P E P T O N E S IN T H E G R O W T H M E D IU M O N T H E A G G L U T I N A T IO N O F T E N S a lm o n e l la S P E C IE S W IT H P O O L E D S P I C E R - E D W A R D S A N T I S E R A . W .J . S T A M P E R & G .J . B A N W A R T . J.Food Sci. 3 9 , 8 0 - 8 2 ( 1 9 7 4 ) - E l e v e n p e p to n e s ( b e e f e x t r a c t , c a s i to n e , g e ly s a te , p e p t o n e , p e p to n iz e d m ilk , p r o t e o s e p e p t o n e # 2 , p r o t o n e , so y - to n e , t r y p t o n e , t r y p t i c a s e a n d y e a s t e x t r a c t ) a d d e d in c o n c e n t r a t i o n s o f 0 .5 , 1 a n d 2 % to a b a s a l g r o w th m e d iu m , w e r e t e s t e d f o r t h e i r e f f e c t o n th e H a g g lu t in a t io n t e s t . T h e b a s a l g r o w th m e d iu m c o n s i s te d o f 2 .5 g d e x t r o s e , 5 g N a C l, 5 g K 2 H P 0 4 a n d 1 l i t e r d i s t i l l e d w a te r . T h e b r o t h s w e re i n o c u la t e d , i n c u b a t e d 2 4 h r a t 3 7 ° C a n d s a m p le d fo r t h e f la g e l la r H a g g lu t in a t io n t e s t . A c o m p a r i s o n w a s m a d e b e tw e e n th e p e p t o n e b r o t h s a n d o t h e r r e c o m m e n d e d b r o t h s (H b r o t n , M b r o t h , t r y p t i c a s e s o y t r y p - to s e b r o t h a n d b r a in h e a r t in f u s io n b r o t h ) . P r o t e o s e p e p t o n e # 2 y ie ld e d s ig n i f i c a n t ly g r e a te r n u m b e r s o f p o s i t iv e a g g lu t in a t io n s t h a n d i d a n y o f th e o t h e r p e p to n e s o r t h a n a n y o f t h e p r e s e n t l y r e c o m m e n d e d b r o th s . T h e n u m b e r o f p o s i t iv e a g g lu t in a t io n s in c r e a s e d w i th in c r e a s in g p e p t o n e c o n c e n t r a t i o n . T h e 2 -h r o b s e r v a t io n s w e re s ig n i f i c a n t ly g r e a te r t h a n th e r e s u l ts o b s e r v e d a f t e r 1 h r . T h e s e r e s u l t s i n d i c a t e t h a t c e r t a in p e p t o n e s in th e g r o w th m e d iu m s ig n i f i c a n t ly in c r e a s e t h e e f f e c t iv e n e s s o f t h e H t e s t .

C A R B O N Y L P R O D U C T IO N F R O M L I P O L Y Z E D M IL K F A T B Y T H E C O N T IN U O U S M Y C E L IA L C U L T U R E O F P é n ic i l l iu m r o q u e f o r t i . B .K . D W IV E D I & J .E . K I N S E L L A . J. Food Sci. 3 9 , 8 3 - 8 7 ( 1 9 7 4 ) - C o n - t i n u o u s c u l tu r e s o f Pénicillium roqueforti m y c e l iu m m e ta b o l i z e d f r e e f a t t y a c id s f r o m m ilk f a t i n t o C 0 2 a n d c a r b o n y l c o m p o u n d s . M e th y l k e to n e s , i . e . , 2 - p e n ta n o n e , 2 - h e p ta n o n e . 2 - n o n a n o n e a n d 2 - u n d e c a n o n e w e re t h e m a jo r c a r b o n y l c o m p o u n d s p r o d u c e d . O c ta n o i c a n d d e c a n o ic a c id s s e e m e d to b e p r e f e r e n t i a l l y u t i l i z e d . F r e e f a t t y a c id le v e l , i n i t i a l p H o f g r o w th m e d iu m , a g e o f m y c e l iu m a n d s a l t c o n c e n t r a t i o n i n f l u e n c e d th e l e n g th o f in i t i a l la g p h a s e b e f o r e f a t t y a c id u t i l i z a t i o n a n d c a r b o n y l p r o d u c t i o n o c c u r r e d . T h e r e la t iv e p r o p o r t i o n o f t h e m a jo r m e t h y l k e to n e s m a y b e a l t e r e d b y v a ry in g t h e f r e e f a t t y a c id le v e l in c u l t u r e m e d i u m .

IV

A C T IV IT Y A N D S T A B I L I T Y O F /3 -G A L A C T O S ID A S E IM M O B IL IZ E D O N P O R O U S G L A S S . E .S . O K O S & W .J . H A R P E R . / . F o o d S c i . 3 9 , 8 8 - 9 3 ( 1 9 7 4 ) - f l - g a l a c to s id a s e (E C 3 .2 .1 .2 3 ) w a s b o u n d to a n a ry l- a m in e d e r iv a t iv e o f p o r o u s g la ss b y a m o d i f i c a t i o n o f t h e a z o - b in d in g m e th o d . C o lu m n r e a c t o r a s s a y s a t 4 0 ° C a n d p H 3 .5 h a v e s h o w n t h a t la c to s e w a s h y d r o l y z e d a t a p p r o x im a te ly 3 .0 x 1 0"4 m o le s /m in p e r g d ry w e ig h t o f b o u n d s u p p o r t . C o m p a r i s o n o f s o lu b le e n z y m e (S E ) a n d im m o b i l iz e d e n z y m e ( I M E ) h y d r o ly s i s r a te s , a t v a r io u s d e g re e s o f s u b s t r a t e c o n v e r s io n , in d i c a t e d t h a t a m a x im u m o f 9 0 % o f t h e e n z y m e a c t iv i ty w a s re c o v e r e d in t h e IM E p r e p a r a t i o n . W ith p u r e l a c to s e s o lu t io n s , o p e r a t io n a l l ife o f t h e IM E in c o lu m n r e a c t i o n a t 4 0 ° C w i t h o u t m ic r o b io lo g ic a l c o n t r o l w a s d e t e r m in e d to b e 3 d a y s . L o s s o f e n z y m e a c t iv i ty b y th e IM E w a s r e l a t e d t o m o ld g r o w th w i th in t h e e n z y m e b e d . T h e k in e t i c s o f t h e IM E r e a c t io n w e re e x p r e s s e d b e s t w i t h p lo t s o f f r a c t i o n a l s u b s t r a t e c o n v e r s io n p l o t t e d a g a in s t t h e w e ig h t o f t h e c a t a l y s t / m o l a r f lo w r a t e o f s u b s t r a te (X vs. W /F ) . W /F p lo t s w e re a f f e c t e d s l ig h t ly b y in i t i a l s u b s t r a t e c o n c e n t r a t i o n s . C o lu m n c o n v e r s io n s o f u p to 8 7 % w e re a c h ie v e d w i th 5% la c to s e s o lu t io n s . W ith s u b s e q u e n t lo n g t e r m c o lu m n r u n s o n w h e y , t h r e e p r o b le m s w e re e n c o u n t e r e d : ( a ) d e v e l o p m e n t o f m o ld in t h e c o lu m n in 3 d a y s a t 4 0 ° C ; ( b ) c lo g g in g o f t h e c o lu m n a t 4 0 ° C , d u e to m o ld a c c u m u la t io n a n d p r o t e i n p r e c i p i t a t i o n ; a n d (c ) a p a r t i a l i r r e v e r s ib le i n a c t iv a t io n o f th e e n z y m e . T h e d e g re e o f i n a c t iv a t io n u n d e r t h e c o n d i t i o n s r e p o r t e d in o n e w h e y s y s te m w a s n o t r e l a t e d t o m o ld g r o w th a n d w a s a p p r o x im a te ly 38% .

B E N Z Y L I S O T H I O C Y A N A T E A S A N A T U R A L L Y O C C U R R I N G P A P A IN I N H I B I T O R . C -S T A N G . / . F o o d S c i. 3 9 , 9 4 - 9 6 ( 1 9 7 4 ) — B e n z y lg lu c o s in o la te w a s f o u n d to b e a g e n e r a l c o n t a m i n a n t o f c o m m e r c ia l p a p a in . U p o n t h e e n z y m a t i c h y d r o ly s i s w i th th io g lu c o s id a s e , th is c o n t a m i n a n t p r o d u c e d b e n z y l i s o t h i o c y a n a t e (B IT C ) w h ic h s u b s e q u e n t ly i n h ib i t e d p a p a in a c t iv i ty . T h e i n h i b i t o r y e f f e c t o f B IT C c o u ld b e p r e v e n te d b y s u b s t r a t e p r o t e c t i o n o f t h e e n z y m e o r b y th e a d d i t i o n o f e x c e s s iv e a m o u n t o f c y s te in e . A K i o f 3 .9 X 1 0 '6 M w a s e s t i m a te d b y a L in e w e a v e r -B u rk p lo t .

Z O N E E L E C T R O P H O R E S I S O F F O O D S T A B I L I Z E R S IN M A L O N A T E B U F F E R . J .C . C H A N G , M .W . R E N O L L & P .M .T . H A N S E N . J . F o o d S c i . 3 9 , 9 7 - 1 0 2 ( 1 9 7 4 ) - M i x t u r e s o f c o m m o n f o o d s ta b i l i z e r s ( p o l y s a c c h a r id e g u m s ) w e re e x a m in e d b y z o n a l e l e c t r o p h o r e s i s o n c e l lu lo s e a c e ta te s t r ip s ( P h o r o S l id e s ) . A n io n ic s ta b i l iz e r s w e re s e p a r a te d in 0 .0 7 5 M s o d iu m m a lo n a t e b u f f e r , p H 2 .9 a n d th e z o n e s w e re s ta in e d b y to lu id in e b lu e . N o n io n ic s ta b i l i z e r s d id n o t m ig r a te in m a lo n a t e b u f f e r b u t w e re s e p a r a te d in b o r a t e b u f f e r , p H 1 0 a n d c o u ld b e v is u a l iz e d b y th e p e r io d ic a c i d - S c h i f f r e a g e n t . T h e s e m e t h o d s w e re u s e d f o r t h e q u a l i t a t i v e d e t e r m in a t io n o f s ta b i l iz e r m ix tu r e s r e c o v e r e d f r o m a t r y p t i c d ig e s t o f p a s t e u r iz e d m ilk p r o d u c t s b y s o lv e n t p r e c i p i t a t i o n o f t h e h y d r o c o l lo id s f r o m th e f r a c t i o n s o lu b le in 1 2 .5 % t r i c h l o r o a c e t i c a c id . T h e r e s u l t s r e v e a le d t h a t a m ix tu r e o f ^ - c a r r a g e e n a n , k -c a r r a g e e n a n , a lg in a te a n d C M C , e a c h p r e s e n t a t 0 .1 % in s k im m ilk o r c h o c o l a t e m i lk , c o u ld b e r e c o v e r e d a n d t h a t th e in d iv id u a l c h a r a c t e r i s t i c s o f t h e z o n e s w e re n o t a l t e r e d b y th e t r e a t m e n t . A m ix tu r e o f lo c u s t b e a n g u m a n d g u a r g u m w e re a lso r e c o v e r e d i n t a c t f r o m s k im m ilk a n d w e r e d i f f e r e n t i a t e d b y e l e c t r o p h o r e s i s in b o r a t e b u f fe r . E l e c t r o p h o r e t i c i d e n t i f i c a t i o n o f m i x tu r e s o f n o n io n ic a n d a n io n ic s ta b i l iz e r s i n b o r a t e b u f f e r w a s n o t d e e m e d p o s s ib le b e c a u s e o f s t r e a k in g z o n e s . I m p r o v e d p a t t e r n s f o r c a r r a g e e n a n w e re o b t a in e d in 0 .0 7 5 M c a l c iu m m a lo n a t e b u f f e r , p H 2 .9 , to w h ic h 1 5 % e t h a n o l ( V /V ) h a d b e e n a d d e d . U n f r a c t i o n a t e d c a r r a g e e n a n (C h o n d r u s c r is p u s ) p r o d u c e d o n e s t a t i o n a r y a n d tw o m ig r a t in g z o n e s . E l e c t r o p h o r e t i c p a t t e r n s in th i s b u f fe r o f t h e h y d r o c o l lo id r e c o v e r e d f r o m s te r i l i z e d m i lk c o n c e n t r a t e s p r o d u c e d e v id e n c e o f in c o m p le t e i s o la t io n o f c a r r a g e e n a n o r p o s s ib ly s t r u c tu r a l a l t e r a t i o n s d u r in g p ro c e s s in g .

b y z o n a l e l e c t r o p h o r e s i s . C a r r a g e e n a n s ta b i l i z e r s ( 0 .4 % ) w e re i n c o r p o r a t e d b y m o d e r a t e h e a t in g ( 6 0 ° C / 3 0 m in ) i n t o a s y n t h e t i c m i lk - s a l t s y s te m o f v a ry in g p H b e tw e e n 4 .6 a n d 6 .7 a n d e x p o s e d to h e a t t r e a t m e n t s a t 1 2 2 ° C f o r u p to 3 0 m in . T h e r e s u l t in g s o lu t io n s w e re t r e a t e d w i th c a t io n e x c h a n g e re s in in t h e s o d iu m f o r m a n d e x a m in e d b y z o n a l e l e c t r o p h o re s is o n c e l lu lo s e a c e t a t e m e m b r a n e s ( 0 .0 7 5 M c a l c iu m m a lo n a t e b u f f e r , p H 2 .9 , w i th 15% E tO H a d d e d , v /v ) f o l l o w e d b y s ta in in g w i th t o lu id in e b lu e . « - C a r r a g e e n a n p r e p a r e d b y p o ta s s iu m f r a c t i o n a t i o n o f C h o n d r u s c r is p u s e x t r a c t s p r o d u c e d o n e s t a t i o n a r y a n d tw o m ig r a t in g z o n e s . H e a t in g c a u s e d a r e d u c t i o n in t h e i n t e n s i t y o f t h e s t a t i o n a r y z o n e a n d a n in c re a s e in t h e m o b i l i t y a n d s e p a r a t i o n o f t h e m ig r a t in g z o n e s . T h e s e c h a n g e s w e re l e a s t a t p H 6 .7 a n d w e re a c c e l e r a t e d w i th a d e c r e a s e in p H . k -C a r r a g e e n a n f r o m p o ta s s iu m f r a c t i o n a t i o n o f C h o n d r u s c r is p u s e x t r a c t s p r o d u c e d a d i f f u s e m ig r a t in g z o n e b u t n o s t a t i o n a r y z o n e . H e a t t r e a t m e n t o f t h i s f r a c t i o n a t p H 6 .7 r e s u l t e d in a n i n c r e a s e d m o b i l i t y o f th e z o n e . A lk a l i - c o n v e r te d , u n f r a c t i o n a t e d c a r r a g e e n a n p r o d u c e d a p a t t e r n s im ila r to t h e k a p p a f r a c t i o n a n d r e s p o n d e d to h e a t a n d p H c h a n g e s in a s im ila r m a n n e r .

M E C H A N IS M O F E M U L S I F I E R A C T IO N IN A N IC E C R E A M S Y S T E M . P . L I N & J .G . L E E D E R . J. F o o d S c i. 3 9 , 1 0 8 - 1 1 1 ( 1 9 7 4 ) - T h e i n t e r a c t io n s o r c o n p l e x i n g s b e tw e e n e m u ls i f i e r s a n d m ilk p r o t e i n s w e re s tu d ie d b y m e a n s c f e l e c t r o p h o r e t i c t e c h n iq u e s . T h e r e s u l t s s h o w e d th a t t h e r e w e re n o s ig n i f i c a n t i n t e r a c t i o n s w i th in t h e s y s te m . T h e e f f e c t o f f r e e z in g o n th e f a t d e e m u l s i f i c a t i o n o f ic e c r e a m m ix e s c o n ta in in g e m u l s if ie rs o f d i f f e r e n t H L B ’s w e re c o m p a r e d a n d f o u n d to b e s ig n i f ic a n t . A m e c h a n is m is p r o p o s e d to e x p la in t h e a c t i o n o f e m u ls i f i e r s a n d th e i r e f f e c t o n f a t d e e m u l s i f i c a t i o n in a n ic e c r e a m s y s te m .

D E C R E A S E O F L I N O L E A T E O X I D A T I O N R A T E D U E T O W A T E R A T IN T E R M E D I A T E W A T E R A C T IV IT Y . T .P . L A B U Z A & H E . C H O U . J . F o o d S c i . 3 9 , 1 1 2 —1 1 3 ( 1 9 7 4 ) - T h e r a t e o f o x i d a t i o n o f m e th y l l i n o l e a t e in i n t e r m e d i a t e m o i s tu r e c o n t e n t m o d e l s y s te m s s h o w e d u n u s u a l b e h a v io r . A s h a d b e e n f o u n d b y o t h e r r e s e a r c h a t lo w t r a c e m e ta l c o n t e n t , in c r e a s in g th e A w o f t h e s y s t e m , a n d th u s t h e w a te r c o n t e n t , i n c r e a s e d t h e o x i d a t i o n r a t e . S y s te m s a t s im i la r A w b u t h ig h e r m o is tu r e c o n t e n t a ls o o x id i z e d f a s te r . T h i s is d u e t o t h e i n c r e a s e d m o b i l i t y in t h e d i l u t e a q u e o u s p h a s e . O n th e o t h e r h a n d , a t h ig h t r a c e m e ta l s ( ~ 1 0 0 0 p p m ) , e x a c t ly t h e o p p o s i t e e f f e c t o c c u r r e d w i th r e s p e c t t o A w s in c e in th i s c a s e , t h e e f f e c t o f d i l u t i o n p r e d o m i n a t e d . I t w a s s h o w n t h a t o x id a t i o n is d i r e c t l y d e p e n d e n t o n t o t a l m o i s tu r e c o n t e n t , s u p p o r t i n g th e a b o v e c o n c lu s io n s .

B O U N D W A T E R C A P A C I T Y O F C O R N S T A R C H A N D IT S D E R I V A T I V E S B Y N M R . I . M O U S S E R I , M .P . S T E IN B E R G , A .I . N E L S O N &L .S . W E I. J . F o o d S c i. 3 9 , 1 1 4 - 1 1 6 ( 1 9 7 4 ) - B o u n d W a te r C a p a c i ty (B W C ) o f c o rn s t a r c h , w a x y m a iz e s t a r c h , p r e g e la t in iz e d c o rn s ta r c h , h y d r o p h i l i c s t a r c h , c o rn s y r u p , m a l to s e a n d d e x t r o s e w a s d e t e r m in e d b y W id e -L in e N M R . C o rn s ta r c h a n d i t s h y d r o l y t i c p r o d u c t s h a d th e s a m e B W C o f 2 4 % o n a w e t b a s is . P r e g e la t in iz e d s ta r c h h a d a B W C o f 2 4 .3 % w h ile w a x y m a iz e s ta r c h a n d h y d r o p h i l i c s t a r c h h a d a B W C o f 2 8 .1 a n d 2 6 .6 % , re s p e c t iv e ly . T h e u s u a l s t r a ig h t l in e r e l a t i o n a t h ig h m o is tu r e s o b t a in e d w i th s ta r c h a n d o t h e r m a c r o m o le c u l e s w a s n o t o b t a in e d w i th th e c o r n s y r u p s a n d m a l to s e . I n s te a d , a c u rv i l in e a r r e l a t i o n w a s o b ta in e d . R e p l o t t i n g th e d a t a o n a D M b a s is s h o w e d t h a t a d d i t i o n o f l g f r e e w a te r c a u s e d e a c h g ra m d r y s u g a r t o lo s e 0 . 0 3 1 2 g b o u n d w a te r . In in v e s t ig a t in g th e N M R s ig n a l o b t a in e d f r o m c o r n s ta r c h , a t a m o i s tu r e c o n t e n t n e a r th e B W C , i t w a s f o u n d t h a t a lo n g e r p e r io d o f t im e w a s r e q u i r e d to a c h ie v e e q u i l ib r iu m t h a n a t lo w e r o r h ig h e r m o i s tu r e c o n te n t s .

H E A T -IN D U C E D C H A N G E S IN T H E Z O N E E L E C T R O P H O R E T I C M IC R O S T R U C T U R E O F M O D I F I E D T A P IO C A S T A R C H -M IL K G E L S . P A T T E R N S O F C A R R A G E E N A N S T A B I L I Z E R S . P .M .T . H A N S E N & L .F . H O O D , A .S . S E I F R I E D & R . M E Y E R . J . F o o d S c i. 3 9 , 1 1 7 - 1 2 0M .W . R E N O L L . J . F o o d S c i. 3 9 , 1 0 3 - 1 0 7 ( 1 9 7 4 ) - T h e p o s s ib i l i ty o f ( 1 9 7 4 ) - T h e u l t r a s t r u c t u r e o f h y d r o x y p r o p y l d i s ta r c h p h o s p h a te - s k im h e a t - in d u c e d a l t e r a t i o n s o f c a r r a g e e n a n in s ta b i l i z e d m i lk w a s e x a m in e d m ilk g e ls w a s e v a l u a te d w i th l ig h t , s c a n n in g (S E M ) a n d tr a n s m is s io n

V


(T E M ) e l e c t r o n m ic r o s c o p y . S E M o f th e u n g e la t in iz e d g r a n u le s s h o w e d t h a t c h e m ic a l m o d i f i c a t i o n d id n o t a f f e c t t h e s u r f a c e n o r th e s ize o f th e g r a n u le . T w o ty p e s o f g e la t in iz e d g r a n u le s w e re o b s e r v e d w i th t h e T E M . O n e h a d a h o m o g e n e o u s g r a n u la r t e x t u r e t h r o u g h o u t w h ile t h e o t h e r h a d a g r a n u la r c o a t a n d a d i s p e r s e d le ss d e n s e c o r e . T h e c o a t - c o r e t y p e g r a n u le m a y b e t h e r e s u l t o f t h e c h e m ic a l m o d i f i c a t i o n . C a s e in m ic e l le s u b u n i t s w e re e v id e n t . M ic e lle s w e re n o t a g g re g a te d t o g e t h e r n o r w a s t h e r e a n y e v id e n c e o f a c o n t i n u o u s n e t w o r k a m o n g th e m ic e l le s , o r t h e m ic e l le s a n d th e s t a r c h g r a n u le s .

E F F E C T O F F R O Z E N S T O R A G E O N T H E M IC R O S T R U C T U R E A N D S Y N E R E S IS O F M O D I F I E D T A P IO C A S T A R C H -M IL K G E L S . L .F .H O O D & A .S . S E I F R I E D . J. Food Sci. 3 9 , 1 2 1 - 1 2 4 ( 1 9 7 4 ) G e ls p r e p a r e d w i th m o d i f i e d t a p io c a s ta r c h ( h y d r o x y p r o p y l d i s t a r c h p h o s p h a te ) in s k im m ilk w e re s to r e d f o r u p to 6 0 d a y s a t —3 to - 3 2 CC . T h e e f f e c t o f c y c l ic f r e e z in g a n d th a w in g a n d a u t o m a t i c d e f r o s t f r e e z e r s o n g e l s t r u c tu r e w a s e v a l u a te d b y e l e c t r o n m ic r o s c o p y . S y n e re s is w a s d e t e r m in e d . C o a t - c o r e t y p e g r a n u le s d i s a p p e a r e d a f t e r s e v e ra l f r e e z e - th a w c y c le s . S ta r c h g r a n u le s w e re r u p t u r e d b y f r e e z in g a n d th a w in g a n d th e n o n g r a n u - la r s t a r c h w a s d is p e r s e d t h r o u g h o u t th e c o n t i n u o u s p h a s e . C a s e in m ic e l le s w e re d i s t o r t e d a n d th e s u b u n i t s w e re a lm o s t c o m p le t e l y d is a g g r e g a te d a f t e r 6 0 d a y s . S y n e re s is i n c r e a s e d w i th t im e b u t t h e a m o u n t o f in c r e a s e v a r ie d w i th t h e c o n d i t i o n s o f f r o z e n s to r a g e .

R A P ID T E C H N I Q U E S F O R S A L T -C U R IN G F I S H . A R e v ie w . J .M . M E N D E L S O H N . / . Food Sci. 3 9 , 1 2 5 - 1 2 7 ( 1 9 7 4 ) - T h e in c re a s in g n e e d f o r h ig h q u a l i t y s a l t e d f is h c o m b in e d w i th th e o b v io u s d is a d v a n ta g e s o f c o n v e n t io n a l s a l t in g p r o c e d u r e s h a v e p r o v id e d a n i m p e tu s t o d e v e lo p r a p id s a l t in g t e c h n iq u e s . I n t h i s a r t i c le th e m a jo r m e t h o d s f o u n d in th e l i t e r a t u r e f o r t h e r a p id s a l t - c u r in g o f f is h a re re v ie w e d in c lu d in g a d i s c u s s io n o f th e a d v a n ta g e s a n d d is a d v a n ta g e s o f e a c h m e th o d .

E V A L U A T IO N O F B E T A L A IN P IG M E N T S A S S U B S T IT U T E S A U S A G E C O L O R A N T S . J .H . v o n E L B E , J .T . K L E M E N T , C .H . A M U N D S O N , R .G . C A S S E N S & R .C . L IN D S A Y . J. Food Sci. 3 9 , 1 2 8 - 1 3 2 ( 1 9 7 4 ) - R e c e n t l y , t h e a d d i t i o n o f s o d iu m n i t r i t e a n d n i t r a t e a s a f o o d a d d i t iv e h a s b e e n q u e s t i o n e d , a n d i f i t s u s e is p r o h i b i t e d , a n e w c o lo r s y s te m to p r o d u c e a t y p i c a l c u r e d m e a t h u e w o u ld b e n e e d e d . B e ta la in p ig m e n ts f r o m b e e t s w e re u s e d a s c o lo r r e p l a c e m e n t s in c o o k e d , s m o k e d b o lo g n a a n d in a s e m i d r y , f e r m e n te d s u m m e r s a u s a g e . P r o d u c t s c o n t a i n in g v a r io u s le v e ls o f b e t a l a in p ig m e n ts w e re c o m p a r e d to c o n t r o l p r o d u c t s c o n ta in in g n o n i t r i t e - n i t r a t e s a l t s o r t h e m a x im u m le g a l p e r m i t t e d le v e ls . A t in te r v a l s d u r in g s to ra g e a t 4°,C f o r 14 d a y s , t h e c o lo r , f la v o r a n d m ic r o b io lo g ic a l c h a r a c t e r i s t i c s w e re e v a lu a te d . C u r e d m e a t c o lo r s w e re s im u la te d to a h ig h d e g re e w i th s o m e le v e ls o f b e t a l a .n p ig m e n t . T h e c o lo r o f b e t a l a in - c o n t a in i n g s a u s a g e s p ro v e d to b e m o r e s ta b l e t o l ig h t e x p o s u r e d u r in g s to r a g e th a n th e c o lo r o f th o s e c o n ta in in g n i t r i t e - n i t r a t e s a l t s . S m a ll p a n e l s ( c a . 3 5 ) in d i c a t e d n o s ig n i f i c a n t o v e ra l l p r e f e r e n c e f o r s a m p le s p r e p a r e d w i th n i t r i t e - n i t r a t e s a l t s o v e r s a m p le s p r e p a r e d w i th o p t i m u m le v e ls o f b e t a l a in p ig m e n t . H o w e v e r , e x p e r t t a s t e r s w e re a b le to d e t e c t s u b t le f la v o r a n d c o lo r d i f f e r e n c e s . M ic r o b ia l b e h a v io r w a s s im ila r f o r e a c h t y p e o f s a u s a g e f o r t o t a l s p o r e a n d p la te c o u n t s d u r in g s to r a g e . R e s u l t s o f th i s s t u d y in d ic a te t h a t t h e p o t e n t i a l a p p l i c a t i o n o f b e ta la in s i n s o m e s a u s a g e s is f e a s ib le .

E F F E C T S O F D IE T A R Y F A T A N D d l-c r -T O C O P H E R Y L O N S T A B I L IT Y C H A R A C T E R I S T I C S O F P R E C O O K E D F R O Z E N B R O I L E R P A R T S . J .E . W E B B , C .C . B R U N S O N & J .D . Y A T E S . J. Food Sci. 39, 1 3 3 - 1 3 6 ( 1 9 7 4 ) - T h e e f f e c t s o f s e v e ra l f a t s o u r c e s a n d d l - a - t o c o p h e r y l a c e t a t e s u p p l e m e n t a t i o n o f b r o i l e r fe e d s o n io d in e n u m b e r s o f d e p o t f a t , t a s t e p a n e l r e s p o n s e s a n d T B A v a lu e s o f p r e c o o k e d , f r o z e n b r o i l e r th ig h

a n d d r u m s t i c k (T a n d D ) p a r t s w e re s tu d i e d . V a r ia b le s e v a l u a te d w e r e 1 8 c o m m e r c ia l f e e d -g ra d e f a t s o f v a r io u s s a t u r a t i o n le v e ls (5 % o f r a t i o n ) w i th a n d w i t h o u t 1 1 .2 I .U . o f a d d e d v i ta m in E /k g o f f e e d . T h e b i r d s w e r e p r o c e s s e d , d e p o t f a t s a m p le s r e m o v e d a n d p a r t s d e e p - f a t f r i e d a n d f r o z e n a t - 4 0 ° C . I o d in e n u m b e r s r e v e a le d t h a t t h e s a t u r a t i o n le v e l o f d i e t a r y f a t in f lu e n c e d d e p o t f a t s a tu r a t i o n . A s ig n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t ( 0 .9 9 ) w a s o b ta in e d fo r t h e d i e t a r y f a t io d in e n u m b e r s vs. t h e d e p o t f a t io d in e n u m b e r s . W h e n v i ta m in E s u p p l e m e n t a t i o n e f f e c t w a s e v a l u a t e d w i th in e a c h s t u d y , T B A n u m b e r s w e re s ig n i f i c a n t ly lo w e r f o r D p a r t s f r o m v i ta m in E s u p p le m e n te d b i r d s th a n f o r D p a r t s f r o m n o n s u p p le - m e n te d b i r d s (P < 0 .0 5 ) . T a s te p a n e l r a n c id i ty s c o r e s d id n o t d i f f e r s ig n i f i c a n t ly d u e t o v i t a m in E s u p p le m e n ta t i o n . O f f - f la v o r s c o r e s w e re v a r ia b le , b u t r e f l e c t e d a b e n e f ic ia l e f f e c t f r o m v i ta m in E s u p p l e m e n t a t io n . S ig n i f i c a n t d i f f e r e n c e s (P < 0 .0 5 ) w e re n o t e d b e tw e e n th e v a r io u s f a t d ie t s f o r T B A n u m b e r s a n d o f f - f la v o r s c o r e s b u t n o t f o r r a n c id i ty s c o re s . W h e n io d in e n u m b e r s f o r d e p c t f a t f r o m in d iv id u a l b i r d s w e r e c o r r e la te d w i th t h e i r c o r r e s p o n d in g T B A n u m b e r s a n d p a n e l s c o r e s , n o c o n s i s t e n t e f f e c t o f s a tu r a t i o n le v e l o f d e p o t f a t o n s t a b i l i t y o f b r o i l e r p a r t s w a s d e t e c t e d .

S O Y A A D D IT I V E S IN B E E F P A T T IE S . M .D . J U D G E , C .G . H A U G H ,G .L . Z A C H A R I A H , C .E . P A R M E L E E & R .L . P Y L E . J. Food Sci. 3 9 , 1 3 7 - 1 3 9 ( 1 9 7 4 ) - R e s e a r c h w a s c o n d u c t e d t o e v a l u a te t h e e f f e c t s o f tw o s o y a p r o d u c t s ( s o y a f l o u r a n d s o y a p r o t e i n c o n c e n t r a t e ) o n s e v e ra l m e a s u re s o f q u a l i t y in g r o u n d b e e f p a t t i e s . T h e r e s u l t s i n d i c a t e d t h a t t h e i n i t i a l le v e ls o f f r e s h m e a t s p o ila g e o r g a n is m s w e re in c r e a s e d s l ig h t ly b y th e s o y a f l o u r . H o w e v e r , n e i t h e r t h e f l o u r n o r t h e s o y a p r o t e i n c o n c e n t r a t e r e s u l t e d in i n c r e a s e d b a c t e r i a l n u m b e r s a t t h e e n d o f a 7 -d a y s to r a g e p e r io d . G r o u n d m e a t c o n ta in in g t h e s o y a p r o d u c t s in c o m b i n a t i o n w i th 2 0 % f a t h a d g r e a t e r l ig h t r e f l e c t i o n th a n c o n t r o l s a m p le s . W ith m e a t c o n ta in in g 3 0 % f a t , t h e s o y a p r o t e i n c o n c e n t r a t e i n c r e a s e d l ig h t r e f l e c t io n in p a t t i e s t h a t w e re s t o r e d f o r a t l e a s t 3 h r in t h e u n f r o z e n s t a t e , w h e re a s t h e s o y a f l o u r d e c r e a s e d th e r e f l e c t i o n u n d e r a ll s to r a g e c o n d i t io n s t e s t e d . T h e s o y a a d d i t iv e s d e c r e a s e d c o o k in g s h r in k a g e in a l l t e s t s c o n d u c t e d . A t h ig h le v e ls o f u s e th e f l o u r r e d u c e d s h r in k a g e t o a g r e a te r e x t e n t t h a n th e c o n c e n t r a t e , b u t a t lo w a d d i t iv e le v e ls , t h e s h r in k a g e w a s n e a r ly i d e n t i c a l b e tw e e n t h e t w o s o y a p r o d u c t s . P a t t i e s t h a t h a d b e e n f r o z e n s h r a n k m o r e t h a n u n f r o z e n p a t t i e s . T h e e a s e o f r e m o v a l o f i n t e r le a v in g p a p e r f r o m f r o z e n p a t t i e s w a s h ig h ly d e p e n d e n t o n t h e f a t c o n t e n t o f t h e m e a t . W h e re a s , t h e s o y a a d d i t iv e s h a d n o e f f e c t o n p a p e r r e le a s e f o r c e in p a t t i e s c o n ta in in g 2 0 % f a t , t h e a d d i t iv e s i n c r e a s e d th e f o r c e r e q u i r e d f o r p a p e r r e m o v a l f r o m t h o s e c o n ta in in g 3 0 % f a t .

H O T B O N IN G A N D V A C U U M P A C K A G IN G O F E I G H T M A J O R B O V IN E M U S C L E S . G .R . S C H M ID T & S. K E M A N . J. Food Sci. 3 9 , 1 4 0 - 1 4 2 ( 1 9 7 4 ) - S i x A n g u s s te e r s w e r e u t i l i z e d in a s t u d y o f h o t b o n in g a n d v a c u u m p a c k a g in g v e r s u s c o ld b o n in g o f e ig h t m a jo r b o v in e m u s c le s . T h e m u s c le s s a m p le d w e re : a n t e r i o r lo n g is s im u s d o rs i ( A L D ) , p o s t e r io r lo n g is s im u s d o r s i (P L D ) , p s o a s m a jo r (P M ) , g lu te u s m e d iu s (G M ), s e m i t e n d in o s u s (S T ) , s e m i m e m b r a n o s u s (S M ) , b ic e p s fe - m o r i s ( B F ) , a n d q u a d r ic e p s f e m o r i s ( Q F ) . T h e r e t a i l y ie ld o f h o t - b o n e d a n d v a c u u m -p a c k a g e d s id e s w a s s ig n i f i c a n t ly h ig h e r (P < 0 .0 5 ) c o m p a r e d w i th t h e c o ld - b o n e d s id e ; h o w e v e r , p e r c e n t f a t a n d b o n e w e re n o t s ig n i f i c a n t ly d i f f e r e n t . T h e d i f f e r e n c e in r e ta i l y ie ld w a s d u e to t h e g r e a te r a m o u n t o f f a t r e m a in in g o n c u t s p r e p a r e d f r o m th e h o t - b o n e d s id e s . T a s te p a n e l e v a lu a t io n a n d W a rn e r B r a tz le r s h e a r f o r c e d e t e r m i n a t i o n f a i le d to d e t e c t a n y s ig n if ic a n t d i f f e r e n c e s b e tw e e n h o t - a n d c o l d - b o n e d s te a k s o r r o a s t s . T w o m e t h o d s o f f ib e r d i a m e te r m e a s u r e m e n t s h o w e d th a t m o s t h o t - b o n e d m u s c le s in c r e a s e d in f ib e r d i a m e te r c o m p a r e d w i th th e c o n t r o l . A s ig n if ic a n t in c r e a s e (P < 0 .0 5 ) o f f ib e r d i a m e te r o f t h e h o t - b o n e d o v e r t h e c o ld - b o n e d m u s c l e w a s o b s e r v e d in t h e P L D , P M , G M a n d S T . A n o n s ig n i f i c a n t (P < 0 .0 5 ) in c r e a s e w a s f o u n d in t h e o t h e r m u s c le s o b s e r v e d .

vi

E F F E C T O F V A C U U M P A C K A G IN G O N W E IG H T L O S S , M IC R O B IA L G R O W T H A N D P A L A T A B I L I T Y O F F R E S H B E E F W H O L E S A L E C U T S . J .H . H O D G E S , V .R . C A H I L L & H .W . O C K E R M A N ./ . Food Sci. 3 9 , 1 4 3 - 1 4 6 ( 1 9 7 4 ) - C a r c a s s e s o f h ig h a n d lo w q u a l i t y g r a d e w e re h e ld f o r 1 a n d 1 5 d a y s p o s t m o r t e m p r i o r t o c u t t i n g , s a m p l in g a n d p a c k a g in g . W h o le s a le c u t s w e r e p a c k a g e d u n d e r v a c u u m a n d s t o r e d f o r 2 8 d a y s . W e ig h t lo s s d e t e r m i n a t i o n , a e r o b ic a n d a n a e r o b i c b a c t e r i a l c o u n t s a n d ta s t e p a n e l e v a l u a t io n s w e re m a d e a t v a r io u s s to r a g e in te rv a ls . P u rg e lo s se s w e re d i f f e r e n t f o r w h o le s a le c u t s . I n c r e a s e d p r e p a c k a g e h o ld in g t im e a n d t im e in v a c u u m s to r a g e in c r e a s e d b o t h a e r o b ic a n d a n a e r o b i c b a c t e r ia l c o u n t s . T e n d e r n e s s i n c r e a s e d d u r in g 15 d a y s h o ld in g p e r io d a n d d u r in g v a c u u m s to r a g e o f p r o d u c t p a c k a g e d 1 d a y p o s t m o r t e m . G e n e r a l a c c e p ta b i l i t y s c o r e s f o l l o w e d te n d e r n e s s s c o r e s e x c e p t f o r t h e lo w g r a d e , 1 5 -d a y p o s t m o r t e m p a c k a g in g s a m p le w h ic h w a s g iv e n a n u n f a v o r a b le r a t in g d u e t o o f f - f l a v o r .

A N A N A L Y S I S O F A T M O S P H E R I C F R E E Z E D R Y I N G . D .R . H E L D - M A N & G .A . H O H N E R . J. Food Sci. 3 9 , 1 4 7 - 1 5 5 ( 1 9 7 4 ) —A m a t h e m a t ic a l m o d e l w h ic h s im u la te s a tm o s p h e r i c f r e e z e d r y in g o f f o o d a n d a llo w s f o r e v a l u a t io n o f a p p r o p r i a t e h e a t a n d m a s s t r a n s f e r p a r a m e te r s b y n o n l in e a r e s t i m a t i o n h a s b e e n d e v e lo p e d . T h e m o d e l is a n u m e r i c a l s o lu t io n o f a p p r o p r i a t e p a r t i a l d i f f e r e n t i a l e q u a t i o n s f o r h e a t a n d v a p o r t r a n s p o r t in t h e s e m i -d ry , p o r o u s p r o d u c t l a y e r w h ic h d e v e lo p s a s t h e p r o d u c t d r ie s . T h e m o d e l h a s b e e n v e r i f i e d b y e x p e r i m e n t a l m e a s u r e m e n t o f m o is t u r e c o n t e n t d u r in g a t m o s p h e r i c f r e e z e d r y in g o f b e e f . T h e r e s u l t s c o n f ir m t h a t t h e r a t e o f a t m o s p h e r i c f r e e z e d r y in g is m a s s t r a n s f e r l im i t e d a n d h e a t t r a n s f e r is b y c o n d u c t i o n t h r o u g h th e m a t r i x o f p r o d u c t s o l id s in t h e s e m i -d ry l a y e r . S im u la t i o n o f p r o d u c t d r y in g in t h r e e - d im e n s io n s in d i c a t e s t h a t t h e r a t e o f a t m o s p h e r i c f r e e z e d r y in g c a n b e i n c r e a s e d m o s t e f f e c t iv e ly b y r e d u c in g p a r t i c l e s iz e a n d in c r e a s in g th e s u r f a c e m a s s t r a n s f e r c o e f f i c i e n t . A d im e n s io n le s s n u m b e r w h ic h r e p r e s e n t s t h e r a t i o o f e x t e r n a l t o i n t e r n a l m a s s t r a n s f e r i n c o r p o r a t e s t h e in f l u e n c e o f t h e p r i m a r y c o n t r i b u t i o n s t o a t m o s p h e r i c f r e e z e - d r y in g r a te .

D E T E R M I N A T IO N O F F R E S H P A P A Y A ’S T E X T U R E B Y P E N E T R A T I O N T E S T S . M . P E L E G . / Food Sci. 3 9 , 1 5 6 - 1 5 9 ( 1 9 7 4 ) - F r e s h C a r ic a p a p a y a f r u i t s w e r e s u b je c t e d t o p e n e t r a t i o n t e s t s u s in g a n I n s t r o n U n iv e r s a l T e s t in g M a c h in e . P lu n g e rs u s e d w e re o f t h e s a m e s h a p e a s t h o s e o f t h e s t a n d a r d F r u i t P r e s s u r e T e s te r s w i th d i a m e te r s r a n g in g f r o m 3 /1 6 in . t o 1 1 /1 6 in . , a n d w e r e a p p l i e d a t p e n e t r a t i o n s p e e d s o f 2 0 , 5 0 a n d 1 0 0 c m /m in . F o r r i p e f r u i t s a c le a r y ie ld p o i n t w a s o b t a i n e d re g a rd le s s o f d i a m e te r a n d r a t e o f p e n e t r a t i o n w i th in a d i s ta n c e o f 1 —5 m m . U n r ip e o r u n p e e l e d f r u i t s s h o w e d a y ie ld p o i n t b e tw e e n 2 - 1 0 m m . T h e a v e ra g e y ie ld f o r c e w a s p r o p o r t i o n a l t o t h e c ro s s - s e c t io n a l a r e a o f th e p lu n g e r i n d ic a t in g t h a t t h e m a jo r m e c h a n is m o f t h e t e x t u r e f a i lu r e w a s o f a c o m p re s s iv e n a t u r e . T h i s f a c t e n a b l e d t h e e v a l u a t io n o f t h e p a p a y a t e x tu r e i n s t r e n g t h u n i t s ( g / c m 2 ). Y ie ld f o r c e i n c r e a s e d w i th t h e p e n e t r a t i o n s p e e d . F r o m a t e x t u r e p o i n t o f v ie w , t h e p a p a y a f r u i t m a t u r e d in a n o n u n i f o r m w a y a n d s o f t e n in g s o m e t im e s o c c u r r e d p r i o r t o t h e fu l l d e v e l o p m e n t o f t h e t o t a l s o lu b le s o l id s .

F L O W P R O P E R T I E S O F T R O P I C A L F R U I T P U R E E S . M .A . R A O , L .N . O T O Y A P A L O M IN O & L .W . B E R N H A R D T . J. Food Sci. 3 9 , 1 6 0 - 1 6 1( 1 9 7 4 ) - T h e f lo w p r o p e r t i e s o f t h e p u r e e s o f b a n a n a , m a n g o , g u a v a a n d p a p a y a w e re s tu d i e d w i th a t u b e v i s c o m e te r t h a t w a s d e s ig n e d t o e l im i n a t e e n t r a n c e a n d e x i t f lo w c o r r e c t io n s . T h e e x p e r i m e n t s w e re p e r f o r m e d a t t h e p r e v a i l in g a m b i e n t t e m p e r a t u r e . A ll t h e f r u i t p u r e e s w e re f o u n d to b e p s e u d o p la s t i c f lu id s . O v e r t h e r a n g e o f s h e a r r a t e s b e tw e e n a p p r o x i m a te ly 2 0 a n d 1 , 0 0 0 s '1 t h e f r u i t p u r e e s o b e y e d t h e p o w e r la w r h e o lo g i c a l m o d e l . T h e s e r e s u l t s a re in g e n e r a l a g r e e m e n t w i th p re v io u s s tu d ie s o n a v a r ie ty o f f r u i t p u r e e s .

ing, the “ freeze-heat” m e th o d gave increased case y ield and b e tte r qualityas ju d g ed by USDA standards. E lim ination o f w aste w ate r, absence o f lyeand chem ical co n tam in an ts are o f env ironm en ta l significance.

H IG H -L Y S IN E C O R N F R A C T I O N S A N D T H E I R C H A R A C T E R I S T I C S .G .N . B O O K W A L T E R , K . W A R N E R , O .L . B R E E K E & E .L . G R I F F I N J r .

J. Food Sci. 3 9 , 1 6 6 - 1 7 0 ( 1 9 7 4 ) - T h e p r o t e i n o f h ig h - ly s in e ( o p a q u e - 2 ) c o r n c o n ta in s h ig h e r le v e ls o f ly s in e a n d t r y p t o p h a n a n d is c o n s id e r e d n u t r i t i o n a l ly s u p e r io r t o t h a t o f o r d in a r y y e l lo w d e n t c o r n . B o th ty p e s w e re d ry - m i l l e d t o g r i t s , m e a l , f l o u r a n d d e g e r m e r f in e s . T h e h ig h - ly s in e c o rn f r a c t i o n s w e re lo w e r in f a t a n d p r o t e i n t h a n o r d i n a r y d e n t c o m f r a c t io n s . T h e s e f r a c t i o n s w e re i n v e s t ig a te d f o r c o lo r , s t o r a g e s t a b i l i t y , a m y lo g r a p h v i s c o s i ty , e x t r u s io n - c o o k in g , f e r m e n t a b i l i t y a n d b a k in g p e r f o r m a n c e . C o lo r v a lu e s o f d e n t f r a c t i o n s w e re m o r e y e l lo w a n d d a r k e r t h a n t h o s e o f h ig h - ly s in e c o r n f r a c t i o n s . A ll f r a c t i o n s d i s p la y e d a d e q u a t e s t a b i l i t y f o r 5 6 d a y s a t 4 9 ° C a n d f o r 1 8 2 d a y s a t e i t h e r 2 5 ° o r 3 7 ° C a s d e t e r m in e d b y c h a n g e s in a v a i la b le ly s in e , f a t a c i d i ty a n d f la v o r o f c o o k e d g ru e ls . H ig h - ly s in e g r i t s h a d lo w e r p a s t i n g t e m p e r a t u r e s t h a n d e n t g r i t s . H o t p a s te a n d s e tb a c k v i s c o s i t ie s w e r e h ig h e r f o r h ig h - ly s in e f r a c t io n s . F e r m e n t a t i o n s w e r e m o r e a c t iv e f o r h ig h - ly s in e t h a n f o r d e n t c o rn f r a c t i o n s , w h ic h w e re e x t r u s i o n p r o c e s s e d . E x t r u s i o n - p r o c e s s e d h ig h - ly s in e f r a c t i o n s e x h i b i t e d le s s e x p a n s io n a n d h a d h ig h e r w a te r s o lu b i l i t y v a lu e s t h a n d e n t . B a k in g c h a r a c t e r i s t i c s w e re s im i la r w h e n c o m p a r i s o n s w e re m a d e w i th f l o u r a n d f in e s f r o m h ig h - ly s in e a n d d e n t in p a n c a k e s a n d h ig h - ly s in e a n d d e n t m e a ls in c o m b r e a d .

A M IN O A C ID C O M P O S IT I O N A N D B IO L O G IC A L Q U A L I T Y O F L IM A B E A N P R O T E I N . S . M A N E E P U N , B .S . L U H & R .B . R U C K E R . J. Food Sci. 3 9 , 1 7 1 - 1 7 4 ( 1 9 7 4 ) - T h e p r o t e i n s in d r y la rg e l im a b e a n s (Phaseolus lunatus, L . v a r B C 6 ) w e re e x t r a c t e d a t p H 7 .2 w i t h a 0 .1 M p h o s p h a t e b u f f e r , a n d th e n p r e c i p i t a t e d a t p H 5 .0 a f t e r a c i d i f i c a t i o n w i th p h o s p h o r i c a c id a n d h e a t in g a t 1 0 0 ° C f o r 1 0 m in . T h e f r e e z e - d r ie d p r o d u c t c o n t a i n e d 5 4 .3 % p r o t e in . T h e a m in o a c id c o n t e n t o f w h o le la rg e l im a b e a n s a n d th e f r e e z e - d r ie d l im a b e a n p r o t e i n c o n c e n t r a t e (L P C ) w e re d e t e r m in e d :n a T e c h n ic o n a m in o a c id a n a l y z e r a f t e r a c id h y d r o ly s i s o f th e p r o t e in s . T h e n u t r i t i v e v a lu e o f t h e L P C w a s d e t e r m i n e d b y f e e d in g to m a le a lb in o r a t s f o r 2 1 d a y s , u s in g c a s e in a n d s o y b e a n p r o t e i n a s r e f e r e n c e s . I t w a s f o u n d t h a t L P C w a s lo w in m e t h i o n i n e , ly s in e , p h e n y l a la n in e a n d v a l in e . W h e n L P C w a s f o r t i f i e d w i t h 0 .5 % m e t h i o n i n e , 0 .3 % ly s in e , 0 .5 % p h e n y la l a n in e a n d 0 .3 % v a l in e , a n d t h e n f e d t o t h e a lb in o r a t s in a n o th e r w is e n o r m a l d i e t c o n t a in in g 1 0 % p r o t e i n , t h e n u t r i t i v e v a lu e o f t h e L P C w a s a s g o o d a s t h a t o f t h e c a s e in s t a n d a r d . T h e f r e e z e - d r ie d L P C h a s a p r o t e i n e f f i c ie n c y r a t i o ( P E R ) o f 1 .6 8 ± 0 .0 7 , f e e d e f f i c ie n c y r a t i o ( F E R ) o f 5 .9 4 ± 0 .2 5 , a n d n e t p r o t e i n u t i l i z a t i o n (N P U ) v a lu e o f 4 8 .4 1 ± 1 .9 7 . F o r t i f i c a t i o n o f t h e L P C w i th e s s e n t ia l a m in o a c id s as d e s c r ib e d a b o v e in c r e a s e d th e P E R to 3 .0 4 ± 0 .0 9 , d e c r e a s e d F E R to 3 .4 2 ± 0 .0 3 , a n d in c r e a s e d th e N P U t o 7 2 .7 0 ± 3 .3 8 . T h e im p o r t a n c e o f e s s e n t ia l a m in o a c id s t o n u t r i t i o n a l v a lu e o f b e a n p r o t e i n s is d is c u s s e d .

A C O M P A R IS O N O F T H E E M U L S I F I C A T I O N C A P A C I T IE S O F S O M E P R O T E I N C O N C E N T R A T E S . D .D . C R E N W E L G E , C .W . D I L L , P .T . T Y B O R & W .A . L A N D M A N N . J. Food Sci. 3 9 , 1 7 5 - 1 7 7 ( 1 9 7 4 ) - T h e e m u l s i f i c a t i o n c a p a c i t i e s o f f o u r p r o t e i n s w e re c o m p a r e d u n d e r e x p e r i m e n ta l c o n d i t i o n s o p t im iz e d f o r b l e n d o r s p e e d , p H , r a t e o f o i l a d d i t i o n a n d p r o t e i n c o n c e n t r a t i o n . T h e e m u l s i f i c a t i o n c a p a c i ty ( o i l p h a s e v o lu m e a t in v e r s io n ) f o r t h e f o u r p r o t e i n s i n d i c a t e d t h a t a ll w e re g o o d e m u ls i f ie r s . T h e a m o u n t s o f p r o t e i n r e q u i r e d t o o b t a i n m a x im u m o il p h a s e v o lu m e d i f f e r e d b e tw e e n s a m p le s . T h e a m o u n t s o f e a c h p r o t e i n r e q u i r e d f o r t h i s m a x im u m e f f e c t w e re a p p r o x i m a t e l y 0 .4 0 % f o r g lo b in , 0 .8 8 % f o r c o t t o n s e e d p r o t e i n , 0 .9 8 % f o r s o y p r o t e i n a n d 1 .1 9 % f o r m i lk p r o t e i n s , a ll e x p r e s s e d a s a p e r c e n t a g e o f t h e a q u e o u s p h a s e . T h e e m u l s i f i c a t i o n c a p a c i ty f o r e a c h p r o t e i n r e l a t e d c lo s e ly t o t h e c o n c e n t r a t i o n o f s o lu b le p r o t e i n in t h e s a m p le .

P I L O T A P P L IC A T IO N O F F R E E Z E - H E A T P E E L I N G O F T O M A T O E S . C O M P O S IT I O N A N D C H A R A C T E R I S T I C S O F G L A N D L E S S A N DS . L E O N A R D & F . W IN T E R . / . Food Sci. 3 9 , 1 6 2 - 1 6 5 ( 1 9 7 4 ) - D a t a L I Q U I D C Y C L O N E P R O C E S S D E G L A N D E D C O T T O N S E E D W H E Y S , w e re o b t a in e d t o d e t e r m in e t h e f e a s ib i l i ty o f a c o m m e r c ia l a p p l i c a t i o n o f S .H .C . L I N , J .T . L a W H O N , C .M . C A T E R & K .F . M A T T I L . J. Food Sci. t h e “ f r e e z e - h e a t” m e t h o d f o r t o m a t o s k in re m o v a l . B y a d a p t in g a c o n - 3 9 , 1 7 8 - 1 8 2 ( 1 9 7 4 ) - C o t t o n s e e d w h e y s a r e l i q u id b y - p r o d u c t s f r o m t ro l le d t im e - t e m p e r a tu r e h o t w a t e r d ip t o a “ F r e o n ” F r e e z e r a n d M ag- c o t t o n s e e d p r o t e i n i s o l a t i o n p r o c e s s e s . T h e s e w h e y s w h e n p r e p a r e d in th e n u s c r u b b e r , t h e v a lu e o f s u c h a s y s te m , a s c o m p a r e d t o e x i s t in g ly e l a b o r a t o r y c o n t a i n e d 2 2 - 3 6 % o f t h e o r ig in a l f l o u r s o l id s o r 1 2 - 2 3 % o f p e e l in g m e th o d s , w a s d e m o n s t r a t e d . C o m m e r c ia l ly m a c h in e - h a r v e s te d t h e f l o u r n i t r o g e n . D i f f e r e n c e s a m o n g v a r io u s w h e y s f r o m d i f f e r e n t p ro c - t o m a to e s w e r e s u r f a c e f r o z e n , c o n v e y e d t h r o u g h h e a t e d w a te r , s k in - s l i t e s s e s w e r e c le a r ly i n d i c a t e d b y t h e i r c h e m ic a l c o m p o s i t i o n s a n d g e l f i l t r a - b y k n i f e e d g e s , a n d d is c h a rg e d i n t o t h e s c r u b b e r . C o m p a r e d t o ly e p e e l - t i o n c h r o m a to g r a m s . W h e y p r o t e i n s w e r e w a t e r s o lu b le , h e a t s t a b l e a n d


c o n t a i n e d u p t o 7 % ly s in e a n d 5 % c y s t i n e . W h e y c a r b o h y d r a t e s w e re m a in ly r a f f in o s e a n d s u c r o s e . M in e ra ls a n d v i t a m in s in c o t t o n s e e d w h e y s w e r e a ls o s h o w n f o r c o m p a r i s o n . R e c o v e ry o f t h e u t i l i z a b l e w h e y c o n s t i t u e n t s t o m in im iz e d i s p o s a l p r o b le m w a s e m p h a s iz e d .

U T I L I Z A T I O N O F C O T T O N S E E D W H E Y P R O T E I N C O N C E N T R A T E S P R O D U C E D B Y U L T R A F I L T R A T I O N . J .T . L A W H O N , S .H .C . L IN , L .W . R O O N E Y , C .M . C A T E R & K .F . M A T T I L . J. Food Sci. 3 9 , 1 8 3 —1 8 7 ( 1 9 7 4 ) - W h e y - t y p e l iq u id b y - p r o d u c t s f r o m t h e m a n u f a c tu r e o f p r o t e i n i s o la te s f r o m g la n d le s s c o t to n s e e d f l o u r w e r e p r o c e s s e d w i th s e m ip e r m e a b le u l t r a f i l t r a t i o n ( U F ) m e m b r a n e s t o f r a c t i o n a t e a n d c o n c e n t r a t e w h e y c o n s t i t u e n t s b e f o r e s p r a y d ry in g . T h r e e d i f f e r e n t c o t t o n s e e d w h e y s r e s u l t i n g f r o m tw o i s o la t io n p r o c e d u r e s w e re p ro c e s s e d . T h e th r e e s p r a y - d r ie d p r o t e in p r o d u c t s o b t a i n e d W ere e v a l u a te d f o r p o t e n t i a l u s e in p r o t e in f o r t i f i c a t i o n o f b r e a d s o r n o n c a r b o n a t e d b e v e ra g e s , a n d a ls o as w h ip p in g p r o d u c t s . S e p a r a t i o n o f c a r b o h y d r a t e s a n d s a l t s ( t h o u g h i n c o m p le te ) f r o m p r o t e in b y th e U F m e m b r a n e y ie ld e d l ig h t c r e a m c o lo r e d p r o te in - r ic h p r o d u c t s g r e a t ly e n h a n c e d in w h ip p a b i l i t y a n d in t h e i r u t i l i t y f o r b e v e r a g e f o r t i f i c a t i o n o v e r t h a t o f u n f r a c t i o n a t e d w h e y s o l id s . M e m b r a n e f r a c t i o n a t i o n s ig n i f i c a n t ly in c r e a s e d m o s t o f t h e e s s e n t ia l a m in o a c id s . A v a ila b le ly s in e in t h e p r o d u c t s w a s in c r e a s e d b y m o r e t h a n 5 0 % o f t h e a v a i la b le ly s in e o f t h e o r ig in a l f lo u r .

V IS C O S I T Y A N D W A T E R A B S O R P T IO N C H A R A C T E R I S T I C S O F S L U R R I E S O F S U N F L O W E R A N D S O Y B E A N F L O U R S , C O N C E N T R A T E S A N D IS O L A T E S . S .E . F L E M IN G , F .W . S O S U L S K I , A . K I L A R A & E .S . H U M B E R T . J. Food Sci. 3 9 , 1 8 8 - 1 9 1 ( 1 9 7 4 ) - T h e v is c o s i ty a n d w a te r a b s o r p t io n c h a r a c t e r i s t i c s o f s lu r r ie s o f s u n f lo w e r f lo u r s , c o n c e n t r a t e s a n d i s o la te s w e re c o m p a r e d w i t h s o y f lo u r s , c o n c e n t r a t e s a n d i s o la te s . T h e c o m m e r c ia l s o y i s o la te s h o w e d h ig h e r w a te r a b s o r p t io n s a n d h ig h e r v is c o s i t ie s a t e a c h c o n c e n t r a t i o n th a n th e s o y f lo u r a n d c o n c e n t r a t e w h ile t h e s u n f lo w e r c o n c e n t r a t e s w e re m u c h m o r e v is c o u s a n d g e n e r a l ly h a d h ig h e r w a te r a b s o r p t io n s t h a n t h e o t h e r s u n f lo w e r p r o d u c t s . T h e s o y f l o u r s lu r r ie s w e r e m o r e v is c o u s t h a n s u n f lo w e r f l o u r , w h e r e a s t h e s u n f lo w e r c o n c e n t r a t e s w e re s u b s t a n t i a l l y m o r e v is c o u s t h a n t h e c o r r e s p o n d in g s o y p r o d u c t . T h e s o y i s o la te s s h o w e d v is c o s i t i e s m u c h h ig h e r t h a n th e s u n f lo w e r is o la te s . In a d d i t i o n , t h e s lu r r ie s o f s u n f lo w e r a n d s o y c o n c e n t r a t e s w h ic h w e re c y c l e d th r o u g h t h e p H t r e a t m e n t h a d v is c o s i t ie s s im i la r t o t h a t o f t h e s o y i s o la te s . T h e v is c o - a m y lo g r a p h c u rv e d e m o n s t r a t e d t h a t t h e s o y i s o la te h a d a h ig h p e a k a n d c o ld v is c o s i ty w h i le c o n c e n t r a t e s f r o m s u n f lo w e r s h o w e d n o p e a k b u t v e ry h ig h c o ld v is c o s i t ie s . B y v a ry in g t h e t e m p e r a tu r e , m ix in g re g im e , s lu r ry m e d iu m a n d s lu r r y c o n c e n t r a t i o n , a n d b y p H -a c t iv a t io n a p r o d u c t c a n b e a l t e r e d to p r o d u c e a w id e v a r ie ty o f w a te r a b s o r p t io n a n d v is c o s i t y c h a r a c te r i s t i c s . T h e r e s u l t s s h o u ld b e c o r r e l a t e d w i th t h e i r f u n c t i o n s in s u c h f o o d s y s te m s a s b e v e ra g e s , g ra v ie s , s a u c e s a n d m e a t p r o d u c ts .

S O L U B L E C O F F E E : S H E L F L I F E S T U D I E S . N .E . H A R R I S , S .J . B IS H O V , A .R . R A H M A N , M .M . R O B E R T S O N & A .F . M A B R O U K . J. Food Sci. 3 9 , 1 9 2 —1 9 5 ( 1 9 7 4 ) - T w o ty p e s o f s o lu b le c o f f e e ( s p r a y d r ie d a n d f r e e z e d r ie d ) w e re p a c k e d in h e r m e t ic a l ly - s e a le d m e ta l c o n ta in e r s t o t e s t t h e e f f e c t o f t h r e e d i f f e r e n t a tm o s p h e r e s ( a i r , 2% o x y g e n in n i t r o g e n a n d “ z e r o ” o x y g e n ) o n th e k e e p in g q u a l i t y o f e a c h p r o d u c t . C o f f e e c o n ta in e r s w e re s to r e d a t 3 7 .8 ° C f o r p e r io d s u p t o 1 2 m o n t h s . I t w a s f o u n d t h a t t h e a ir p a c k h a d le s s d e l e t e r io u s e f f e c t o n t h e f la v o r o f f r e e z e - d r i e d c o f f e e t h a n w a s e x p e c t e d . In f a c t , n i t r o g e n p a c k in g o f f r e e z e - d r ie d c o f f e e m a y n o t b e e s s e n t ia l . C o n v e r s e ly t h e f la v o r o f a i r - p a c k e d s p ra y - d r ie d c o f f e e c h a n g e d s ig n i f i c a n t ly a f t e r 9 m o n t h s s to r a g e a t 3 7 .8 ° C . A f te r 1 2 m o n t h s t h e c o n s u m e r p a n e l f o u n d th i s s a m p le s ig n i f i c a n t ly le s s a c c e p t a b l e t h a n a ll o th e r s . T h i s s u g g e s ts t h a t lo w o x y g e n p a c k s m a y b e

u s e f u l in p r o t e c t i n g h ig h m o i s tu r e s o lu b le c o f f e e s . C a r b o n d i o x id e c o n c e n t r a t i o n in t h e h e a d s p a c e g a se s w a s i n d e p e n d e n t o f o x y g e n c o n t e n t a n d w a s a f u n c t i o n o f t im e . A f t e r 2 m o n t h s s to r a g e a t 3 7 .8 ° C , s o lu b le c o f f e e s p a c k e d in t h e a b s e n c e o f o x y g e n d e v e lo p e d u n d e s i r a b l e o d o r s r e m in i s c e n t o f s h e l la c . T h e s e o d o r s , h o w e v e r , w e r e n o t p e r c e p t ib l e in t h e r e c o n s t i t u t e d p r o d u c t s .

D E H Y D R A T E D C O C O N U T S K IM M IL K A S A F O O D P R O D U C T : C O M P O S IT IO N A N D F U N C T IO N A L IT Y . R . H A G E N M A I E R , K .F . M A T T I L & C .M . C A T E R . J. Food Sci. 3 9 , 1 9 6 - 1 9 9 ( 1 9 7 4 ) - T h e m in e r a l a n a ly s is o f d e h y d r a t e d c o c o n u t s k im m ilk i n d ic a te s t h e p r e s e n c e o f m o r e p o ta s s iu m a n d le s s c a l c iu m t h a n is f o u n d in c o w ’s m i lk . T h e a m in o a c id a n a ly s is s u g g e s ts t h a t i s o le u c in e a n d m e t h io n in e a r e t h e l im i t in g a m in o a c id s . O th e r d a t a i n d ic a te t h a t c o c o n u t s k im m ilk h a s g o o d s o lu b i l i t y a b o v e p H 6 .0 , b u t lo w s o lu b i l i ty a t p H 3 . 0 - 6 . 0 . T h e p r o d u c t is n o t e a s ily h e a t c o a g u la te d a t p H h ig h e r t h a n 6 .0 . A 1 2 % s o lu t io n o f c o c o n u t s k im m ilk is a n a c c e p ta b l e b e v e r a g e . D r ie d c o c o n u t s k im m i lk is v e ry h y g r o s c o p ic .

S E N S O R Y R A T IO S C A L E S R E L A T I N G H A R D N E S S A N D C R U N C H I N E S S T O M E C H A N IC A L P R O P E R T I E S O F S P A C E C U B E S . H R . M O S K O W IT Z , R .A . S E G A R S , J .G . K A P S A L IS & R .A . K L U T E R . J. Food Sci. 3 9 , 2 0 0 - 2 0 2 ( 1 9 7 4 ) - P a n e l i s t s e v a l u a te d b o t h t e x t u r a l h a r d n e s s a n d c r u n c h in e s s b y th e m e t h o d o f m a g n i tu d e e s t i m a t i o n ( r a t i o s c a l in g ) , in w h ic h t h e r a t io s a m o n g e s t i m a te s r e f l e c t e d r a t i o s a m o n g p e r c e iv e d h a r d n e s s a n d c ru n c h in e s s o f f o u r f la v o r s o f s p a c e c u b e s . T h e ju d g m e n t s w e re f i t t e d b y p o w e r f u n c t i o n s o f t h e f o r m S = k l m (S = s e n s o ry j u d g m e n t , 1 = i n s t r u m e n t a l m e a s u r e ) . T h e e x p o n e n t m is le s s t h a n 1 .0 , i n d ic a t in g t h a t t h e s e n s o r y s y s te m c o m p r e s s e s t h e r a n g e o f p h y s ic a l m a g n i tu d e s t o a s m a l le r r a n g e o f p e r c e iv e d m a g n i tu d e s . A d i s c u s s io n is p r o v id e d a b o u t t h e p o t e n t i a l u s e s o f t h e s c a l in g p r o c e d u r e f o r ( a ) d e v e l o p in g a p s y c h o lo g y o f t e x t u r e a n d (b ) u s in g s e n s o r y s c a l in g f o r q u a l i t y a n d p r o c e s s c o n t r o l .

P R E D I C T IO N O F H E D O N IC R A T IN G S O F R IC E B Y S E N S O R Y A N A L Y S IS . H .G . S C H Ü T Z & J .D . D A M R E L L . J. Food Sci. 3 9 , 2 0 3 - 2 0 6 ( 1 9 7 4 ) —T h e s tu d y w a s c o n d u c t e d in o r d e r t o d e t e r m i n e th e r e la t i o n s h ip b e tw e e n s e n s o r y a t t r i b u t e s o f c o o k e d r i c e a n d p a l a t a b i l i t y . 2 0 s a m p le s o f c o o k e d r ic e w e re e v a lu a te d o n 15 s e n s o r y d im e n s io n s b y 6 t r a in e d p a n e l i s t s a n d 1 0 0 c o n s u m e r s o n a h e d o n i c s c a le . T h e d a t a w e re a n a ly z e d b y c o m p u t in g a v e ra g e s f o r e a c h s e n s o r y d im e n s io n a n d h e d o n i c v a lu e . C o r r e l a t i o n s w e re c a l c u la te d a m o n g a ll v a r ia b le s a n d a s te p w is e m u l t ip l e r e g r e s s io n a n a ly s is c o n d u c t e d u s in g t h e s e n s o r y v a r ia b le s a s i n d e p e n d e n t v a r ia b le s a n d h e d o n ic v a lu e a s t h e d e p e n d e n t v a r ia b le . A v e ry h ig h m u l t i p l e R ( 0 .9 7 ) w a s o b t a i n e d a n d p e r c e n t c o n t r i b u t i o n o f t h e s e n s o r y d im e n s io n s w a s d e t e r m in e d . F o r u s e a s a m o r e p r a c t i c a l s e t o f p r e d i c to r s , 5 s e n s o r y d im e n s io n s w e r e u s e d in a m u l t i p l e r e g r e s s io n a n a l y s is t o p r e d i c t h e d o n ic v a lu e ( R = 0 .9 0 ) . T h e m o r e i m p o r t a n t s e n s o r y a t t r i b u t e s t o p a l a t a b i l i t y w e re , “ d r y n e s s , ” “ s t i c k in e s s , ” “ r u b b e r in e s s , ” “ s t a r c h i n e s s , ” “ r ic e f l a v o r ” a n d “ c o m p a c tn e s s . ” A t t r i b u t e s o f l i t t l e im p o r t a n c e w e re “ s iz e ,” “ c h a lk in e s s ,” “ o t h e r f l a v o r ” a n d “ f i r m n e s s .” T h e s tu d y c le a r ly d e m o n s t r a t e d t h e a b i l i t y t o p r e d i c t h e d o n i c v a lu e o f c o o k e d r ic e f r o m s e n s o r y a t t r i b u t e s a s w e ll a s i d e n t i f y in g i m p o r t a n t c h a r a c t e r i s t ic s t o p a la t a b i l i t y .

D E G R A D A T IO N O F T H I A M IN E A N D R I B O F L A V I N D U R I N G E X T R U S I O N P R O C E S S IN G . G . B E E T N E R , T . T S A O , A . F R E Y & J . H A R P E R . J. Food Sci. 3 9 , 2 0 7 —2 0 8 ( 1 9 7 4 ) —E x t r u s i o n p ro c e s s in g o f c o r n g r i t s f o r t i f i e d w i th t h i a m in e a n d r ib o f la v in a t 1 0 0 m g p e r k g w a s

v iii

d o n e o n a B r a b e n d e r p l a s t i c o r d e r w i th 3 /4 - in . d ia m b a r r e l a n d 3 :1 f l i t e d e p th r a t io s c r e w . T h e v i t a m in d e g r a d a t io n w a s m e a s u r e d b y m ic r o b io lo g ic a l a s s a y . T h e e f f e c t o f e x t r u d e r b a r r e l t e m p e r a t u r e , e x t r u d e r s c r e w rp m a n d in i t i a l m o i s tu r e c o n t e n t o f t h e c o r n g r i t s w a s m e a s u r e d . T h e a v e ra g e r e t e n t i o n o f t h i a m in e w a s 5 4 % w i th 2 1 % d e c r e a s e f o r 4 0 ° F in c re a s e i n t e m p e r a t u r e a n d 1 5 % d e c r e a s e f o r 2 5 r p m in c r e a s e in s c r e w s p e e d . T h e c o r r e s p o n d in g f ig u r e s f o r r ib o f l a v in w e re 9 2 % w i t h 1 5 % d e c re a s e d u e t o r p m a n d 2 1 % d e c r e a s e f o r 1 .5 % in c r e a s e in m o i s tu r e c o n te n t . M o is tu r e h a d n o e f f e c t o n th ia m in e , a n d t e m p e r a t u r e h a d l i t t l e e f f e c t o n r ib o f la v in .

D A T A A N A L Y S I S : R E G R E S S I O N A N A L Y S I S W IT H R E P E T I T I O N S O F T H E IN D E P E N D E N T V A R I A B L E . J . J . K U B A L A , M .C . G A C U L A J R . & M .J . M O R A N . J. Food Sci. 3 9 , 2 0 9 - 2 1 0 ( 1 9 7 4 ) - T h r e e m e t h o d s f o r a n a ly z in g d a t a b y re g re s s io n a re s tu d i e d f o r c a s e s w h e re t h e r e a re m o r e o b s e r v a t i o n s i n t h e d e p e n d e n t v a r ia b le (Y ) t h a n in t h e i n d e p e n d e n t v a r ia b le ( X ) . S u c h d a t a a p p e a r in t im e - s t a b i l i t y e x p e r im e n t s . M e th o d 1 u s e s r e p e a t e d v a lu e s o f X f o r e a c h Y . M e th o d 2 re g re s s e s t h e m e a n Y o nX . M e th o d 3 re g re s s e s t h e s u m £ Y o n X . I f t h e c o e f f i c i e n t o f v a r i a t i o n p e r e v a l u a t io n is h ig h a n d t h e n u m b e r o f e v a l u a t io n p e r io d s a re la rg e , t h e b e s t c h o ic e is m e t h o d 2 , o th e r w is e m e t h o d 1 is r e c o m m e n d e d . W h e n m e t h o d 2 is u s e d , i t is s u g g e s te d t h a t t h e c o e f f i c i e n t o f v a r i a t i o n w i th in e v a l u a t io n p e r io d s h o u ld b e p r o v id e d t o g iv e t h e r e a d e r i n f o r m a t i o n a b o u t t h e t r u e v a r ia b i l i ty o f t h e d a t a e x c l u d e d in t h e r e g r e s s io n a n a ly s is .

E F F E C T O F M IC R O W A V E H E A T I N G O F P R E C O O K E D C H IC K E N O N C lo s t r id iu m p e r f r in g e n s . S .E . C R A V E N & H .S . L I L L A R D . / . Food Sci. 3 9 , 2 1 1 - 2 1 2 ( 1 9 7 4 ) - P r e c o o k e d c h ic k e n th ig h s i n o c u l a t e d w i th Clostridium perfringens v e g e t a t iv e c e l ls o r w i th s p o r e s w e r e h e a t e d b y m ic ro - w a v e s t o i n t e r n a l t e m p e r a t u r e s w h ic h r a n g e d f r o m 4 9 - 8 4 ° C . R e c o v e r y o f v e g e t a t iv e c e l ls a f t e r m ic r o w a v e t r e a t m e n t d e c r e a s e d m a r k e d ly w i th i n c r e a s in g in t e r n a l t e m p e r a tu r e s . V ia b le c e l ls w e r e n o t r e c o v e r e d f r o m s a m p le s h e a t e d t o 7 0 ° C o r m o r e e i t h e r b y d i r e c t p l a t i n g o n s u l f i t e - p o ly - m y x in - s u l f a d ia z in e ( S P S ) a g a r o r b y e n r i c h m e n t t e c h n iq u e s . G e r m in a t io n o f s p o r e s w a s s t im u la t e d w h e n s a m p le s w e re h e a t e d t o m a x im u m i n t e r n a l t e m p e r a t u r e s o f 4 9 - 8 4 ° C .

M Y C O T O X IN S P R O D U C E D B Y F U N G I IS O L A T E D F R O M IN S H E L L P E C A N S . A .F . S C H I N D L E R , A .N . A B A D I E , J .S . G E C A N , P .B . M IS L IV E C & P .M . B R IC K E Y . / . Food Sci. 3 9 , 2 1 3 - 2 1 4 ( 1 9 7 4 ) - P e c a n s f r o m v a r io u s s o u r c e s w e r e e x a m in e d . P o t e n t i a l m y c o t o x i n p r o d u c in g m o ld s f o u n d w e re s tu d i e d f o r t o x i n p r o d u c t i o n b y g r o w in g th e m o n v a r io u s s u b s t r a te s . P e c a n s w e re d e m o n s t r a t e d t o b e s u i ta b le f o r t h e p r o d u c t i o n o f s t e r ig m a to c y s t in , a f l a t o x i n a n d o c h r a t o x i n . T h e m o s t p r e v a l e n t m o ld s p e c ie s r e c o v e r e d , w h ic h a r e a ls o k n o w n m y c o t o x i n p r o d u c e r s , w e re Pénicillium expansum a n d Aspergillus versicolor. A ls o r e c o v e r e d w e re i s o la te s o f A. ochraceus, A. parasiticus a n d A. sydowi. A p p r o x i m a te ly 8 6 % o f t h e A. versicolor i s o la te s t e s t e d p r o d u c e d s t e r ig m a to c y s t in . T h e s in g le i s o l a t e o f A. parasiticus p r o d u c e d a f l a t o x i n . A ll o t h e r c u l tu r e s t e s t e d f a i le d t o p r o d u c e e i t h e r p a t u l i n , o c h r a t o x i n o r s t e r ig m a to c y s t in .

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• J . F o o d S c i . , 3 8 ( 7 ) i i - i i i , B . J . L i s k a : M e m o F r o m T h e S c i e n t i f i c E d i t o r . O n p a g e i i , E d i t o r a l B o a r d C h a n g e s a s o f M a y 1 9 7 3 , D r . Y . P o m e r a n z ’ n a m e w a s i n a d v e r t e n t l y o m i t t e d f r o m t h e l i s t o f N e w B o a r d M e m b e r s . O u r s i n c e r e a p o l o g i e s f o r t h i s o m i s s i o n .

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IX

I n t e r n a t i o n a l F o o d I n f o r m a t i o n S e r v i c e ( I F I S ) p r e s e n t s . . .

FOOD SCIENCE AND TECHNOLOGY ABSTRACTS. . . a m o n t h l y c o m p i l a t i o n o f a b s t r a c t s i n 1 9 s u b j e c t a r e a s .

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I n s t i t u t e o f F o o d T e c h n o lo g is t s / 2 2 1 N . L a S a l le S t. / C h ic a g o , I L . 6 0 6 0 1

/ b a s i c s c i e n c e

H . E . G A N T H E R

D e p t , o f N u t r i t i o n a l S c i e n c e s , U n i v e r s i t y o f W i s c o n s i n , M a d i s o n , W l 5 3 7 0 6

a n d M . L . S U N D E

D e p t , o f P o u l t r y S c i e n c e , U n i v e r s i t y o f W i s c o n s i n , M a d i s o n , W l 5 3 7 0 6

EFFECT OF TUNA FISH AND SELENIUM ON THE TOXICITY OF METHYLMERCURY: A PROGRESS REPORT

INTRODUCTIONC O N C E R N a b o u t t h e p o s s i b l e d a n g e r f r o m m e r c u r y i n f i s h h a s r e c e i v e d m u c h

a t t e n t i o n . O n t h e o n e h a n d , l o c a l i z e d p o l l u t i o n o f i n l a n d w a t e r w a y s w i t h H g r e s u l t s i n H g b e i n g a c c u m u l a t e d b y f i s h f a r

i n e x c e s s o f t h e m a x i m u m a l l o w a b l e c o n c e n t r a t i o n e s t a b l i s h e d b y F D A ( 0 . 5 p p m ) . F i s h t a k e u p H g i n t h e f o r m o f m e t h y l - m e r c u r y ( C H 3 H g + ) , b o t h f r o m t h e i r f o o d a n d d i r e c t l y f r o m t h e w a t e r . M e t h y l - m e r c u r y i s r e a d i l y a b s o r b e d a n d t a k e n u p b y m u s c l e a n d o t h e r t i s s u e s , a n d o n l y s l o w l y e x c r e t e d , r e s u l t i n g i n H g c o n c e n t r a t i o n s i n e x p o s e d f i s h r a n g i n g f r o m s e v e r a l p p m u p t o 2 0 p p m ( N e l s o n e t a l . ,

1 9 7 1 ) . S u c h l e v e l s p o s e a c l e a r c o n c e r n t o

p e r s o n s c o n s u m i n g t h e s e f i s h . O n t h e o t h e r h a n d , m a r i n e f o o d f i s h , e s p e c i a l l y

t h e l a r g e r s p e c i e s o f t u n a a n d s w o r d f i s h , a l s o t e n d t o a c c u m u l a t e H g . T h e p o t e n t i a l

h a z a r d h e r e i s n o t s o c l e a r b e c a u s e t h e l e v e l s o f H g a r e n o t m u c h d i f f e r e n t f r o m

t h e 0 . 5 p p m l i m i t . M o r e o v e r , t h e a v a i l a b l e e v i d e n c e s u g g e s t s t h a t m e r c u r y l e v e l s i n t h e o c e a n h a v e r e m a i n e d n e a r l y c o n s t a n t

o v e r m a n y y e a r s a n d t h a t m a n h a s l o n g c o n s u m e d t u n a c o n t a i n i n g e l e v a t e d l e v e l s

o f m e r c u r y ( M i l l e r e t a l . , 1 9 7 2 ) .T h e s e f a c t s r a i s e i m p o r t a n t q u e s t i o n s

i n r e g a r d t o ( 1 ) h o w t o x i c a r e l o w l e v e l s o f m e r c u r y , a s i n g e s t e d i n t h e f o r m o f t u n a ? a n d ( 2 ) w h a t o t h e r f a c t o r s i n t u n a m i g h t b e p r e s e n t t h a t m o d i f y t h e t o x i c i t y o f m e r c u r y ? T h e a c u t e l a c k o f i n f o r m a

t i o n b e a r i n g o n t h e s e p o i n t s h a s l e d t o e c o n o m i c h a r d s h i p s f o r s o m e s e g m e n t s o f

t h e f o o d i n d u s t r y .A s t u d y w a s t h e r e f o r e b e g u n a t t h e

U n i v e r s i t y o f W i s c o n s i n i n m i d - 1 9 7 1 o n t h e l o n g - t e r m b i o l o g i c a l e f f e c t s o f l o w le v e l s o f m e r c u r y . T u n a o f h i g h m e r c u r y c o n t e n t w a s c h o s e n a s t h e s o u r c e o f m e r

c u r y i n t h e d i e t . J a p a n e s e q u a i l , w i d e l y u s e d i n t o x i c o l o g i c a l s t u d i e s , w e r e c h o s e n a s t h e e x p e r i m e n t a l a n i m a l b e c a u s e t h e y e a t a l o t i n r e l a t i o n t o t h e i r s i z e , t o l e r a t e

f a i r l y h i g h l e v e l s o f t u n a i n t h e d i e t , a n d r e p r o d u c e s u b s e q u e n t g e n e r a t i o n s i n a m i n i m u m o f t i m e . T h e y a l s o e x c e e d t h e

a b i l i t y o f o t h e r s p e c i e s t o p r o d u c e h i g h n u m b e r s o f o f f s p r i n g t o b e o b s e r v e d f o r p o s s i b l e d e v e l o p m e n t a l a b n o r m a l i t i e s .

T h e e x p e r i m e n t a l p r o t o c o l c a l l e d f o r a f i v e - g e n e r a t i o n s t u d y t o i n c l u d e e f f e c t s o f m e r c u r y o n t h e g e n e r a l a p p e a r a n c e ,

g r o w t h , s u r v i v a l a n d r e p r o d u c t i o n , a l o n g

w i t h a n a l y s i s o f t i s s u e s f o r t o t a l m e r c u r y . T h i s s t u d y i s m o s t l y c o m p l e t e d a n d w i l l b e r e p o r t e d i n d e t a i l e l s e w h e r e . T h e p r e s

e n t p a p e r i s a p r o g r e s s r e p o r t e x t e n d i n g o u r i n i t i a l f i n d i n g s o n t h e f i r s t g e n e r a t i o n ,

d e s c r i b e d i n a p r e l i m i n a r y r e p o r t ( G a n -

t h e r e t a l . , 1 9 7 2 ) , p l u s a d d i t i o n a l d a t a o n c e r t a i n a s p e c t s o f t h e f i v e - g e n e r a t i o n s t u d y a n d t h e p r o t e c t i v e e f f e c t o f s e l e n

i u m i n m e r c u r y t o x i c i t y .

EXPERIMENTALT H E C O M P O S IT I O N o f t h e d ie t s is g iv e n in T a b le 1 . A p i l o t s t u d y e s t a b l i s h e d t h a t ly o p h i l i - z a t i o n o f t u n a p r o d u c e d a f o r m s u i t a b l e f o r i n c o r p o r a t i o n i n t o d i e t s w i t h o u t s ig n i f i c a n t lo s s o f m e r c u r y a n d t h a t 1 7 % o f t h i s t u n a c o u ld b e a d d e d t o t h e d i e t w i t h o u t d i f f i c u l ty , p ro v id e d a d j u s t m e n t s w e re m a d e f o r s a l t c o n t e n t a n d C a /P r a t i o . C a n n e d t u n a h a v in g a H g c o n t e n t o f 0 . 7 - 1 . 0 p p m ( t o t a l c o n t e n t s o f c a n , f r e s h b a s is ) w a s ly o p h i l i z e d t o a m o i s t u r e c o n t e n t o f a p p r o x i m a t e l y 2 % , g iv in g a p r o d u c t c o n ta in in g a n a v e ra g e o f 2 .9 p p m H g ( “ h ig h -H g t u n a ” ). T u n a o f lo w -H g c o n t e n t d r ie d in t h e s a m e w a y

Table 1—Composition of Diets—Generation 1

Group: 1 2 3 4 5 6 7 8Diet C-S T T T T T T C-S

Hg (ppm) 0 0.05 0.25 0.5 1 10 20 20

g/kgBasal mixa 405 405 405 405 405 405 405 405Corn 300 - 40 80 80 80 80 280Oats — 382 352 322 322 322 322 -Soybean meal 290 40 30 20 20 20 20 290Salt 3 - - - - - - 3DiCal (20.5% Ca,

18.5% P) 2.5 - - - - - - 2.5CaC03 - 3 3 3 3 3 3 -Low Hg tuna - 170 87 - - - - -High Hg tuna - - 83 170 170 170 170 -Hg pre-mixb - - - 0.05 0.61I 10.7 21.9 22.5a C o r n 8 , 0 0 0 , s o y 4 , 0 0 0 , o a t s 1 , 0 0 0 , a l f a l f a m e a l 1 , 2 0 0 , s k i m m i l k 8 0 0 ,

B r e w e r s y e a s t 4 0 0 , v i t a m i n s + m i n e r a l s ( G a n t h e r e t a l . , 1 9 7 2 )

b 8 9 2 p p m H g a s m e t h y l m e r c u r y h y d r o x i d e a d d e d t o h i g h H g t u n a

Volume 39 (19741-JOURNAL OF FOOD SCIENCE—"\

2 - J O U R N A L OF FOOD S C IE N C E- Vo lu m e 3 9 (1974)

Table 2—Delayed onset of mortality in Japanese quail fed 20 ppm Hg in tuna diet compared to corn-soy diet

WeekPeriod(days)

Cumulative mortality (%)a

Generation 1 Generati on 2

Tuna Corn-soy Tuna Corn-soy

5 29-35 0 4 (1) 0 06 36-42 7 (2) 54 (13) 0 12 (3)7 43-49 41 (9) 77 (6) 4 (1) 54 (10)8 50-56 63 (6) 92 (4) 29 (6) 83 (7)9 57-63 81 (5) 100 (2) 50 (2) 83 (0)

10 64-70 96 (4) 100 79 (7) 87 (1)a F i g u r e in p a r e n t h e s e s is n u m b e r o f d e a t h s p e r g r o u p d u r i n g p e r i o d ,

o u t o f a p p r o x i m a t e l y 2 5 i n e a c h g r o u p r e t a i n e d a t 4 w k . H g ( 2 0 p p m )

w a s p r e s e n t i n d i e t s i n t h e f o r m o f m e t h y l m e r c u r y c o n t i n u o u s l y f r o m

d a y 1 .

Table 3—Decreased toxicity of methyl mercury in corn-soy diets supplemented with sodium selenite

Added to C-S diet3

Hg (ppm) Se (ppm)

Percentage of quail surviving

0—4 wk 4—8 wkb

M F

4-12 wkb

M F

4 -1 6 wkb

M F

0 0 95 100 100 100 100 100 1000 6 100 100 100 100 91 100 91

20 0 55 0 3 0 0 0 020 0.35 95 36 35 7 12 0 620 0.70 100 67 60 17 40 8 3020 1.50 100 88 100 41 50 6 2520 3.00 95 83 85 28 77 11 4620 6.00 98 100 90 67 50 25 15

a B a s a l d i e t c o n t a i n s a b o u t 0 . 1 5 p p m S e

b B a s e d o n n u m b e r p r e s e n t i n g r o u p a t 4 w k

y i e l d e d “ lo w - H g t u n a ” ( 0 . 3 p p m H g ) . B y a d d in g 1 7 % o f l o w - o r h ig h - H g t u n a , o r m i x t u r e s , d i e t s c o n t a i n i n g n o m i n a l H g l e v e l s o f 0 . 0 5 , 0 . 2 5 a n d 0 .5 p p m w e r e o b t a i n e d in w h i c h a l l t h e H g c a m e f r o m t u n a . H i g h e r l e v e l s o f H g w e r e o b t a i n e d b y a d d i n g m e t h y l m e r c u r y h y d r o x i d e in t h e f o r m o f a p r e m i x c o n t a i n i n g a p p r o x i m a t e l y1 , 0 0 0 p p m H g t o d i e t s c o n t a i n i n g 1 7 % o f t u n a , g iv i n g t o t a l H g l e v e l s o f 1 , 1 0 a n d 2 0 p p m . I n t h e i n i t i a l e x p e r i m e n t s t h e p r e m i x w a s p r e p a r e d b y s p r a y i n g a s o l u t i o n o f m e t h y l m e r c u r y h y d r o x i d e o n t o d r i e d t u n a . I n l a t e r e x p e r i m e n t s i t w a s f o u n d m o r e c o n v e n i e n t t o a d d t h e

m e t h y l m e r c u r y s o l u t i o n t o a s u s p e n s i o n o f c a s e i n i n 9 5 % e t h a n o l , f o l l o w e d b y d r y i n g . C a s e i n h a s a l o w c o n t e n t o f s e l e n i u m ( a b o u t 0 .1 p p m ) s o u s e o f t h e c a s e i n p r e m i x m a d e i t p o s

s i b l e t o v a r y m e t h y l m e r c u r y c o n t e n t o f t h e d i e t s w i t h o u t a p p r e c i a b l y a l t e r i n g s e l e n i u m le v e l s . A s a c o n t r o l f o r p o s s i b l e e f f e c t s o f t h e t u n a i t s e l f o n m e t h y l m e r c u r y t o x i c i t y , a p r a c t i c a l t y p e d i e t b a s e d o n c o r n - s o y b e a n m e a l w a s a l s o p r e p a r e d c o n t a i n i n g 2 0 p p m H g , a s w e l l a s t h e s a m e d i e t w i t h o u t H g . T h e s e d i e t s w e r e f e d t o d a y - o l d J a p a n e s e q u a i l d i v i d e d i n t o e i g h t g r o u p s o f 4 5 e a c h . A t 4 w e e k s , a p p r o x i m a t e l y 2 5 b i r d s f r o m e a c h g r o u p w e r e r e t a i n e d f o r b r e e d i n g a n d t h e r e m a i n d e r s a c r i f i c e d f o r t i s s u e

H g a n a l y s i s . M e r c u r y w a s a n a l y z e d b y f l a m e l e s s a t o m i c a b s o r p t i o n ( M u n n s a n d H o l l a n d , 1 9 7 1 ) a n d s e l e n i u m b y a f l u o r i m e t r i c p r o c e d u r e ( H o f f m a n e t a l . , 1 9 6 8 ) .

RESULTST H E M O S T I N T E R E S T I N G f i n d i n g f r o m

o u r s t u d i e s w a s t h a t t u n a d i d i n d e e d m o d i f y t h e t o x i c i t y o f m e t h y l m e r c u r y . Q u a i l r e c e i v i n g 2 0 p p m H g i n t h e c o r n - s o y a d i e t s h o w e d s i g n s o f H g t o x i c i t y a t 4 w k a n d h a d a 5 4 % m o r t a l i t y r a t e b e t w e e n t h e4 —6 w k p e r i o d . I n c o n t r a s t , t h o s e q u a i l g i v e n t h e s a m e a m o u n t o f H g i n t h e d i e t c o n t a i n i n g 1 7 % o f t u n a w e r e f r e e o f s i g n s o f p o i s o n i n g f o r a l o n g e r t i m e a n d o n l y 7 % d i e d b e t w e e n t h e 4 - 6 w k p e r i o d . T h e s e e x p e r i m e n t s w e r e r e p e a t e d i n t h e s e c o n d g e n e r a t i o n a n d a s i m i l a r p r o l o n g a t i o n o f s u r v i v a l w i t h t h e t u n a d i e t s w a s o b s e r v e d ( T a b l e 2 ) . E x p r e s s e d a s t i m e t o

r e a c h 5 0 % m o r t a l i t y , t h i s i n c r e a s e i n s u r

v iv a l a m o u n t e d t o a p p r o x i m a t e l y 2 w k w h e n 2 0 p p m H g ( 1 9 . 5 p p m a d d e d a s

m e t h y l m e r c u r y ) w a s p r e s e n t i n t h e d i e t .

I n l a t e r g e n e r a t i o n s w h e r e 1 0 p p m o f H g

w a s u s e d , s u r v i v a l w a s i n c r e a s e d b y 5 w k ( d a t a n o t s h o w n ) .

F u r t h e r w o r k e s t a b l i s h e d t h a t s e l e n

i u m i s p r o b a b l y t h e f a c t o r i n t u n a t h a t r e d u c e s m e t h y l m e r c u r y t o x i c i t y . T h e

a d d i t i o n o f 0 . 5 p p m s e l e n i u m a s s o d i u m s e l e n i t e t o p u r i f i e d d i e t s i m p r o v e d g r o w t h

a n d d e l a y e d t h e o n s e t o f m o r t a l i t y i n r a t s g i v e n u p t o 2 5 p p m o f H g a s m e t h y l m e r

c u r y i n t h e d r i n k i n g w a t e r ( G a n t h e r e t a l . , 1 9 7 2 ; 1 9 7 3 ) . I t w a s n e x t s h o w n t h a t t h e a d d i t i o n o f s e l e n i t e t o t h e c o r n - s o y d i e t s o f J a p a n e s e q u a i l d u p l i c a t e d t h e p r o t e c t i v e e f f e c t o f t u n a i n d e c r e a s i n g m e t h y l m e r c u r y t o x i c i t y ( E l - B e g e a r m i e t a l . ,1 9 7 3 ) . B y c h o o s i n g a p p r o p r i a t e l e v e l s o f m e r c u r y a n d s e l e n i u m , i t h a s b e e n p o s s i b l e t o a c h i e v e q u i t e r e m a r k a b l e e f f e c t s o f s e l e n i u m r e d u c i n g m e r c u r y t o x i c i t y a n d a l s o m e r c u r y r e d u c i n g s e l e n i u m t o x i c i t y .

T a b l e 3 s h o w s t h e e f f e c t o f a d d i n g i n

c r e a s i n g l e v e l s o f s e l e n i u m t o c o r n - s o y d i e t s c o n t a i n i n g 2 0 p p m o f H g .

T a b l e 4 s h o w s t h e s e l e n i u m a n d m e r c u r y c o n t e n t o f l y o p h i l i z e d t u n a . F i s h o f a l l t y p e s h a v e a h i g h e r c o n t e n t o f s e l e n

i u m t h a n m o s t f o o d s ( M o r r i s a n d L e v a n - d e r , 1 9 7 0 ; A r t h u r , 1 9 7 2 ) a n d t u n a is n o e x c e p t i o n . T h e a d d i t i o n o f 1 7 % o f h i g h -

H g t u n a ( S e c o n t e n t 2 . 9 p p m ) i n t r o d u c e s 0 . 4 9 p p m o f S e i n t o t h e d i e t . A s s h o w n i n

t h e o t h e r e x p e r i m e n t s , t h i s i s a l e v e l s u f f i c i e n t t o g iv e s u b s t a n t i a l p r o t e c t i o n a g a i n s t H g . I n t e r e s t i n g l y , o u r d a t a i n d i

c a t e t h a t t h e s e l e n i u m c o n t e n t o f h i g h - H g t u n a i s g r e a t e r t h a n t h a t o f l o w - H g t u n a .

I n d e e d , o n a m o l a r b a s i s , t h e i n c r e m e n t i n S e i s i n a n a p p r o x i m a t e 1 :1 r a t i o w i t h t h e

i n c r e m e n t i n H g . T h i s s u g g e s t s t h a t t u n a

a c c u m u l a t e a d d i t i o n a l s e l e n i u m , e v e n b e y o n d t h e r a t h e r s u b s t a n t i a l b a s a l l e v e l , a s t h e y a c c u m u l a t e H g ( b o t h e l e m e n t s a r e

c o n c e n t r a t e d s u b s t a n t i a l l y f r o m a l e v e l o f a p p r o x i m a t e l y 0 .1 p p b i n o c e a n w a t e r ) . W e o b s e r v e d t h i s t r e n d w h e n l a r g e l o t s o f

l y o p h i l i z e d t u n a w e r e s a m p l e d , b u t t h e t r e n d w a s n o t s o a p p a r e n t w h e n i n d i v i d

u a l c a n s w e r e a n a l y z e d . I t c a n n o t b e s a i d t h a t S e i n t u n a i s t h e p r i m a r y c a u s e o f H g

a c c u m u l a t i o n b e c a u s e o t h e r s p e c i e s o f

Table 4—Mercury and selenium content of lyophilized tunaa

ppm Hg range mean

ppm Se Hg/Se range mean (molar basis)

(a) Low-Hg tuna 0.25-0.44 0.32 1.71-2.16 1.91 0.066(b) High-Hg tuna 2.62-3.31 2.87 2.43-3.40 2.91 0.39

(b) - (a)atom. wt. A 'mo ar■ basis): AHg _ 2.55

200 "= 0.0128 ASe = : 79 = 0.0127

AHg 0.0128= 1.0ASe 0.0127

a T h r e e s a m p le s o f l o w - H g t u n a a n d t h r e e s a m p le s o f h i g h - H g t u n a w e r e

a n a l y z e d . T w o o f t h e t h r e e s a m p le s o f e a c h t y p e o f t u n a w e r e t a k e n

f r o m 5 g a l c o n t a i n e r s o f t u n a f r e e z e d r i e d a t a c o m m e r c i a l p l a n t . T h e

t h i r d s a m p l e w a s t a k e n f r o m a 2 k g l o t o f t u n a f r e e z e d r i e d o n

c a m p u s . T h e H g / S e i n c r e m e n t w a s 1 . 1 4 f o r t h e c o m m e r c i a l l y d r i e d

s a m p le s a n d 0 . 9 1 f o r t h e l o c a l l y d r i e d s a m p le .

TOXICITY OF ME THY LM ERC URY —2

Table 5—Effect of mercury level on survival and reproductive performance in Japanese quail (Generation 1)

DietTotal Hg

(ppm)

No. quail surviving3 (47 wk)

13-16 wk Egg prod

(% hen day)Fertility

%Hatchability

%

C-S 0 14 74 91 82T 0.05 13 84 85 81T 0.25 14 69 85 79T 0.5 16 77 88 75T 1 16 77 80 84T 10 4 63 43 46T 20 0 12 — —C-S 20 0 - - -a O u t o f a p p r o x i m a t e l y 2 5 p e r g r o u p ( f r o m 4 w k )

f i s h a n d f i s h m e a l s c o n t a i n e q u a l l y h i g h l e v e l s o f S e b u t l i t t l e H g ( L u n d e , 1 9 6 8 ) . N e i t h e r c a n i t b e e x p e c t e d t h a t f i s h i n

i n l a n d b o d i e s o f w a t e r w i l l n e c e s s a r i l y s h o w t h e s a m e t e n d e n c y f o r p a r a l l e l a c c u m u l a t i o n o f H g a n d S e a s t u n a r a n g i n g i n t h e o p e n o c e a n .

T h e f i r s t g e n e r a t i o n e x p e r i m e n t w a s c o n t i n u e d f o r 4 7 w k . S u r v i v a l a n d r e p r o d u c t i v e p e r f o r m a n c e d a t a a r e s h o w n i n T a b l e 5 . A l l b i r d s f e d t h e 2 0 p p m H g d i e t s e v e n t u a l l y d i e d , w h e t h e r t h e y r e c e i v e d t h e c o r n - s o y a d i e t o r t h e t u n a d i e t . E g g p r o d u c t i o n i n t h e s e g r o u p s w a s v e r y p o o r a n d t h e f e w e g g s t h a t w e r e p r o d u c e d

w h i c h w e r e f e r t i l e g e n e r a l l y f a i l e d t o h a t c h . A t t h e i n t e r m e d i a t e l e v e l o f 1 0

p p m H g i n t h e t u n a d i e t , n o m a l e s s u r v i v e d , a n d r e p r o d u c t i v e p e r f o r m a n c e i n t h e f e m a l e s w a s i m p a i r e d . A t l e v e l s o f m e r c u r y i n t h e t u n a d i e t s o f 1 p p m o r

l e s s , s u r v i v a l a n d r e p r o d u c t i v e p e r f o r m a n c e w a s c o m p a r a b l e t o t h e c o n t r o l g r o u p r e c e i v i n g t h e c o r n - s o y d i e t d e v o i d o f m e r

c u r y . T h e s e d a t a s h o w t h a t l e v e l s o f m e t h y l m e r c u r y u p t o 1 p p m w e r e w e l l t o l e r a t e d . M o r e o v e r , t h e p r e s e n c e o f 1 7 % o f t u n a i t s e l f i s s h o w n t o b e w e l l t o l e r

a t e d , i n d i c a t i n g a l a c k o f p r o b l e m s r e l a t e d t o l i p i d p e r o x i d a t i o n o r o t h e r p o s s i b l e

c o m p l i c a t i o n s . F e e d c o n s u m p t i o n p e r b i r d p e r d a y w a s s i m i l a r f o r a l l g r o u p s . B i r d s f e d h i g h l e v e l s o f m e r c u r y c o n t i n u e t o e a t n o r m a l l y u n t i l s h o r t l y b e f o r e d e a t h .

T h e f a t e o f m e r c u r y i n q u a i l f e d t h e

t u n a d i e t s f o r 4 7 w k i s s h o w n i n T a b l e 6 . I n t h e c a s e o f t h o s e b i r d s f e d u p t o 0 . 5 p p m H g , t h e t i s s u e H g r e p r e s e n t s t h a t

w h i c h w a s d e r i v e d f r o m t h e t u n a i t s e l f . A t i s s u e d i s t r i b u t i o n p a t t e r n t y p i c a l o f m e t h y l m e r c u r y , r a t h e r t h a n H g + + ( s e e S t o e w s a n d e t a l . , 1 9 7 1 ) , w a s o b s e r v e d , i . e . , h i g h c e l l / p l a s m a r a t i o i n b l o o d a n d a n e v e n d i s t r i b u t i o n i n t h e t i s s u e s . B y a n d l a r g e , m e r c u r y w a s a c c u m u l a t e d i n r a t h e r c o n s t a n t p r o p o r t i o n t o d i e t a r y m e r c u r y . F o r g r o u p 4 f e d t h e d i e t c o n t a i n i n g 0 . 5

p p m H g d e r i v e d f r o m t u n a , t h e r a t i o o f t i s s u e H g : d i e t H g ( d i e t H g 0 . 5 5 p p m b y a n a l y s i s ) w a s a s f o l l o w s : b r a i n 1 .4 , b l o o d1 . 5 , l i v e r 2 . 0 , k i d n e y 3 . 0 , f e a t h e r s 1 7 a n d e g g s 1 .2 . T h e c o n c e n t r a t i o n i n t h e b r a i n o f q u a i l f e d 0 . 5 p p m H g f o r 4 7 w k w a s 0 . 7 7 p p m , a p p r o x i m a t e l y 1 0 % o f t h e l o w

e s t l e v e l e x p e c t e d t o c a u s e n o t i c e a b l e s y m p t o m s i n m o s t s p e c i e s . S i n c e t h e b i r d s w e r e p r o b a b l y i n s t e a d y s t a t e a t 4 7 w k r e l a t i v e t o H g i n t a k e a n d e x c r e t i o n , t h e s e a r e p r o b a b l y t h e m a x i m u m v a l u e s t o b e

Table 7—Effect of mercury level in thediet on percentage of abnormal embryos

DietHg

(ppm)Abnormal

Fertile %

Corn-soy 0 39/3501 1.11Tuna 0.05 19/2580 0.73Tuna 0.25 49/3277 1.50Tuna 0.5 39/3659 1.06Corn-soy 1.0 18/649 2.77Tuna 1.0 45/4025 1.12Tu na 2.5 15/1064 1.41Corn-soy 5.0 5/769 0.65Tuna 5.0 22/1893 1.16Tuna 7.5 5/973 0.51Tuna 10 15/941 1.59

271/23,331

e x p e c t e d i n t h e t i s s u e s f r o m t h i s l e v e l o f m e r c u r y i n t u n a .

T h e a v a i l a b i l i t y o f t i s s u e s f r o m q u a i l

f e d v a r i o u s l e v e l s o f m e t h y l m e r c u r y f o r p r o l o n g e d p e r i o d s p r o m p t e d u s t o e x a m i n e t h e p o s s i b l e r o l e o f m e t a l l o t h i o n e i n i n

m e t h y l m e r c u r y m e t a b o l i s m . M e t a l l o t h i o n e i n i s a s m a l l p r o t e i n ( 1 0 , 0 0 0 m o l e c u l a r w e i g h t ) i s o l a t e d f r o m k i d n e y ( K a g i a n d V a l l e e , 1 9 6 0 ) a n d l a t e r f r o m l i v e r ( N o r d - b e r g e t a l . , 1 9 7 1 ) . I t h a s a n u n u s u a l l y h i g h c o n t e n t o f c y s t e i n e , a p p r o x i m a t i n g 3 0 % o f t h e t o t a l a m i n o a c i d r e s i d u e s . I t h a s a h i g h a f f i n i t y f o r h e a v y m e t a l s s u c h a s C d + + a n d H g + + a n d i s k n o w n t o b e i n d u c e d b y f e e d i n g h e a v y m e t a l s ( N o r d - b e r g e t a l . , 1 9 7 1 ) , s u g g e s t i n g a r o l e i n m e t a l d e t o x i f i c a t i o n . A s d e s c r i b e d e l s e

w h e r e ( C h e n e t a l . , 1 9 7 3 ) w e s u b j e c t e d t h e s o l u b l e f r a c t i o n o f l i v e r a n d k i d n e y t o S e p h a d e x G - 7 5 c h r o m a t o g r a p h y a n d a n a l y z e d t h e 1 0 , 0 0 0 m o l e c u l a r w e i g h t p e a k f o r t o t a l m e r c u r y . A l t h o u g h t h e s o l u b l e f r a c t i o n s c o n t a i n e d a p r o t e i n t h e s i z e o f m e t a l l o t h i o n e i n w h i c h h a d a h i g h a f f i n i t y f o r 1 0 9 C d + + a d d e d a s a t r a c e r , o n l y a f e w p e r c e n t o f t h e m e r c u r y i n t h e s o l u b l e

f r a c t i o n w a s p r e s e n t i n t h i s p r o t e i n f r a c t i o n . T h u s , a l t h o u g h m e t a l l o t h i o n e i n i s k n o w n t o b e i n v o l v e d i n t h e m e t a b o l i s m o f H g + + a n d C d + + , o u r s t u d i e s i n q u a i l , a s

w e l l a s r a t s , i n d i c a t e t h a t t h i o n e i n d o e s n o t p l a y a s i g n i f i c a n t r o l e i n d e t o x i f y i n g m e t h y l m e r c u r y , s i n c e v e r y l i t t l e H g is b o u n d t o t h i s p r o t e i n w h e n t o x i c l e v e l s o f m e t h y l m e r c u r y a r e f e d . I n r a t s , t h i s m i g h t b e b e c a u s e t h e b i o t r a n s f o r m a t i o n o f m e t h y l m e r c u r y t o H g + + i s i n h i b i t e d a t h i g h e r d o s e s o f m e t h y l m e r c u r y ( N o r s e t h ,

1 9 7 2 ) . W h e n l o w l e v e l s o f m e t h y l m e r c u r y a r e f e d , h o w e v e r , t h e r e m a y b e s o m e H g + + f o r m e d w h i c h t h e n b i n d s t o t h i o n e i n .

T h e a b i l i t y o f m e t h y l m e r c u r y t o p r o d u c e d e v e l o p m e n t a l a b n o r m a l i t i e s h a s b e e n r e p o r t e d b y v a r i o u s w o r k e r s a n d w a s a s s o c i a t e d w i t h i n g e s t i o n o f H g - c o n t a m i -

Table 6—Total Hg levels in tissues of quail fed tuna diets for 47 wk

Diet

Total Hg in diet (ppm)

Blood Total Hg (ppm. fresh basis)

Whole Cells/plasma Liver Kidney Brain Feathers Eggs

C-S 0 0.01 — 0.01 0.01 0.01 0.17 0.01T 0.05 0.06 17 0.13 0.14 0.09 1.67 o o

T 0.25 0.26 27 0.48 0.63 0.34 4.10 0.28T 0.5a 0.84 67 1.11 1.63 0.77 9.23 0.66T 1 1.94 72 2.63 4.02 1.73 22.3 1.46T 10 6.73 32 22.9 25.7 9.38 196 15.5

a A l l o f H g In d i e t s c o n t a i n i n g 0 . 5 p p m o r le s s c a m e f r o m t u n a

4 - JO UR NA L OF FOOD S CI EN C E- Vo lu m e 3 9 (1974)

n a t e d f i s h i n t h e M i n a m a t a d i s a s t e r . T h e

i n c i d e n c e o f a b n o r m a l e m b r y o n i c d e v e l o p m e n t i n q u a i l i s b e i n g e x a m i n e d i n t h e

f i v e g e n e r a t i o n s t u d y b y b r e a k i n g o u t a l l e g g s t h a t f a i l t o h a t c h a n d t a b u l a t i n g a b

n o r m a l i t i e s b y s t a n d a r d p r o c e d u r e s . T o d a t e , o v e r 2 0 , 0 0 0 e g g s h a v e b e e n s e t ( T a b l e 7 ) . T h e i n g e s t i o n o f m e r c u r y i n t u n a d o e s n o t a p p e a r t o i n c r e a s e t h e i n c i d e n c e o f d e v e l o p m e n t a l a b n o r m a l i t i e s . W e h a v e o b t a i n e d s o m e e v i d e n c e t h a t m e t h y l m e r c u r y a d d e d t o d i e t s a t c e r t a i n l e v e l s m a y i n c r e a s e t h e p r o p o r t i o n o f o f f s p r i n g t h a t d i e i n t h e f i r s t w e e k o r t w o a f t e r h a t c h i n g . A s s h o w n i n T a b l e 8 , q u a i l h a t c h e d f r o m h e n s f e d 5 p p m H g a s m e t h y l m e r c u r y i n a c o r n - s o y d i e t s h o w e d a 5 5 % m o r t a l i t y d u r i n g t h e f i r s t 4 w k . I n

t h e c a s e o f q u a i l h a t c h e d f r o m e g g s o f h e n s f e d t h e t u n a d i e t , t h i s e f f e c t w a s l a r g e l y s u p p r e s s e d a t 5 p p m H g , b u t s h o w e d u p a t 7 . 5 p p m . T h e s e l e v e l s o f H g

c a n b e f e d t o n o r m a l c u a i l f r o m d a y 1 w i t h l i t t l e e f f e c t o n m o r t a l i t y d u r i n g t h e f i r s t 4 w k . T h e o n l y c o m p a r a b l e o b s e r v a t i o n o f e a r l y m o r t a l i t y i n o f f s p r i n g o f

m e r c u r y - f e d a n i m a l s t h a t w e a r e a w a r e o f i s t h a t o f K h e r a w i t h m i c e , c i t e d b y C l e g g a n d b r i e f l y m e n t i o n e d i n t h e m o n o g r a p h

b y F r i b e r g a n d V o s t a l ( 1 9 7 2 ) . W e a r e i n v e s t i g a t i n g t h i s a p p a r e n t e f f e c t o f H g f u r t h e r t o s e e i f t h e e f f e c t i s i n d e e d r e l a t e d t o m e r c u r y , o r t o s o m e o f s e v e r a l o t h e r c o n d i t i o n s t h a t h a v e b e e n k n o w n t o i n

c r e a s e e a r l y m o r t a l i t y i n c h i c k s .

DISCUSSIONT H E R E S U L T S o f o u r l o n g - t e r m s t u d y t h u s f a r i n d i c a t e t h a t g r o w t h , s u r v i v a l , a n d r e p r o d u c t i v e p e r f o r m a n c e o f J a p a n e s e q u a i l f e d d i e t s c o n t a i n i n g t u n a a r e c o m p a r a b l e t o t h o s e o f c o n t r o l b i r d s f e d a c o r n - s o y d i e t . W e w e r e a b l e t o a c h i e v e a m e r c u r y c o n c e n t r a t i o n o f 0 . 5 p p m i n t h e t o t a l d i e t b y l y o p h i l i z i n g c o m m e r c i a l c a n n e d t u n a h a v i n g a m e r c u r y c o n t e n t i n e x c e s s o f 0 . 5 p p m ( f r e s h b a s i s ) , a n d a d d i n g 1 7 % t o t h e d i e t . A t t h i s l e v e l t h e t u n a i s s e r v i n g a s t h e m a i n p r o t e i n s o u r c e a n d i s p r o v i d i n g e s s e n t i a l l y a l l o f t h e m e r c u r y . W h e n o n e c o n s i d e r s t h e d i e t a r y l e v e l o f H g a n d t h e r a t e a t w h i c h q u a i l c o n s u m e t h e s e d i e t s , t h e i n t a k e o f t u n a i s e q u i v a l e n t t o a t l e a s t s e v e r a l d o z e n c a n s o f t u n a p e r d a y i n m a n . W h i l e o u r s t u d i e s a r e t h e f i r s t u s i n g t u n a a s a s o u r c e o f m e r c u r y a s f a r a s w e a r e a w a r e , o t h e r s h a v e n o t e d t h e a b s e n c e o f p r o b l e m s i n r a t s f e d d i e t s c o n t a i n i n g 2 0 % o f f i s h p r o t e i n c o n c e n t r a t e ( m e r c u r y c o n t e n t 0 . 2 2 p p m ) f o r f i v e g e n e r a t i o n s ( N e w b e r n e e t a h , 1 9 7 3 ) . A p a r t f r o m t h e s t u d i e s w i t h m a r i n e f o o d f i s h , B i r k e e t a l . ( 1 9 7 2 ) r e c e n t l y r e p o r t e d a s t u d y i n c l u d i n g e i g h t S w e d i s h i n d i v i d u a l s

c h r o n i c a l l y i n g e s t i n g l a r g e a m o u n t s o f f r e s h - w a t e r f i s h c o n t a m i n a t e d w i t h m e t h y l m e r c u r y . T h e y s h o w e d n o d e f i n i t e i n d i c a t i o n s o f m e t h y l m e r c u r y p o i s o n i n g . O n t h e o t h e r h a n d , t h e J a p a n e s e M i n a

Table 8—Early mortality in offspring of quail fed methyl mercuryHg in diet of Avg mortality 0—4 wk

hen and chick {3 hatches)Diet (ppm) (%iCorn-soy 0 5Corn-soy 5 55Tuna 5 16Tuna 7.5 48

m a t a B a y e p i s o d e s h o w e d t h a t a m o u n t s o f m e r c u r y l e t h a l t o m a n c a n b e a t t a i n e d i n f i s h f r o m b a d l y p o l l u t e d w a t e r s .

A m a j o r f i n d i n g e a r l y i n o u r s t u d y w a s t h a t t h e t o x i c i t y o f m e t h y l m e r c u r y a d d e d t o d i e t s w a s l e s s i n t h e c a s e o f q u a i l f e d t u n a c o m p a r e d t o q u a i l f e d a c o r n - s o y d i e t . S e v e r a l l i n e s o f e v i d e n c e s u g g e s t t h a t s e l e n i u m i s p r o b a b l y t h e f a c t o r i n t u n a t h a t r e d u c e s m e r c u r y t o x i c i t y . F i r s t , t h e a b i l i t y o f s e l e n i u m t o d e c r e a s e t h e t o x i c i t y o f m e r c u r y w a s d e m o n s t r a t e d b y

P a r i z e k a n d O s t a d a l o v a ( 1 9 6 7 ) i n a c u t e s t u d i e s w i t h m e r c u r i c c h l o r i d e i n r a t s . M o r t a l i t y w a s p r e v e n t e d b y i n j e c t i n g

e q u i m o l a r l e v e l s o f s o d i u m s e l e n i t e . O u r w o r k s h o w s t h a t t h e c h r o n i c t o x i c i t y o f

m e t h y l m e r c u r y , t h e e n v i r o n m e n t a l l y s i g n i f i c a n t f o r m o f m e r c u r y , c a n b e c o n s i d e r a b l y d e c r e a s e d b y f e e d i n g s o d i u m s e l e n i t e i n t h e d i e t a t l e v e l s c o m p a r a b l e t o t h e

n u t r i t i o n a l r e q u i r e m e n t f o r s e l e n i u m .

C o n f i r m a t i o n o f t h e e f f e c t i v e n e s s o f d i e t a r y s e l e n i t e a g a i n s t m e r c u r y t o x i c i t y h a s b e e n r e p o r t e d b y o t h e r s u s i n g m e t h y l m e r

c u r y o r m e r c u r i c c h l o r i d e ( S t i l l i n g s e t a l . , 1 9 7 2 ; P o t t e r a n d M a t r o n e , 1 9 7 3 ) . I t i s f u r t h e r s h o w n t h a t t h e c o n t e n t o f s e l e n

i u m i n t u n a i s s u f f i c i e n t l y h i g h t o h a v e a p r o t e c t i v e e f f e c t . W h i l e a l l e v i d e n c e t h u s

p o i n t s t o s e l e n i u m a s t h e p r o t e c t i v e f a c t o r i n t u n a , w e h a v e n o t a t t e m p t e d t o

i s o l a t e t h e f a c t o r d i r e c t l y , n o r h a s t h e p r e s e n c e o f o t h e r p r o t e c t i v e f a c t o r s i n t u n a b e e n c o m p l e t e l y e l i m i n a t e d .

L i t t l e i s k n o w n a b o u t t h e f o r m s o f s e l e n i u m i n t u n a . T h e w o r k o f L u n d e ( 1 9 7 0 , 1 9 7 2 ) i n d i c a t e s t h a t s e l e n i u m i n f i s h i s a s s o c i a t e d w i t h p r o t e i n s . A l t h o u g h t u n a i s r i c h i n s e l e n i u m , i t s b i o l o g i c a l

a v a i l a b i l i t y m a y b e s o m e w h a t l o w . I n t h e p r e v e n t i o n o f e x u d a t i v e d i a t h e s i s i n c h i c k s , t h e b i o l o g i c a l a c t i v i t y o f t u n a m e a l ( m a d e f r o m p r o c e s s i n g w a s t e s ) i s o n l y 4 0 % o f t h a t f o u n d f o r o t h e r s o u r c e s o f s e l e n i u m , e v e n t h o u g h c o m p a r a b l e b l o o d l e v e l s o f s e l e n i u m a r e a t t a i n e d r e g a r d l e s s o f t h e s o u r c e ( S c o t t a n d C a n t o r ,1 9 7 1 ) . S i n c e t h e l e v e l o f H g i n t u n a , o n a

m o l a r b a s i s , a p p r o a c h e s 5 0 % o f t h a t f o r S e , t h e p o s s i b i l i t y w a s c o n s i d e r e d t h a t H g m i g h t b e r e d u c i n g t h e b i o l o g i c a l a v a i l a b i l i t y o f s e l e n i u m ( G a n t h e r e t a l . , 1 9 7 2 ) . I t n o w a p p e a r s , h o w e v e r , t h a t t h e a v a i l a b i l i t y o f s e l e n i u m i s l o w e v e n i n f i s h m e a l s

t h a t a r e l o w i n m e r c u r y ( S c o t t , p e r s o n a l c o m m u n i c a t i o n ) , s o s o m e o t h e r f a c t o r

s u c h a s p r o c e s s i n g o f t h e f i s h m a y b e r e s p o n s i b l e .

T o c l o s e t h e c i r c l e o n t h e m e r c u r y - s e l e n i u m a n t a g o n i s m , i t h a s b e e n s h o w n t h a t t h e c h r o n i c t o x i c i t y o f s e l e n i u m i s d e c r e a s e d b y m e r c u r y . H i l l ( 1 9 7 2 ) s h o w e d t h a t H g C l 2 d e c r e a s e d t h e t o x i c i t y o f s e l e n i u m i n t h e c h i c k a n d w e h a v e

f o u n d t h a t m e t h y l m e r c u r y d e c r e a s e s t h e t o x i c i t y o f s o d i u m s e l e n i t e i n J a p a n e s e q u a i l . L e v a n d e r a n d A r g r e t t ( 1 9 6 9 ) , h o w e v e r , d i d n o t o b s e r v e p r o t e c t i o n b y H g C l 2

a g a i n s t t h e c h r o n i c t o x i c i t y o f s o d i u m s e l e n i t e i n r a t s . I t s h o u l d a l s o b e n o t e d

t h a t u n d e r c e r t a i n u n u s u a l c i r c u m s t a n c e s t h e t o x i c i t y o f a s e l e n i u m m e t a b o l i t e , d i m e t h y l s e l e n i d e , c a n a p p a r e n t l y b e i n

c r e a s e d b y m e r c u r i c c h l o r i d e ( P a r i z e k e t a l . , 1 9 7 1 ) .

C l e a r l y , i n c o n s i d e r i n g t h e t o x i c i t y o f

m e r c u r y , o n e m u s t t a k e i n t o a c c o u n t t h e m o d i f y i n g e f f e c t o f s e l e n i u m . I t h a p p e n s t h a t t h e v e r y f o o d w h i c h i s a m o n g t h e

h i g h e s t i n m e r c u r y i s a l s o a m o n g t h e h i g h

e s t i n s e l e n i u m , a n d t h u s i s b l e s s e d w i t h a b u i l t - i n s a f e t y f a c t o r . O b v i o u s l y t h i s g r a

t u i t o u s s a f e t y f a c t o r c a n b e o v e r w h e l m e d i f t h e m e r c u r y l e v e l i s h i g h e n o u g h . I n d e e d , t h e s e l e n i u m c o n t e n t o f t h e f i s h

f r o m M i n a m a t a B a y w a s h i g h a n d a p p a r e n t l y s e l e n i u m i t s e l f w a s o n c e s u s p e c t e d t o b e t h e t o x i c f a c t o r ( K u r l a n d e t a l . , 1 9 6 0 ; U z i o k a , 1 9 6 0 ) . T h e l e v e l s o f m e r c u r y i n m a r i n e f o o d f i s h , h o w e v e r , a r e f a r l e s s t h a n t h o s e f o u n d i n t h e f i s h f r o m M i n a m a t a B a y .

I t i s h o p e d t h a t t h o s e w h o m u s t a t t e m p t t o d e f i n e t h e s a f e l e v e l o f m e r c u r y f o r t h e h u m a n p o p u l a t i o n w i l l c o n s i d e r t h e e f f e c t o f s e l e n i u m . I t w o u l d n o t b e v a l i d , f o r e x a m p l e , t o e s t i m a t e t h e m i n i m u m t o x i c l e v e l o f H g i n t a k e f r o m f i s h o n t h e b a s i s o f a h u m a n p o p u l a t i o n p o i s o n e d f r o m e a t i n g m e r c u r y - t r e a t e d s e e d . E s s e n t i a l l y r . o s e l e n i u m w o u l d a c c o m p a n y t h e m e r c u r y i n g e s t e d i n t r e a t e d s e e d , w h e r e a s s e l e n i u m f a i t h f u l l y a c c o m p a n i e s m e r c u r y i n t u n a . T h e p r e s e n t g u i d e l i n e s , i t s h o u l d b e p o i n t e d o u t , a r e b a s e d o n d a t a f r o m J a p a n e s e i n g e s t i n g m e r c u r y i n f i s h , s o t h e e f f e c t o f s e l e n i u m w o u l d p r e s u m a b l y b e a l r e a d y d i s c o u n t e d .

T h e o b s e r v a t i o n t h a t i n d i v i d u a l s c a n

TOXICITY OF M E T H Y L M E R C U R Y -b

h a v e e l e v a t e d c o n c e n t r a t i o n s o f m e r c u r y i n t h e b l o o d a n d y e t r e m a i n f r e e o f

s y m p t o m s ( B i r k e e t a l . , 1 9 7 2 ) s u g g e s t s

t h a t a g e n t s s u c h a s s e l e n i u m t h a t m o d i f y m e r c u r y t o x i c i t y m a y i n d e e d b e o p e r a t i v e , a n d a c c o u n t f o r s o m e o f t h e i n d i v i d

u a l v a r i a t i o n . I t i s h o p e d t h a t t h o s e s t u d y i n g p o p u l a t i o n s c o n s u m i n g l a r g e a m o u n t s o f f i s h w i l l i n c l u d e m e a s u r e m e n t s o f

s e l e n i u m i n t h e p r o t o c o l , a s w e l l a s m e r c u r y , i n o r d e r t o e v a l u a t e t h i s p o s s i b i l i t y .

REFERENCESA r t h u r , D . 1 9 7 2 . S e l e n i u m c o n t e n t o f C a n a d i a n

f o o d s . C a n a d . I n s t . F o o d S c i . T e c h n o l . J . 5 : 1 6 5 .

B i r k e , G . , J o h n e l s , A .G . , P l a n t i n , L . - O . , S jo s - t r a n d , B . , S k e r f v i n g , S . a n d W e s t e r m a r k , T .1 9 7 2 . S t u d i e s o n h u m a n s e x p o s e d t o m e t h - y l m e r c u r y t h r o u g h f i s h c o n s u m p t i o n . A r c h . E n v i r o n . H e a l t h 2 5 : 7 7 .

C h e n , R .W . , G a n t h e r , H .E . a n d H o e k s t r a , W .G .1 9 7 3 . S t u d i e s o n t h e b i n d i n g o f m e t h y l m e r - c u r y b y t h i o n e i n . B i o c h e m . B i o p h y s . R e s . C o m m . 5 1 : 3 8 3 .

E l - B e g e a r m i , M .M ., G o u d i e , C . , G a n t h e r , H .E . a n d S u n d e , M .L . 1 9 7 3 . A t t e m p t s t o q u a n t i t a t e t h e p r o t e c t i v e e f f e c t o f s e l e n i u m a g a i n s t m e r c u r y t o x i c i t y u s i n g J a p a n e s e q u a i l . F e d . P r o c . 3 2 : 8 8 6 .

F r i b e r g , L . a n d V o s t a l , J . , E d . 1 9 7 2 . “ M e r c u r y i n t h e E n v i r o n m e n t , ” p . 1 4 3 . C h e m i c a l R u b b e r C o . , C le v e l a n d .

G a n t h e r , H .E . , G o u d i e , C . , S u n d e , M .L . , K o - p e c k y , M . J . , W a g n e r , P . , O h , S . - H . a n d H o e k s t r a , W .G . 1 9 7 2 . S e l e n i u m : R e l a t i o n t o d e c r e a s e d t o x i c i t y o f m e t h y l m e r c u r y a d d e d t o d i e t s c o n t a i n i n g t u n a . S c i e n c e 1 7 5 : 1 1 2 2 .

G a n t h e r , H . E . , W a g n e r , P .A . , S u n d e , M .L . a n d H o e k s t r a , W .G . 1 9 7 3 . P r o t e c t i v e e f f e c t s o f s e l e n i u m a g a i n s t h e a v y m e t a l t o x i c i t i e s . I n “ T r a c e S u b s t a n c e s i n E n v i r o n m e n t a l H e a l t h , ” E d . H e m p h i l l , D .D . , p . 2 4 7 . U n i

v e r s i t y o f M i s s o u r i , C o l u m b i a , M d .H i l l , C .H . 1 9 7 2 . I n t e r a c t i o n s o f m e r c u r y a n d

s e l e n i u m i n c h i c k s . F e d . P r o c . 3 1 : 6 9 2 .H o f f m a n , I . , W e s t e r b y , R . J . a n d H i d i r o g l o u , M .

1 9 6 8 . P r e c i s e f l u o r o m e t r i c m i c r o d e t e r m i n a t i o n o f s e l e n i u m i n a g r i c u l t u r a l m a t e r i a l s . J . A s s o c . O f f i c . A n a l . C h e m . 5 1 : 1 0 3 9 .

K a g i , J . R . H . a n d V a l l e e , B .L . 1 9 6 0 . M e t a l l o - t h i o n e i n : A c a d m i u m - a n d z i n c - c o n t a i n i n g p r o t e i n f r o m e q u i n e r e n a l c o r t e x . J . B io l . C h e m . 2 3 5 : 3 4 6 0 .

K u r l a n d , L .T . , F a r o , S .N . a n d S i e d l e r , H . 1 9 6 0 . M i n a m a t a D i s e a s e . W o r l d N e u r o l o g y 1 : 3 7 0 .

L e v a n d e r , O .A . a n d A r g r e t t , L .C . 1 9 6 9 . E f f e c t s o f a r s e n i c , m e r c u r y , t h a l l i u m , a n d l e a d o n s e l e n i u m m e t a b o l i s m i n r a t s . T o x i c o l . A p p l . P h a r m a c o l . 1 4 : 3 0 8 .

L u n d e , G . 1 9 6 8 . A c t i v a t i o n a n a l y s i s o f t r a c e e l e m e n t s i n f i s h m e a l . J . S c i . F o o d A g r . 1 9 : 4 3 2 .

L u n d e , G . 1 9 7 0 . A n a l y s i s o f a r s e n i c a n d s e l e n i u m i n m a r i n e r a w m a t e r i a l s . J S c i . F o o d A g r ic . 2 1 : 2 4 2 .

L u n d e , G . 1 9 7 2 . L o c a t i o n o f l i p i d - s c l u b l e s e l e n i u m i n m a r i n e f i s h t o t h e l i p o p r o t e i n s . J . S c i . F o o d A g r i c . 2 3 : 9 8 7 .

M i l l e r , G .E . , G r a n t , P .M . , K i s h o r e , R . , S t e i n k r u - g e r , F . J . , R o w l a n d , F . S . a n d G u n n , V .P .1 9 7 2 . M e r c u r y c o n c e n t r a t i o n s i n m u s e u m s p e c i m e n s o f t u n a a n d s w o r d f i s h . S c i e n c e 1 7 5 : 1 1 2 1 .

M o r r i s , V .C . a n d L e v a n d e r , O .A . 1 9 7 0 . S e l e n i u m c o n t e n t o f f o o d s . J . N u t r i t i o n 1 0 0 : 1 3 8 3 .

M u n n s , R .K . a n d H o l l a n d , D .C . 1 9 7 1 . D e t e r m i n a t i o n o f m e r c u r y i n f i s h b y f l a m e l e s s a t o m i c a b s o r p t i o n : a c o l l a b o r a t i v e s t u d y . J . A s s o c . O f f i c . A n a l . C h e m . 5 4 : 2 C 2 .

N e l s o n , N . , B y e r l y , T .C . , K o l b y e , A .C . J r . , K u r l a n d , L . T . , S h a p i r o , R . E . , S h i b k o , S . I . , S t i c k e l , W .H . , T h o m p s o n , J . E . , V a n D e n B e r g , L .A . a n d W e i s s l e r , A . 1 9 7 1 H a z a r d s o f m e r c u r y . S p e c i a l R e p o r t t o t h e U S H E W , S e c r e t a r y ’s P e s t i c i d e A d v i s o r y C o m m i t t e e . E n v i r o n m e n t a l R e s . 4 : 1 .

N e w b e r n e , P .M . , G l a s e r , O . , F r i e d m a n , L . a n d S t i l l i n g s , B . 1 9 7 3 . S a f e t y e v a l u a t i o n o f f i s h p r o t e i n c o n c e n t r a t e o v e r f iv e g e n e r a t i o n s o f r a t s . T o x i c o l . A p p l . P h a r m a c o l . 2 4 : 1 3 3 .

N o r d b e r g , G . F . , P i s c a t o r , M . a n d L i n d , B . 1 9 7 1 . D i s t r i b u t i o n o f c a d m i u m a m o n g p r o t e i n

f r a c t i o n s o f m o u s e l i v e r . A c t a P h a r m a c o l . T o x i c o l . 2 9 : 4 5 6 .

N o r s e t h , T . 1 9 7 2 . B i o t r a n s f o r m a t i o n o f m e t h y l - m e r c u r i c s a l t s i n t h e r a t w i t h c h r o n i c a d m i n i s t r a t i o n o f m e t h y l m e r c u r i c c y s t e i n e . A c t a P h a r m a c o l . T o x i c o l . 3 1 : 1 3 8 .

P a r i z e k , J . a n d O s t a d a l o v a , I . 1 9 6 7 . T h e p r o t e c t i v e e f f e c t o f s m a l l a m o u n t s o f s e l e n i t e i n s u b l i m a t e i n t o x i c a t i o n . E x p e r i e n t i a 2 3 : 1 4 2 .

P a r i z e k , J . , O s t a d a l o v a , I . , K a l o u s k o v a , J . , B a b i c k y , A . a n d B e n e s , J . 1 9 7 1 . I n “ N e w e r T r a c e E l e m e n t s i n N u t r i t i o n , ” E d . M e r t z , W . a n d C o r n a t z e r , W .E . , p . 8 5 . M a r c e l D e k k e r , N e w Y o r k .

P o t t e r , S .D . a n d M a t r o n e , G . 1 9 7 3 . E f f e c t o f s e l e n i t e o n t h e t o x i c i t y a n d r e t e n t i o n o f d i e t a r y m e t h y l m e r c u r y a n d m e r c u r i c c h l o r i d e . F e d . P r o c . 3 2 : 9 2 9 .

S c o t t , M .L . a n d C a n t o r , A .H . 1 9 7 1 . T i s s u e s e l e n i u m l e v e l s i n c h i c k s r e c e i v i n g g r a d e d a m o u n t s o f d i e t a r y s e l e n i u m . F e d . P r o c . 3 0 : 2 3 7 .

S t i l l i n g s , B . , L a g a l l y , H . , S o a r e s , J . a n d M i l l e r , D . 1 9 7 2 . E f f e c t o f c y s t i n e a n d s e l e n i u m o n t h e t o x i c o l o g i c a l e f f e c t s o f m e t h y l m e r c u r y i n r a t s . N i n t h I n t e r n a t i o n a l C o n g r e s s o f N u t r i t i o n , M e x i c o C i t y , S u m m a r i a , p . 2 0 6 .

S t o e w s a n d , G .S . , A n d e r s o n , J . L . , G u t t e n m a n n ,W .H . , B a c h e , C .A . a n d L i s k , D . J . 1 9 7 1 . E g g s h e l l t h i n n i n g i n J a p a n e s e q u a i l f e d m e r c u r i c c h l o r i d e . S c i e n c e 1 7 3 : 1 0 3 0 .

U z i o k a , T . 1 9 6 0 . A n a l y s i s o f m e r c u r y i n f o o d a n d a n i m a l o r g a n s ( i n J a p a n e s e ) . K u m a m o t o I g a k k a i Z a s s h i 3 4 : 3 8 3 .

M s r e c e i v e d 6 / 2 8 / 7 3 ; r e v i s e d 8 / 2 1 / 7 3 ; a c c e p t e d 8 / 2 4 / 7 3 . __________________________________________________

P r e s e n t e d a t t h e 3 3 r d A n n u a l M e e t i n g o f t h e I n s t i t u t e o f F o o d T e c h n o l o g i s t s i n M ia m i B e a c h .

R e s e a r c h w a s s u p p o r t e d b y t h e C o l l e g e o f A g r i c u l t u r a l & L i f e S c i e n c e s , U n i v e r s i t y o f W is c o n s i n , M a d i s o n , a n d b y g r a n t s f r o m t h e F o o d R e s e a r c h I n s t i t u t e , U n i v e r s i t y o f W i s c o n s i n , a n d t h e N a t i o n a l I n s t i t u t e s o f H e a l t h ( A M 1 4 1 8 9 ) .

T h e c a p a b l e t e c h n i c a l a s s i s t a n c e o f C a r o l G o u d i e i n t h e q u a i l e x p e r i m e n t s a n d M a r y J o K o p e c k y i n t h e m e r c u r y a n a l y s i s is a p p r e c i a t e d . T h e e x p e r i m e n t d e m o n s t r a t i n g p r o t e c t i o n b y s e l e n i t e a d d e d t o c o r n - s o y d i e t s w a s c o n d u c t e d b y M r . E l - B e g e a r m i .

m m u k mum«-'*'«»1•»

RONALD R. EITENMILLERDept, o f Food Science, University o f Georgia, Athens, GA 30602

CATHEPSIN

INTRODUCTIONI T I S G E N E R A L L Y B E L I E V E D t h a t

m u s c l e s o f m o s t a q u a t i c s p e c i e s c o n t a i n i n h e r e n t p r o t e o l y t i c e n z y m e s c a p a b l e o f a c t i n g o n t h e m u s c l e a f t e r d e a t h a n d l e a d i n g t o u n d e s i r a b l e f l a v o r a n d t e x t u r e c h a n g e s . H o w e v e r , r e l a t i v e l y f e w a q u a t i c f o o d s p e c i e s h a v e b e e n s t u d i e d t o i d e n t i f y t h e t y p e s o f e n z y m e s p r e s e n t i n t h e m u s c l e w h i c h c o u l d p l a y a r o l e i n i t s d e g r a d a

t i o n .S o m e o f t h e m o s t e x t e n s i v e r e s e a r c h

o n f i s h m u s c l e h y d r o l y t i c e n z y m e a c t i v i t y h a s b e e n d o n e b y S i e b e r t a n d c o - w o r k e r s

( 1 9 5 8 , 1 9 6 3 , 1 9 6 5 ) . S i e b e r t ( 1 9 5 8 ) r e p o r t e d t h a t t h e m u s c l e s o f a v a r i e t y o f f i s h i n c l u d i n g c o d , h e r r i n g , s o l e , f l o u n d e r ,

t r o u t a n d c a r p c o n t a i n e d a s m u c h a s 1 0 t i m e s t h e a m o u n t o f c a t h e p s i n a c t i v i t y a s f o u n d i n m a m m a l i a n m u s c l e t i s s u e s . S i e b e r t e t a l . ( 1 9 6 3 ) f o u n d n o c a t h e p s i n

A , B , o r C a c t i v i t y i n c o d m u s c l e a n d s h o w e d t h a t t h e s p e c i f i c i t y o f p u r i f i e d

c o d s p l e e n c a t h e p s i n a g a i n s t t h e B c h a i n o f i n s u l i n w a s d i f f e r e n t f r o m t h a t o b s e r v e d f o r a n y m a m m a l i a n p r o t e a s e .

T h e p r o t e o l y t i c a c t i v i t y o f c a r p

(Cyprinus carpio) m u s c l e h a s b e e n s t u d i e d i n d e t a i l b y M a k i n o d a n a n d I k e d a ( 1 9 6 9 a , b ; 1 9 7 1 ) . T h e s e a u t h o r s f o u n d a

c a t h e p s i n D t y p e e n z y m e i n t h e m u s c l e w i t h a p H o p t i m u m o f 3 . 0 a n d a n a l k a l i n e p r o t e a s e w i t h a p H o p t i m u m o f 8 . 0 . U n l i k e c o d m u s c l e , c a r p m u s c l e c o n t a i n e d m e a s u r a b l e a m o u n t s o f c a t h e p s i n A , B a n d C a c t i v i t y . T h e p r e s e n c e o f t h e a l k a l i n e p r o t e a s e a n d o f c a t h e p s i n s A , B a n d C w i t h p H o p t i m a n e a r n e u t r a l i t y w a s t h o u g h t t o b e o f g r e a t e r i m p o r t a n c e t h a n t h e c a t h e p s i n D i n r e l a t i o n t o p r o d u c t d e g r a d a t i o n .

G r o n i n g e r ( 1 9 6 4 ) p a r t i a l l y p u r i f i e d a n d c h a r a c t e r i z e d a n a c i d p r o t e a s e f r o m a l b a c o r e m u s c l e w i t h a p H o p t i m u m o f2 . 4 — 2 . 5 . T h e e n z y m e w a s a c t i v e a g a i n s t h e m o g l o b i n , b u t i n a c t i v e a g a i n s t b o v i n e s e r u m a l b u m i n a n d s y n t h e t i c s u b s t r a t e s o f c a t h e p s i n s A , B a n d C . R e c e n t l y ,

R e d d i e t a l . ( 1 9 7 2 ) s t u d i e d a l y s o s o m a l c a t h e p s i n i n t h e m u s c l e o f w i n t e r f l o u n d e r w h i c h w a s o p t i m a l l y a c t i v e a t p H 4 . 0 f o r b o t h h e m o g l o b i n a c t i v i t y a n d b r e a k d o w n o f e n d o g e n o u s p r o t e i n s .

S a l e m e t a l . ( 1 9 7 0 ) i n v e s t i g a t e d g e n e r a l p r o t e o l y t i c d e c o m p o s i t i o n o f s e v e r a l s h e l l f i s h a n d o b s e r v e d a n i n c r e a s e i n t h e

t y r o s i n e v a l u e i n m u s c l e s o f p r a w n , b l u e c r a b , c o a r s e c l a m a n d c u t t l e f i s h . W h i l e s p e c i f i c e n z y m e a c t i v i t i e s w e r e n o t d e f i n e d i n t h i s w o r k , t h e a u t h o r s d i d i n d i c a t e t h a t t h e r a p i d i n c r e a s e i n t y r o s i n e

ACTIV ITY OF PENAEUS SETIFERUS MUSCLE

v a l u e w a s d u e i n p a r t t o e n d o g e n o u s p r o t e o l y t i c a c t i v i t y .

T h e p u r p o s e o f t h e p r e s e n t s t u d y w a s t o a s s e s s t h e c a t h e p t i c a c t i v i t y o f m u s c l e

o f w h i t e s h r i m p , Penaeus setiferus, a n d

i d e n t i f y t h e s p e c i f i c e n z y m e s i n v o l v e d .

EXPERIMENTALS o u r c e o f s h r i m p

L i v e w h i t e s h r i m p (P e n a e u s s e t i f e r u s ) w e r e p r o v i d e d b y t h e S k i d a w a y I n s t i t u t e o f O c e a n o g r a p h y , S a v a n n a h , G a . a n d e i t h e r t r a n s p o r t e d l iv e t o t h e U n i v e r s i t y o f G e o r g i a a t A t h e n s o r d e h e a d e d a n d i m m e d i a t e l y i c e d b e f o r e t r a n s p o r t a t i o n . C o m m e r c i a l s h r i m p w e r e p u r c h a s e d a t s h r i m p d o c k s l o c a t e d a t B r u n s w i c k , G a . o r a t l o c a l w h o l e s a l e o u t l e t s . S h r i m p w e r e u s e d f r e s h

o r h e l d a t - 3 0 ° C u n t i l u s e .

P r e p a r a t i o n o f s h r i m p m u s c l e e x t r a c t

S h r i m p w e r e d e v e i n e d a n d t h o r o u g h l y w a s h e d w i t h a r u n n i n g s t r e a m o f d i s t i l l e d H 2 O .

2 5 g o f s h r i m p m u s c l e w e r e b l e n d e d f o r 3 0 s e c w i t h 7 5 m l o f c o l d 2 .0 % K C 1 i n a W a r i n g B l e n d o r . T h e h o m o g e n a t e w a s c e n t r i f u g e d a t1 0 , 0 0 0 x G f o r 1 0 m i n a t 4 ° C , a n d t h e i n s o l u b l e r e s i d u e w a s d i s c a r d e d . E n z y m e a s s a y s w e r e c a r r i e d o u t o n t h e s u p e r n a t a n t i m m e d i a t e l y o r

a f t e r s t o r a g e a t - 3 0 ° C . C o m p a r i s o n o f c a t h e p s in D a c t i v i t y i n t h e 2 .0 % K C 1 s u p e r n a t a n t t o t h a t i n e x t r a c t s p r e p a r e d w i t h 2 .0 % K C 1 c o n t a i n i n g 0 .1 % T r i t o n X - 1 0 0 i n d i c a t e d t h a t t h e b l e n d i n g p r o c e d u r e w i t h 2 .0 % K C 1 r e s u l t e d in c o m p l e t e r e l e a s e o f a c t i v i t y .

A n a l y t i c a l m e t h o d s

P r o t e i n c o n t e n t o f s h r i m p m u s c l e e x t r a c t w a s d e t e r m i n e d b y u s i n g t h e b i u r e t m e t h o d a s g iv e n b y G o r n a l l e t a l . ( 1 9 4 9 ) a n d b y t h e F o l i n - C i o c a l t e a u r e a g e n t m e t h o d o f L o w r y e t a l . ( 1 9 5 1 ) . B o v in e s e r u m a l b u m i n ( N u t r i t i o n a l B i o c h e m i c a l C o r p . ) w a s t h e s t a n d a r d p r o t e i n .

C a t h e p s i n A a c t i v i t y w a s m e a s u r e d b y a m o d i f i e d m e t h o d o f L i c h t e n s t e i n a n d F r u t o n ( 1 9 6 0 ) . T h e r e a c t i o n m i x t u r e c o n t a i n e d 0 . 1 5 m l o f 0 . 0 3 4 M c a r b o b e n z o x y - L - g l u t a m y l - L - t y r o s i n e i n 0 .2 M c i t r a t e b u f f e r , p H 5 . 6 , a n d 0 . 1 5 m l o f s h r i m p m u s c l e e x t r a c t . A f t e r i n c u b a t i o n f o r 1 h r a t 3 7 ° C , 1 .7 m l o f 1 .0 % p i c r i c a c id w a s a d d e d t o t h e r e a c t i o n m i x t u r e . F o l l o w i n g f i l t r a t i o n t h r o u g h W h a t m a n # 1 f i l t e r p a p e r , d u p l i c a t e 0 . 0 2 5 m l a l i q u o t s o f t h e f i l t r a t e w e r e a s s a y e d b y t h e Y e m m a n d C o c k i n g n i n h y d r i n m e t h o d a s m o d i f i e d b y M a t h e s o n a n d T a t t r i ( 1 9 6 4 ) . F o r t h e c o n t r o l , s h r i m p e x t r a c t w a s a d d e d t o t h e s u b s t r a t e f o l l o w i n g a d d i t i o n o f t h e p i c r i c a c i d .

C a t h e p s i n B a c t i v i t y w a s m e a s u r e d b y t h e m e t h o d o f G r e e n b a u m a n d F r u t o n ( 1 9 5 7 ) . T h e r e a c t i o n w a s c a r r i e d o u t i n t h e o u t e r w e l l o f a C o n w a y M i c r o d i f f u s i o n d i s h f o r 2 a n d 2 4 h r a t 3 7 ° . T h e r e a c t i o n m i x t u r e c o n t a i n e d 0 .5 m l o f 1 0 m M b e n z o y l - L - a r g i n i n a m i d e in 0 .2 M c i t r a t e b u f f e r , p H 5 . 0 , 0 .5 m l o f m u s c l e e x t r a c t , a n d 0 .5 m l o f 0 .2 M c i t r a t e b u f f e r , p H 5 . 0 , c o n t a i n in g 0 .0 8 M c y s t e i n e . A f t e r i n c u b a t i o n , 1 .0 m l o f 5 . 0 % t r i c h l o r o a c e t i c a c i d a n d 1 .0 m l o f 4 5 . 0 %

K 2 C 0 3 w e r e i n j e c t e d i n t o t h e r e a c t i o n m i x t u r e . L i b e r a t e d N H 3 w a s a b s o r b e d i n t o 1 .0 m l o f

0 . 0 3 N H 2 S O „ c o n t a i n e d in t h e c e n t e r w e l l o f t h e C o n w a y d i s h . A f t e r s h a k i n g f o r 1 h r i n a D u b n o f f m e t a b o l i c s h a k e r , t h e c o n t e n t s o f t h e c e n t e r w e l l w a s q u a n t i t a t i v e l y t r a n s f e r r e d , a n d

N H 3 w a s d e t e r m i n e d a c c o r d i n g t o t h e N e s s l e r r e a c t i o n ( J o h n s o n , 1 9 4 1 ) .

C a t h e p s i n B a c t i v i t y w a s a l s o d e t e r m i n e d u t i l i z i n g t h e c h r o m o g e n i c s u b s t r a t e , b e n z o y l - L - a r g i n i n a m i d e - ( 3 - n a p h t h y l a m i n e , a c c o r d i n g t o t h e p r o c e d u r e o f M c D o n a l d e t a l . ( 1 9 6 9 ) .

C a t h e p s i n C a c t i v i t y w a s a s s a y e d b y t h e m e t h o d o f D e L a H a b a e t a l . ( 1 9 5 9 ) w i t h 1 0 m M g l y c y l - L - t y r o s i n a m i d e in 0 .1 M c i t r a t e b u f f e r , p H 5 . 0 , a s s u b s t r a t e . T h e r e a c t i o n m i x t u r e c o n t a i n e d 0 .0 2 M c y s t e i n e , a n d q u a n t i t a t i o n o f a c t i v i t y f o l l o w e d t h e p r o c e d u r e d e s c r i b e d f o r

c a t h e p s i n B . C a t h e p s i n C a c t i v i t y w a s a l s o m e a s u r e d w i t h g l y c y l - L - p h e n y l a l a n i n e - j 3 - n a p h t h y l -

a m i d e ( M c D o n a l d e t a l . , 1 9 6 9 ) .C a t h e p s i n D a c t i v i t y w a s a s s a y e d b y a m o d i

f i c a t i o n o f t h e m e t h o d o f A n s o n ( 1 9 3 8 ) u s i n g a c i d d e n a t u r e d a n d a c i d - u r e a d e n a t u r e d h e m o g l o b i n s u b s t r a t e s . T h e a c i d d e n a t u r e d h e m o g l o b i n w a s p r e p a r e d b y a d j u s t i n g a 4 . 4 % h e m o g l o b i n s o l u : i o n t o p H 1 .0 w i t h c o n c e n t r a t e d H C 1 a n d a l l o w i n g t h e s o l u t i o n t o s t a n d a t a m

b i e n t t e m p e r a t u r e f o r 2 h r . T h e s o l u t i o n w a s a d j u s t e d t o t h e d e s i r e d p H w i t h 2 . O N N a O H

a n d d i l u t e d ( 1 : 1 v / v ) w i t h 0 .4 M c i t r a t e o r 0 .4 M p h o s p h a t e b u f f e r o f t h e d e s i r e d p H .

T h e a c i d - u r e a d e n a t u r e d h e m o g l o b i n w a s

p r e p a r e d b y a d j u s t i n g a 4 . 4 % h e m o g l o b i n s o l u t i o n t o p H 1 .0 w i t h c o n c e n t r a t e d H C 1 a n d a l l o w i n g i t t o s t a n d a t a m b i e n t t e m p e r a t u r e f o r

1 0 m i n . S o l i d u r e a w a s t h e n a d d e d t o m a k e t h e h e m o g l o b i n s o l u t i o n 5 .4 M i n u r e a . A f t e r 3 0 m i n , t h e s o l u t i o n w a s a d j u s t e d t o t h e d e s i r e d p H w i t h 2 . O N N a O H o r 2 . O N H C 1 a n d d i l u t e d

( 1 : 1 v / v ) w i t h 0 .4 M c i t r a t e o r 0 .4 M p h o s p h a t e b u f f e r s c o n t a i n i n g 5 .4 M u r e a . F o r p H o p t i m a

s t u d i e s , c i t r a t e b u f f e r w a s u s e d b e t w e e n p H 2 .2 a n d 5 .0 a n d p h o s p h a t e b u f f e r f o r t h e p H r a n g e

5 . 0 - 8 . 0 .C a t h e p s i n D a c t i v i t y w a s d e t e r m i n e d b y i n

c u b a t i n g e q u a l v o l u m e s o f s u b s t r a t e a n d s h r i m p m u s c l e e x t r a c t i t 3 7 ° C f o r 4 h r . A l l c a t h e p s i n D r e a c t i o n m i x t u r e s ( 1 0 m l ) c o n t a i n e d 0 .1 m l o f t o l u e n e .

F o l l o w i n g t h e i n c u b a t i o n , 2 . 0 m l a l i q u o t s o f t h e r e a c t i o n m i x t u r e w e r e r e m o v e d a n d p i p e t t e d i n t o t e s t t u b e s c o n t a i n i n g 5 .0 m l o f 1 0 .0 % t r i c h l o r o a c e t i c a c i d . A f t e r 3 0 m i n , t h e p r e c i p i t a t e d p r o t e i n w a s r e m o v e d b y f i l t r a t i o n t h r o u g h W h a t m a n # 1 f i l t e r p a p e r . L i b e r a t e d t y r o s i n e i n t h e s u p e r n a t a n t w a s q u a n t i t a t e d b y t h e L o w r y p r o c e d u r e , a n d a c t i v i t y w a s d e t e r m i n e d f r o m a s t a n d a r d c u r v e a n d e x p r e s s e d a s n m o l e s t y r o s i n e / m g p r o t e i n / h r .

A c t i v i t y a g a i n s t 2 .0 % K C 1 s o l u b l e p r o t e i n w a s d e t e r m i n e d b y a d j u s t i n g t h e e x t r a c t t o p H3 .0 w i t h 1 .0 N H C 1 a n d a d d i n g a n e q u a l v o l u m e o f 0 .4 M c i t r a t e b u f f e r , p H 3 . 0 . T h e a s s a y w a s c a r r i e d o u t a s d e s c r i b e d f o r t h e h e m o g l o b i n a s s a y . C o n t r o l s w e r e o b t a i n e d b y a d d i t i o n o f t h e t r i c h l o r o a c e t i c a c i d t o t h e r e a c t i o n c o m p o n e n t s b e f o r e t h e a d d i t i o n o f s u b s t r a t e .

6-JO U R N AL OF FOOD SCIENCE-Volume 39 (1974)

SHRIMP MUSCLE CATHEPS IN-7

T o d i f f e r e n t i a t e b e t w e e n f r e e a n d b o u n d

c a t h e p s i n D in t h e m u s c l e , t o t a l c a t h e p s i n D a c t i v i t y w a s d e t e r m i n e d in a s u p e r n a t a n t p r e

p a r e d f r o m 2 0 .0 % h o m o g e n a t e o f f r e s h u n f r o z e n s h r i m p m u s c l e p r e p a r e d b y b l e n d i n g t h e m u s c l e i n a W a r i n g B l e n d o r f o r 1 5 s e c a t 4 ° C in

0 .2 5 M s u c r o s e c o n t a i n i n g 0 .1 % T r i t o n X - 1 0 0 . T h e h o m o g e n a t e w a s c e n t r i f u g e d f o r 3 0 m i n in

a r e f r i g e r a t e d M o d e l L S p i n c o c e n t r i f u g e a t5 0 , 0 0 0 x G . T o d e t e r m i n e f r e e e n z y m e t h e s a m e p r o c e d u r e w a s u s e d e x c e p t T r i t o n X - 1 0 0 w a s o m i t t e d . B o u n d a c t i v i t i e s w e r e c a l c u l a t e d b y s u b t r a c t i n g f r e e a c t i v i t i e s f r o m t o t a l a c t i v i t i e s . C a t h e p s i n a c t i v i t y in t h e s u p e r n a t a n t s w a s

d e t e r m i n e d a s p r e v i o u s l y d e s c r i b e d . T h e e n z y m e a s s a y s in a l l e x p e r i m e n t s w e r e d o n e in d u p l i c a t e .

RESULTS & DISCUSSIONActivity against various substrates

T a b l e 1 s u m m a r i z e s t h e a c t i v i t y o f t h e s h r i m p e x t r a c t a g a i n s t v a r i o u s c a t h e p s i n s u b s t r a t e s . A c t i v i t y w a s o n l y d e t e c t e d

a g a i n s t a c i d d e n a t u r e d h e m o g l o b i n , a c i d - u r e a d e n a t u r e d h e m o g l o b i n , a n d a g a i n s t 2 . 0 % K C 1 s o l u b l e m u s c l e p r o t e i n i n t h e a c i d p H r a n g e . T h e r e w a s n o a c t i v i t y a g a i n s t t h e s e s u b s t r a t e s a t p H 8 . 0 , i n d i c a t i n g t h a t t h e e n z y m e i s o f t h e c a t h e p s i n D t y p e . T h e e x t r a c t w a s i n a c t i v e a g a i n s t c a s e i n , a z o c a s e i n a n d b o v i n e s e r u m a l b u m i n w h i c h a r e a t t a c k e d t o v a r y i n g d e g r e e s b y m a m m a l i a n c a t h e p s i n D e n z y m e s ( L e b e z e t a l . , 1 9 7 1 ) a n d b y c a t h e p - t i c e n z y m e s o b t a i n e d f r o m s o m e a q u a t i c s o u r c e s ( G r o n i n g e r , 1 9 6 4 ; R e d d i e t a l . , l ' 9 7 2 ; M a k i n o d a n a n d I k e d a , 1 9 6 9 a ) .

T h e e x t r a c t w a s i n a c t i v e a g a i n s t c a r b o -

b e n z o x y - L - g l u t a m y l - L - t y r o s i n e ( C a t h e p s i n A s u b s t r a t e ) , b e n z o y l - L - a r g i n i n a m i d e

a n d b e n z o y l - L - a r g i n i n a m i d e - ( 3 - n a p h t h y l - a m i d e ( C a t h e p s i n B s u b s t r a t e s ) a n d a g a i n s t g l y c y l - L - t y r o s i n a m i d e a c e t a t e a n d g l y c y l - L - p h e n y l a l a n i n e - ' j 3 (- n a p h t h y l a m i d e

Fig. 1—E f f e c t o f h e m o g lo b in o n t h e p r o t e o l y t i c

a c t i v i t y Ip H 3 .0 ) o f m u s c l e p r o t e i n e x t r a c t a s a f u n c t i o n o f in c u b a t io n t im e .

( C a t h e p s i n C s u b s t r a t e s ) , i n d i c a t i n g t h e a b s e n c e o f t h e s e e n z y m e s i n t h e m u s c l e . C a t h e p s i n A , B a n d C h a v e b e e n r e p o r t e d

i n c a r p m u s c l e ( M a k i n o d a n a n d I k e d a ,1 9 7 1 ) b u t n o t i n c o d m u s c l e ( S i e b e r t e t

a l . , 1 9 6 3 ) a n d a l b a c o r e m u s c l e ( G r o n i n g e r , 1 9 6 4 ) .

W h e n b a s e d o n a 4 - h r r e a c t i o n t i m e , t h e l e v e l o f e n z y m e a c t i v i t y a g a i n s t 2 . 0 % K C 1 s o l u b l e p r o t e i n s w a s s l i g h t l y l o w e r t h a n w h e n a c i d d e n a t u r e d o r a c i d - u r e a d e n a t u r e d h e m o g l o b i n w a s i n c l u d e d i n t h e

r e a c t i o n m i x t u r e ( 8 . 5 n m o l e s t y r o s i n e / m g p r o t e i n / h r c o m p a r e d t o 1 2 . 5 a n d I n m o l e s t y r o s i n e / m g p r o t e i n / h r , r e s p e c t i v e l y ) . H o w e v e r , a s s h o w n i n F i g u r e 1 , t h e r e a c t i o n a g a i n s t t h e e n d o g e n o u s p r o t e i n s

1 5 . 0 - o ---------o A C I D D E N A T U R E D

K • — • a c i d - u r e a d e n a t u r e d

p H

F ig. 2 —E f f e c t o f p H o n a c t i v i t y a g a in s t a c id

d e n a t u r e d a n d a c id -u r e a d e n a t u r e d h e m o g lo b in .

i n t h e e x t r a c t s u b s i d e d q u i c k l y a n d w a s o n l y p r e s e n t a t a l o w r a t e a f t e r 6 h r o f i n c u b a t i o n . T h e a c t i v i t y i n t h e r e a c t i o n c o n t a i n i n g h e m o g l o b i n c o n t i n u e d w i t h t y r o s i n e s t i l l b e i n g l i b e r a t e d a f t e r 2 4 h r o f i n c u b a t i o n . T h e r a p i d l o s s o f a c t i v i t y w h e n t h e e x t r a c t w a s i n c u b a t e d a t 3 7 ° C c o m p a r e d t o t h e r e a c t i o n i n t h e p r e s e n c e o f h e m o g l o b i n p r o b a b l y r e p r e s e n t s a

p r o t e c t i v e a c t i o n o f t h e h e m o g l o b i n s u b s t r a t e t o w a r d t h e e n z y m e .

Effect o f pH and temperature on catheptic activity

T h e e f f e c t o f p H o n p r o t e o l y t i c a c t i v i

t i e s a g a i n s t a c i d d e n a t u r e d a n d a c i d - u r e a d e n a t u r e d h e m o g l o b i n i s s h o w n i n F i g u r e

Table 1—Cathepsin a c tiv ity against several substrates

Substrate pH ActivityAcid denatured hemoglobin 3.0 12.5a

8.0 0Acid-urea denatured hemoglobin 3.5 1 1 .0a

8.0 0Soluble shrimp muscle protein 3.0 8.5a

8.0 0Casein 3.0 0

8.0 0Azocasein 3.0 0

8.0 0Bovine serum albumin 3.0 0

8.0 0Carbobenzoxy-L-glutamy I-L-tyrosine 5.6 0Benzoyl-L-argininamide 5.0 0Benzoyl-L-argininamide-0-naphthylamide 6.0 0Glycyl-L-tyrosinamide acetate 5.0 0Glycyl-L-phenylalanine-fi-naphthylamide 6.0 0

a n M ole ty ro s in e /m g p ro te in /h r

Table 2—Effect of several chemicals on cathepsin D activityConcentration % Inhibition

Treatment (mM) (% Activation)Control - 0

CaCl2 10 9ZnCI2 10 40ZnCI2 1 37CoClj 1 0 44CoCI2 1 25HgCI2 10 69HgCI2 1 37EDTA 10 50EDTA 1 25Cysteine 10 (18)Cysteine 1 0KCN 1 0 23N-ethylmaleimice 10 0N-ethylmaleimice 1 0P-chloromercurioenzoate 10 0P-chloromercuribenzoate 1 0

8 - JO UR NA L OF FOOD SCIE NC E- Vo lum e 3 9 (1974)

TIME MIN

F ig. 3 —E f f e c t o f t e m p e r a tu r e o n s h r im p m u s c l e c a th e p s in . F ig. 4 —H e a t s t a b i l i t y a t 4 5 a n d 5 0 ° C o f c a t h e p

s in a c t i v i t y in 2 .0 % K C I e x t r a c t , p H 7 .2 .

2 . T h e p H o p t i m u m w a s 3 . 0 f o r t h e a c i d d e n a t u r e d s u b s t r a t e a n d 3 . 5 f o r t h e a c i d -

u r e a d e n a t u r e d h e m o g l o b i n . A p H o p t i m u m s h i f t i s c o m m o n l y o b s e r v e d w h e n a c i d d e n a t u r e d a n d a c i d - u r e a d e n a t u r e d h e m o g l o b i n s u b s t r a t e s a r e u s e d f o r

c a t h e p s i n D c h a r a c t e r i z a t i o n . B o v i n e s p l e e n c a t h e p s i n D s h o w s a n o p t i m u m p H

o f 3 . 0 f o r a c i d d e n a t u r e d a n d 3 . 8 f o r u r e a d e n a t u r e d ( K e i l o v a , 1 9 7 1 ) .

T h e o p t i m u m t e m p e r a t u r e o f t h e

s h r i m p m u s c l e c a t h e p s i n d u r i n g a 4 - h r i n - e u b a t i o n w a s 4 2 ° C ( F i g . 3 ) . A b o v e 4 2 ° C t h e a c t i v i t y r a p i d l y d e c r e a s e d . F i g u r e 4 s h o w s t h e e f f e c t o f h e a t i n g t h e m u s c l e e x t r a c t a t 4 5 ° C a n d 5 0 ° C f o r p e r i o d s u p t o 3 0 m i n . T h e e n z y m e w a s c o m p l e t e l y i n a c t i v a t e d w i t h i n 1 0 m i n a t 5 0 ° C . C a t h e p s i n D e n z y m e s f r o m b o t h m a m m a l i a n a n d a q u a t i c s o u r c e s h a v e o p t i m a l t e m p e r a t u r e s n e a r t h e o p t i m u m o b s e r v e d f o r t h e s h r i m p m u s c l e e n z y m e . T h e a l b a - c o r e m u s c l e p r o t e i n a s e s t u d i e d b y G r o n - i n g e r ( 1 9 6 4 ) h a d a n o p t i m u m o f 4 2 ° C , a n d t h e a c t i v i t y o f b o v i n e m u s c l e c a t h e p s i n D w a s o p t i m a l a t 4 0 ° C ( L u t a l o - B o s a a n d M a c R a e , 1 9 6 9 ) . T h e s e e n z y m e s w e r e o b s e r v e d t o b e h e a t - l a b i l e w i t h l o s s o f a c t i v i t y o c c u r r i n g r a p i d l y a t t e m p e r a t u r e s a b o v e t h e o p t i m u m .

E f f e c t o f i n h i b i t o r s a n d

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I n o r d e r t o s t u d y t h e e f f e c t o f s e v e r a l

s u b s t a n c e s o n t h e c a t h e p s i n D a c t i v i t y o f

t h e m u s c l e e x t r a c t , t h e 2 . 0 % K C I e x t r a c t

w a s d i l u t e d 1 : 1 w i t h t h e m o d i f i e r s o l u

t i o n s a t c o n c e n t r a t i o n s n e c e s s a r y t o g i v e t h e f i n a l c o n c e n t r a t i o n s s h o w n i n T a b l e2 . T h e e x t r a c t c o n t a i n i n g t h e a c t i v a t o r o r

i n h i b i t o r w a s a l l o w e d t o s t a n d a t a m b i e n t t e m p e r a t u r e f o r 1 5 m i n b e f o r e t h e a c i d d e n a t u r e d h e m o g l o b i n w a s a d d e d .

O f t h e v a r i o u s c o m p o u n d s t e s t e d o n l y 1 0 m M c y s t e i n e a c t i v a t e d t h e c a t h e p s i n a c t i v i t y . N o m e a s u r a b l e c y s t e i n e e f f e c t

w a s n o t e d a t a c o n c e n t r a t i o n o f 1 m M . C h l o r i d e s a l t s o f C a + + , Z n + + , C o + + a n d H g + + i n h i b i t e d t h e a c t i v i t y t o v a r y i n g d e g r e e s w i t h C a C l 2 s h o w i n g t h e l e a s t i n h i b i t o r y e f f e c t , i n h i b i t i n g t h e a c t i v i t y b y o n l y 9 . 0 % a t a c o n c e n t r a t i o n o f 1 0 m M ; w h e r e a s , H g C l 2 ( 1 0 m M ) w a s t h e s t r o n g e s t i n h i b i t o r , i n h i b i t i n g t h e a c t i v i t y b y 6 9 . 0 % . K C N ( 1 0 m M ) a n d E D T A ( 1 m M a n d 1 0 m M ) a l s o i n h i b i t e d t h e c a t h e p s i n a c t i v i t y , b u t N - e t h y l m a l e i m i d e a n d p - c h l o r o m e r c u r i b e n z o a t e d i d n o t a l t e r t h e a c t i v i t y .

I t i s i m p o r t a n t t o n o t e t h a t a c t i v a t i o n o r i n h i b i t i o n o c c u r r e d o n l y a t r e l a t i v e l y h i g h m o d i f i e r c o n c e n t r a t i o n s o f 1 m M

a n d 1 0 m M . C y s t e i n e a c t i v a t i o n i s n o t a c o m m o n o c c u r r e n c e f o r c a t h e p s i n D t y p e e n z y m e s ; h o w e v e r , t h e c a t h e p s i n D f r o m c h i c k e n m u s c l e ( C a l d w e l l a n d G r o s j e a n ,1 9 7 1 ) a n d t h e a l k a l i n e p r o t e i n a s e o f c a r p m u s c l e w e r e a c t i v a t e d b y c y s t e i n e ( M a k i n o d a n a n d I k e d a , 1 9 6 9 b ) . M e t a l i n a c t i v a t i o n v a r i e s f o r c a t h e p s i n D e n z y m e s

s t u d i e d f r o m d i f f e r e n t s p e c i e s . A c t i v i t y o f a p u r i f i e d p o r c i n e c a t h e p s i n w a s i n h i b i t e d b y 1 0 m M c a l c i u m c h l o r i d e a n d z i n c a c e t a t e ( P a r r i s h a n d B a i l e y , 1 9 6 6 ) . K o s z a l k a a n d M i l l e r ( 1 9 6 0 ) a l s o f o u n d t h a t r a t

m u s c l e p r o t e a s e o p t i m a l l y a c t i v e a t p H 8 . 5 ^ 9 . 0 w a s i n h i b i t e d b y 1 0 m M c o n c e n t r a t i o n s o f c a l c i u m c h l o r i d e a n d z i n c a c e

t a t e .

F r e e a n d b o u n d c a t h e p s i n a c t i v i t y

D i f f e r e n t i a t i o n o f f r e e a n d b o u n d c a t h e p s i n D a c t i v i t y i n t h r e e l o t s o f f r e s h w h i t e s h r i m p c o l l e c t e d a t d i f f e r e n t t i m e s i n d i c a t e d t h a t 8 1 ± 8 . 1 % o f t h e c a t h e p s i n D i n t h e m u s c l e e x i s t e d i n a b o u n d s t a t e

a n d i s p o s s i b l y l y s o s o m a l i n n a t u r e . C a t h e p s i n D e n z y m e s f r o m m o s t s o u r c e s

a r e f o u n d i n l y s o s o m e s . S h i b k o e t a l . ( 1 9 6 3 ) r e p o r t e d t h a t c a t h e p s i n s o f s e v e r a l i n v e r t e b r a t e s p e c i e s i n c l u d i n g a p r a w n

s p e c i e s w e r e l y s o s o m a l i n n a t u r e . A l s o , f i s h m u s c l e c a t h e p s i n s h a v e b e e n r e p o r t e d t o e x i s t i n l y s o s o m a l p a r t i c l e s ( B i r d e t a l . , 1 9 6 9 ; R e d d i e t a l . , 1 9 7 2 ) .

CONCLUSIONC A T H E P S I N D t y p e e n z y m e a c t i v i t y h a s b e e n d e m o n s t r a t e d i n t h e m u s c l e o f t h e w h i t e s h r i m p . T h e a b s e n c e o f c a t h e p s i n s

A , B a n d C s u g g e s t s t h a t t h e s h r i m p m u s c l e h a s a p e p t i d a s e p r o f i l e d i f f e r e n t f r o m t h a t o f m a m m a l i a n t i s s u e . J o i n t a c t i o n o f c a t h e p s i n D w i t h o t h e r c a t h e p s i n s h a s b e e n p o s t u l a t e d b y H u a n g a n d T a p p e l

SHRIMP MUSCLE CA TH EP SIN -9

0 9 7 1 ) a s a m e c h a n i s m f o r o r d e r l y p r o t e i n d e g r a d a t i o n i n m a m m a l i a n t i s s u e .

T h e a b s e n c e o f e n z y m e s w i t h s p e c i f i c i t i e s s i m i l a r t o c a t h e p s i n A , B a n d C d o e s n o t p r e c l u d e t h e p r e s e n c e o f o t h e r e n z y m e s

w i t h d i f f e r e n t s p e c i f i c i t i e s b e i n g p r e s e n t i n t h e s h r i m p m u s c l e t h a t c o u l d p a r t i c i p a t e i n f u r t h e r p r o t e i n d e g r a d a t i o n .

REFERENCESA n s o n , M .L . 1 9 3 8 . T h e e s t i m a t i o n o f p e p s i n ,

t r y p s i n , p a p a i n a n d c a t h e p s i n w i t h h e m o g l o b i n . J . G e n . P h y s i o l . 2 2 : 7 9 .

B i r d , J .W .C . , B e r g , T . , M i l a n e s i , A . a n d S t a u b e r , W .T . 1 9 6 9 . L y s o s o m a l e n z y m e s i n a q u a t i c s p e c i e s . 1 . D i s t r i b u t i o n a n d p a r t i c l e p r o p e r t i e s o f m u s c l e l y s o s o m e s o f t h e g o l d f i s h . C o m p . B i o c h e m . P h y s i o l . 3 0 : 4 5 7 .

C a l d w e l l , K .A . a n d G r o s j e a n , O .K . 1 9 7 1 . L y s o s o m a l c a t h e p s i n s o f c h i c k e n s k e l e t a l m u s c l e : D i s t r i b u t i o n a n d p r o p e r t i e s . J . A g r . F o o d C h e m . 1 9 : 1 0 8 .

D e L a H a b a , G . , C a m m a r a t a , P .S . a n d T i m a - s h e f f , S .N . 1 9 5 9 . T h e p a r t i a l p u r i f i c a t i o n a n d s o m e p h y s i c a l p r o p e r t i e s o f c a t h e p s i n C f r o m b e e f s p l e e n . J . B io l . C h e m . 2 3 4 : 3 1 6 .

G o r n a l l , A .G . , B a r d a w i l l , C . J . a n d D a v i d , M .M . 1 9 4 9 . D e t e r m i n a t i o n o f s e r u m p r o t e i n s b y m e a n s o f b i u r e t r e a c t i o n . J . B io l . C h e m . 1 7 7 : 7 5 1 .

G r e e n b a u m , L .M . a n d F r u t o n , J . S . 1 9 5 7 . P u r i f i c a t i o n a n d p r o p e r t i e s o f b e e f s p l e e n c a t h e p s i n B . J . B io l . C h e m . 2 2 6 : 1 7 3 .

G r o n i n g e r , H .S . J r . 1 9 6 4 . P a r t i a l p u r i f i c a t i o n a n d s o m e p r o p e r t i e s o f a p r o t e i n a s e f r o m A l b a c o r e ( G e r m o a l a l u n g a ) m u s c l e . A r c h , o f B i o c h e m . B i o p h y s . 1 0 8 : 1 7 5 .

H u a n g , F . L . a n d T a p p e l , A .L . 1 9 7 1 . A c t i o n o f c a t h e p s i n s C a n d D i n p r o t e i n h y d r o l y s i s . B i o c h i m . B i o p h y s . A c t a 2 3 6 : 7 3 9 .

J o h n s o n , M . 1 9 4 1 . I s o l a t i o n a n d p r o p e r t i e s o f a p u r e y e a s t p o l y p e p t i d a s e . J . B io l . C h e m . 1 3 7 : 5 7 5 .

K e i l o v a , H . 1 9 7 1 . O n t h e s p e c i f i c i t y a n d i n h i b i t i o n o f c a t h e p s i n D a n d B . I n “ T i s s u e P r o t é i n a s e s , ” E d . B a r r e t t , A . J . a n d D i n g l e , J . T . , p . 4 5 . A m e r i c a n E l s e v i e r P u b l i s h i n g C o . ,N .Y .

K o s z a l k a , T . R . a n d M i l l e r , L .L . 1 9 6 0 . P r o t e o l y t i c a c t i v i t y o f r a t m u s c l e . P u r i f i c a t i o n a n d p r o p e r t i e s o f a n e n z y m e a c t i v e o p t i m a l l y a t p H 8 . 5 t o 9 . 0 . J . B io l . C h e m . 2 3 5 : 6 6 9 .

L e b e z , D . , K o p i t a r , M . , T u r k , V . a n d K r e g a r , I . 1 9 7 1 . C o m p a r i s o n o f p r o p e r t i e s o f c a t h e p s i n s D a n d E w i t h s o m e n e w c a t h e p s i n s . I n “ T i s s u e P r o t e i n a s e s , ” E d . B a r r e t t , A . J . a n d D i n g l e , J . T . , p . 1 6 7 . A m e r i c a n E l s e v i e r P u b l i s h i n g C o . , N .Y .

L i c h t e n s t e i n , N . a n d F r u t o n , J . S . 1 9 6 0 . S t u d i e s o n b e e f s p l e e n c a t h e p s i n A . P r o c . N a t l . A c a d . S c i . U .S . 4 6 : 7 8 7 .

L o w r y , O .H . , R o s e b r o u g h , N . , F a r r , A . a n d R a n d a l l , J . 1 9 5 1 . P r o t e i n m e a s u r e m e n t w i t h t h e F o l i n p h e n o l r e a g e n t . J . B i e l . C h e m . 1 9 3 : 2 6 5 .

L u t a l o - B o s a , A . J . a n d M a c R a e , H . F . 1 9 6 9 . H y d r o l y t i c e n z y m e s i n b o v i n e s k e l e t a l m u s c l e . 3 . A c t i v i t y o f s o m e c a t h e p t i c e n z y m e s . J . F o o d S c i . 3 4 : 4 0 1 .

M a k i n o d a n , Y . a n d I k e d a , S . 1 9 6 9 a . S t u d i e s o n f i s h m u s c l e p r o t e a s e . I I . P u r i f i c a t i o n a n d p r o p e r t i e s o f a p r o t e i n a s e a c t i v e m s l i g h t l y a l k a l i n e p H r a n g e . B u l l . J a p . S o c . S c i . F i s h . 3 5 : 7 4 9 .

M a k i n o d a n , Y . a n d I k e d a , S . 1 9 6 9 b . S t u d i e s o n f i s h m u s c l e p r o t e a s e . I I I . P u r i f i c a t i o n a n d p r o p e r t i e s o f a p r o t e a s e a c t i v e i n a c i d p H r a n g e . B u l l . J a p . S o c . S c i . F i s h . 3 5 : 7 5 8 .

M a k i n o d a n , Y . a n d I k e d a , S . 1 9 7 1 . S t u d i e s o n

f i s h m u s c l e p r o t e a s e . V . O n t h e e x i s t e n c e o f c a t h e p s i n s A , B a n d C . B u l l . J a p . S o c . S c i . F i s h . 3 7 : 1 0 0 2 .

M a t h e s o n , A .T . a n d T a t t r i , B .L . 1 9 6 4 . A m o d i f i e d Y e m m a n d C o c k l i n g n i n h y d r i n r e a g e n t f o r p e p t i d a s e a s s a y . C a n . J . B i o c h e m . 4 2 : 9 5 .

M c D o n a l d , J . K . , Z e i t m a n , B . , R e i l l y , T . a n d E l l i s , S . 1 9 6 9 . N e w o b s e r v a t i o n s o n t h e s u b s t r a t e s p e c i f i c i t y o f c a t h e p s i n C ( a m i n o p e p - t i d a s e I ) . I n c l u d i n g d e g r a d a t i o n o f B - c o r t i c o - t r o p i n a n d o t h e r p e p t i d e h o r m o n e s . J . B io l . C h e m . 2 4 4 : 2 6 9 3 .

P a r r i s h , F .C . J r . a n d B a i l e y , M .E . 1 9 6 6 . P h y s i c o c h e m i c a l p r o p e r t i e s a n d p a r t i a l p u r i f i c a t i o n o f p o r c i n e m u s c l e c a t h e p s i n . J . A g r . F o o d C h e m . 1 4 : 2 3 2 .

R e d d i , P . E . , C o n s t a n t i n i d e s , S . a n d D y m s z a , H .1 9 7 2 . C a t h e p t i c a c t i v i t y o f f i s h m u s c l e . J . F o o d S c i . 3 7 : 6 4 3 .

S a l e m , H . , Y o u s s e f , A . , E l - N a k k a d i , A . a n d B e k h e i t , M . 1 9 7 0 . P r o t e o l y t i c d e c o m p o s i t i o n o f s h e l l f i s h m u s c l e p r o t e i n s u n d e r d i f f e r e n t c o n d i t i o n s . A l e x a n d r i a J . A g r i . R e s . 1 8 : 6 1 .

S h i b k o , S . , C a l d w e l l , K . A . , S a w a n t , P . I . a n d T a p p e l , A .L . 1 9 6 3 . D i s t r i b u t i o n o f l y s o s o m a l e n z y m e s i n a n i m a l t i s s u e s . J . C e l l . C o m p . P h y s i o l . 6 1 : 8 5 .

S i e b e r t , G . 1 9 5 8 . P r o t e i n - s p l i t t i n g e n z y m e a c t i v i t y o f f i s h f l e s h . E x p e r i e n t i a 1 4 : 6 5 .

S i e b e r t , G . a n d S c h m i t t . A . 1 9 6 5 . F i s h t i s s u e e n z y m e s a n d t h e i r r o l e i n t h e d e t e r i o r a t i v e c h a n g e s i n f i s h . I n “ T h e T e c h n o l o g y o f F i s h U t i l i z a t i o n , ” E d . K r u e g e r , R . , p . 4 7 . F i s h i n g N e w s ( B o o k s ) L t d . , L o n d o n .

S i e b e r t , G . , S c h m i t t , A . a n d T r a x l e r , G . 1 9 6 3 . R e i n i g u n g u n d p r o t e o l y t i s c h e s p e z i f i t a t e i n e s n e u e n k a t h e p s i n s a u s d o r s c h m i l z . Z . P h y s i o l . C h e m . 3 3 2 : 1 6 0 .

M s r e c e i v e d 6 / 2 8 / 7 3 ; r e v i s e d 8 / 1 5 / 7 3 ; a c c e p t e d8 / 2 0 / 7 3 .

BEVERLY A BAUER and R. R. EITENMILLER'Dept, o f Food Science, University o f Georgia, Athens, GA 30602

A STUDY OF SOME KINETIC PROPERTIES OF PARTIALLY PURIFIED Penaeus setiferus ARYLAMIDASE

INTRODUCTIONA R Y L A M I D A S E S a r e e n z y m e s t h a t c a t a l y z e t h e i n v i t r o h y d r o l y s i s o f a m i n o a c i d - ) 3 - n a p h t h y l a m i d e s . R e s e a r c h o n t h e s e e n z y m e s b e g a n a f t e r t h e d e v e l o p m e n t o f t h e c h r o m o g e n i c s u b s t r a t e , l e u c y l - ( 3 -

n a p h t h y l a m i d e ( G o m o r i , 1 9 5 4 ) , w h i c h

w a s o r i g i n a l l y d e v e l o p e d a s a s u b s t r a t e f o r l e u c i n e a m i n o p e p t i d a s e . A l t h o u g h t h e i n v i v o f u n c t i o n a n d s u b s t r a t e a r e n o t y e t k n o w n , s e v e r a l s t u d i e s s u g g e s t a r o l e i n

i n t r a c e l l u l a r c a t a b o l i s m o f p r o t e i n s ( E l l i s a n d N u e n k e , 1 9 6 7 ; S y l v é n a n d L i p p i , 1 9 6 5 ; R i l e y a n d B e h a i , 1 9 7 1 ; R i i s t o w e t

a l . , 1 9 7 1 a n d M á k i n e n a n d R a e k a l l i o ,

1 9 6 8 ) . T h e p o s s i b i l i t y o f a r o l e i n p r o t e

o l y s i s m a k e s a r y l a m i d a s e a p o t e n t i a l l y i n f l u e n t i a l f a c t o r i n t h e s p o i l a g e o f m u s c l e f o o d s . T a p p e l ( 1 9 6 8 ) h a s s u g g e s t e d t h a t t h e r e m a y b e a n o r d e r l y d e g r a d a t i o n o f

p r o t e i n s , p r o c e e d i n g i n a d e f i n e d s e q u e n c e . C o l l a g e n a s e a n d c a t h e p s i n s D a n d

E p a r t i a l l y h y d r o l y z e p r o t e i n s t o p e p t i d e s

a n d s m a l l e r p r o t e i n s . C a t h e p s i n s A a n d B t h e n r e d u c e t h e s e i n t e r m e d i a t e p r o d u c t s t o p e p t i d e s . C a t h e p s i n C , d i p e p t i d a s e , t r i

p e p t i d a s e a n d p r o b a b l y a r y l a m i d a s e f u n c t i o n a t t h e p e p t i d e s t a g e t o c o m p l e t e t h e b r e a k d o w n o f p r o t e i n s t o f r e e a m i n o

a c i d s .

S i n c e a n u n d e r s t a n d i n g o f t h e r e l a t i o n s h i p o f a r y l a m i d a s e t o p r o t e i n d e g r a d a t i o n m a y h e l p t o c l a r i f y r e a c t i o n s t a k i n g p l a c e d u r i n g a u t o l y t i c b r e a k d o w n o f m u s c l e f o o d s , t h i s p a p e r r e p o r t s a p r o c e d u r e f o r t h e p a r t i a l p u r i f i c a t i o n o f Penaeus setiferus ( w h i t e s h r i m p ) m u s c l e

a r y l a m i d a s e a n d d i s c u s s e s s o m e p r o p e r t i e s o f t h e p a r t i a l l y p u r i f i e d e n z y m e .

EXPERIMENTALP a r t i a l p u r i f i c a t i o n o f s h r i m p m u s c l e a r y l a m i d a s e

D e t e r m i n a t i o n o f o p t i m a l e x t r a c t i o n m e d i u m . T h e s h r i m p w e r e o b t a i n e d f r e s h f r o m d o c k s a t B r u n s w i c k , G a . , o r f r o m s e a f o o d w h o l e s a l e h o u s e s i n A t h e n s , G a , a n d k e p t f r o z e n a t - 3 0 ° C u n t i l u s e d . T h e h o m o g e n a t e s ( 1 : 4 w / v ) w e r e p r e p a r e d b y b l e n d i n g e x c i s e d m u s c l e i n a W a r i n g B l e n d o r f o r 3 0 s e c i n 0 .1 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , 2 .0 % K C 1 , d e i o n i z e d w a t e r , 0 .2 5 M s u c r o s e a n d 2 .0 % N a C l . T h e h o m o g e n a t e s w e r e c e n t r i f u g e d a t 1 7 , 5 0 0 X G

f o r 1 0 m i n a t 4 . 0 ° C a n d t h e r e s i d u e d i s c a r d e d .

A m m o n i u m s u l f a t e f r a c t i o n a t i o n . S o l i d ( N H 4 ) 2 S 0 4 w a s s l o w l y a d d e d w i t h s t i r r i n g to t h e c r u d e e x t r a c t p r e p a r e d in 0 .1 M s o d i u m

p h o s p h a t e b u f f e r , p H 7 . 0 , a t 4 . 0 ° C t o t h e 5 0 . 0 % s a t u r a t i o n l e v e l . A f t e r 3 0 m i n , t h e e x t r a c t w a s c e n t r i f u g e d a t 1 7 , 5 0 0 x G f o r 1 0 m i n

a n d t h e p r e c i p i t a t e d i s c a r d e d . T h e s u p e r n a t a n t

w a s t h e n b r o u g h t t o 7 0 .0 % s a t u r a t i o n a n d t h e

p r e c i p i t a t e w a s c o l l e c t e d b y c e n t r i f u g a t i o n .

D i e t h y l a m i n o e t h y l ( D E A E ) c e l l u l o s e c h r o

m a t o g r a p h y . D E A E c e l l u l o s e ( W h a t m a n ) c h r o m a t o g r a p h y w a s p e r f o r m e d a t b o t h p H 8 . 2 a n d

8 . 6 . I n e i t h e r c a s e , t h e p H w a s h e l d c o n s t a n t t h r o u g h o u t . T h e 5 0 . 0 - 7 0 . 0 % ( N H 4 ) 2 S 0 4 f r a c t i o n w a s d i s s o l v e d i n 0 . 0 0 5 M s o d i u m p h o s p h a t e b u f f e r , p H 8 .2 o r 8 .6 ( a c c o r d i n g t o t h e p H o f t h e c o l u m n ) , c o n t a i n i n g 4 . 0 m M m e r c a p t o e t h a - n o l w h i c h h a d b e e n a d d e d a f t e r p r e p a r a t i o n o f t h e b u f f e r , a n d d i a l y z e d f o r 6 h r a g a i n s t 0 .5 l i t e r o f t h e s a m e b u f f e r w i t h o n e c h a n g e o f t h e d i a l y s i s m e d i u m . M e r c a p t o e t h a n o l w a s a d d e d t o

a l l b u f f e r s a n d w a s f o u n d t o p a r t i a l l y p r o t e c t t h e e n z y m e f r o m i n a c t i v a t i o n d u r i n g p u r i f i c a t i o n . T h e d i a l y z e d s o l u t i o n , c o n t a i n i n g a p p r o x i m a t e l y 3 0 0 m g o f p r o t e i n , w a s a p p l i e d t o a 2 0 0 m m x 1 9 m m D E A E c e l l u l o s e c o l u m n e q u i l i b r a t e d w i t h 0 . 0 0 5 M s o d i u m p h o s p h a t e b u f f e r c o n t a i n i n g 4 . 0 m M m e r c a p t o e t h a n o l . A c o n v e x N a C l g r a d i e n t w a s s e t u p a c c o r d i n g t o t h e m e t h o d o f C h e r k i n e t a l . , ( 1 9 5 3 ) . I n i t i a l a n d l i m i t i n g N a C l c o n c e n t r a t i o n s w e r e 0 .0 M a n d 0 . 3 5 M , r e s p e c t i v e l y . A t a f l o w r a t e o f 0 .9

m l / m i n , 6 5 0 m l o f e l u a n t w a s p a s s e d t h r o u g h t h e c o l u m n , a n d f r a c t i o n s c o n t a i n i n g 9 . 0 m l w e r e c o l l e c t e d . T h e a c t i v e f r a c t i o n s w e r e p o o l e d a n d c o n c e n t r a t e d t o a v o l u m e o f 3 0 m l b y u s e o f a n A m i c o n U l t r a f i l t r a t i o n U n i t u s in g a D i a f l o P M - 3 0 f i l t e r in p r e p a r a t i o n f o r a d s o r p t i o n c h r o m a t o g r a p h y .

H y d r o x y l a p a t i t e c h r o m a t o g r a p h y . T h e c o n c e n t r a t e d e l u a n t f r o m t h e D E A E c e l l u l o s e c o l u m n w a s d i a l y z e d f o r 6 h r a g a i n s t 0 .5 l i t e r o f 0 .0 1 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , c o n t a i n i n g 4 . 0 m M m e r c a p t o e t h a n o l , w i t h o n e c h a n g e o f t h e d i a l y s i s s o l u t i o n a f t e r 3 h r . T h e e n z y m e s o l u t i o n , c o n t a i n i n g a p p r o x i m a t e l y 5 0 m g o : p r o t e i n , w a s a p p l i e d t o a 1 7 5 m m x 1 7 m m b e d o f h y d r o x y l a p a t i t e ( B i o - G e l H T , B io R a d L a b o r a t o r i e s ) , w h i c h h a d b e e n p r e v i o u s l y e q u i l i b e r a t e d w i t h 0 .0 1 s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , c o n t a i n i n g 4 . 0 m M m e r c a p o e t h - a n o l . A t a f l o w r a t e o f 0 . 3 5 m l p e r m i n , 1 5 0 m l o f 0 .0 5 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , w a s

p a s s e d t h r o u g h t h e c o l u m n . A c o n v e x g r a d i e n t w a s s e t u p u s i n g 0 .2 5 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , a s t h e l i m i t i n g c o n c e n t r a t i o n , 0 .1 M s o d i u m p h o s p h a t e a s t h e c o n c e n t r a t i o n o f t h e f i x e d v o l u m e c h a m b e r . T h e e n z y m e w a s

e l u t e d w i t h 3 .5 m l o f b u f f e r , a n d f r a c t i o n s c o n t a i n i n g 3 .5 m l w e r e c o l l e c t e d .

A c r y l a m i d e g e l e l e c t r o p h o r e s i s . E l e c t r o p h o r e s i s w a s r u n u s i n g 6 % a c r y l a m i d e g e l s ( 3 . 0 g a c r y l a m i d e a n d 0 . 0 8 5 g N , N ° - m e t h y l e n e b i s - a c r y l a m i d e m a d e u p t o 5 0 m l ) . A p p r o x i m a t e l y 0 .1 m g o f p u r i f i e d e n z y m e p r o t e i n i n 4 0 % s u c r o s e w a s a p p l i e d t o t h e g e l a n d o v e r l a y e r e d w i t h b u f f e r s o l u t i o n . F o r r u n s u s i n g 4 . 5 M u r e a g e l s , t h e e n z y m e w a s p r e i n c u b a t e d in 4 .5 M u r e a f o r a t l e a s t 2 h r . G e l s w e r e r u n a t 5 - 8 ° C a t 5 . 0 m a p e r t u b e f o r 2 .0 h r , u s i n g 0 . 0 5 M s o d i u m p h o s p h a t e , p H 7 . 0 , a s t h e c h a m b e r b u f f e r P r o

t e i n b a n d s w e r e s t a i n e d w i t h a m i d o b l a c k f o r 5 m i n , t h e n d e s t a i n e d w i t h a m e t h a n o l - g l a c i a l

a c e t i c a c i d - w a t e r s o l u t i o n ( 5 : 1 : 5 ) . H i s t o c h e m - i c a l s t a i n s o f a r y l a m i d a s e a c t i v e b a n d s w e r e

m a d e b y i n c u b a t i n g t h e g e l s in s u b s t r a t e ( 6 . 8 5

x 1 0 ‘4 M ) a t 3 7 ° C f o r 1 h r b e f o r e p l a c i n g t h e m in a C . 1 4 % F a s t G a r n e t ( S i g m a ) s o l u t i o n in 0 .2 M a c e t a t e b u f f e r , p H 4 . 5 , f o r 1 h r . A c t i v e

b a n d s a p p e a r e d a s b r i c k - r e d b a n d s a g a i n s t a c l e a r b a c k g r o u n d . G e l s w e r e b o t h w a s h e d w i t h a n d s t o r e d in 7 .0 % a c e t i c a c i d .

T h e b i u r e t r e a c t i o n ( G o r n a l l e t a l . , 1 9 4 9 ) w a s u s e d f o r s a m p l e s c o n t a i n i n g m o r e t h a n 1 .0 m g o f p r o t e i n p e r m l , a n d t h e m e t h o d o f L o w r y

e t a l . ( 1 9 5 1 ) w a s u s e d f o r s a m p l e s c o n t a i n i n g le s s t h a n 1 .0 m g / m l o f p r o t e i n . S t a n d a r d c u r v e s w e r e p r e p a r e d u s in g b o v i n e s e r u m a l b u m e n

( N u t r i t i o n a l B i o c h e m i c a l s C o r p . ) P r o t e i n e l u

t i o n p r o f i l e s f r o m c o l u m n c h r o m a t o g r a p h y w e r e m o n i t o r e d b y t h e a b s o r b a n c e o f t h e f r a c t i o n s a t 2 8 0 n m .

E n z y m e a s s a y

A c t i v i t y o f a r y l a m i d a s e w a s d e t e r m i n e d b y t h e m e t h o d o f G o l d b a r g a n d R u t e n b u r g ( 1 9 5 8 ) . U n l e s s o t h e r w i s e s t a t e d , 0 .1 m l o f e n z y m e s o l u t i o n a n d 0 . 9 m l o f 0 .1 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , w e r e p r e i n c u b a t e d f o r 1 0 m i n a t 3 7 ° C , a f t e r w h i c h , 1 .0 m l o f p r e w a r m e d 6 . 8 5 x 1 0 " 4 M a m i n o a c i d - j 3 - n a p h t h y l a m i d e in 0 .1 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , w a s a d d e d .

Table 1—Comparison of activity of the enzyme in various extraction mediaExtraction medium Relative activity (%)0.1 M sodium phosphate, pH 7.0 100 .02.0% KCI (0.27M) 75.0Deionized water 66.70.25M sucrose 61.52.0% NaCl (0.34M) 57.2

10-JO URNAL OF FOOD SCIENCE-Volume 39 (1974)

P A R T I A L L Y PURIFIED SHRIMP A R Y L A M ID A S E - 11

Fig. 1—E l u t io n o f t h e 5 0 —7 0 % a m m o n i u m s u l f a t e f r a c t io n o n a D E A E c e l lu lo s e c o lu m n , p H 8 .2 . T h e a b s o r b a n c e a t 5 6 0 n m w a s o b t a i n e d a f t e r t h e r e a c t io n o f 1 .0 m l o f e a c h f r a c t io n w i th 1 .0 m l o f a m in o a c id -ß -

n a p h t h y l a m i d e fo r i h r a t 3 7 ° C.

Fig. 2 —E l u t io n o f t h e 5 0 — 7 0 % a m m o n i u m s u l f a t e f r a c t io n o n a D E A E

c e l lu lo s e c o lu m n , o H 8 .6 . T h e a b s o r b a n c e a t 5 6 0 n m w a s o b t a i n e d a f t e r t h e r e a c t io n o f 1 .0 m l o f e a c h f r a c t io n w i th 1 .0 m l o f a m in o a c id -fi- n a p h t h y l a m i d e fo r 1 h r a t 3 7 ° C.

R e a c t i o n t i m e v a r i e d f r o m 1 5 - 2 0 m i n a n d a l l r e a c t i o n s w e r e t e r m i n a t e d b e f o r e 5 0 % s u b s t r a t e h y d r o l y s i s w a s r e a c h e d . T h e v o l u m e o f e n z y m e

s o l u t i o n u s e d w a s c o n s t a n t w h e n e v e r p o s s i b l e s i n c e a d i l u t i o n o f t h e e n z y m e a t a n y s t a g e o f t h e p u r i f i c a t i o n w a s f o u n d t o s l i g h t l y i n c r e a s e a c t i v i t y . S t a n d a r d c u r v e s w e r e p r e p a r e d u s i n g ( ¡ - n a p h t h y l a m i n e ( S i g m a ) a t c o n c e n t r a t i o n s f r o m 1 0 t o 1 0 0 ju g /m l ( 0 . 6 8 5 x 1 0 '4 t o 6 . 8 5 x

1 0 '4 M ) . A u n i t o f a c t i v i t y w a s d e f i n e d a s 1 .0 jug o f |3 - n a p h t h y l a m i n e r e l e a s e d p e r h r .

pH OptimaO p t i m a l p H s t u d i e s w e r e c o m p l e t e d u s i n g

0 .1 M s o d i u m p h o s p h a t e b u f f e r f o r p H v a l u e s f r o m 5 . 5 - 8 . 5 a n d 0 .1 M T R I S b u f f e r f r o m8 . 5 - 9 . 5 . A l a n y l - a n d l y s y l - 0 - n a p h t h y l a m i d e s w e r e u s e d a s s u b s t r a t e s .

Substrate SpecificityT h e e n z y m e ’ s s p e c i f i c i t y f o r d i f f e r e n t s u b

s t r a t e s w a s t e s t e d u s i n g t h e / 3 - n a p h t h y l a m i d e s o f c y s t i n e d i h y d r o c h l o r i d e , h i s t i d i n e , i s o l e u c i n e h y d r o b r o m i d e , m e t h i o n i n e , a s p a r t a t e , g l y c i n e h y d r o c h l o r i d e , l y s i n e c a r b o n a t e , t r y p t o p h a n ( S c h w a r z - M a n n ) a n d a l a n i n e a n d l e u c i n e h y d r o c h l o r i d e ( S i g m a ) . S u b s t r a t e s o l u t i o n s w e r e p r e p a r e d b y m i x i n g e q u a l a m o u n t s o f 0 . 0 0 1 3 7 M s u b s t r a t e a n d 0 .2 M s o d i u m p h o s p h a t e b u f f e r , p H 7 .0 . A s s a y s w e r e d e t e r m i n e d a s d e s c r i b e d

p r e v i o u s l y .

Puromycin inhibitionI n h i b i t i o n b y p u r o m y c i n w a s s t u d i e d b y

r e a c t i n g t h e p a r t i a l l y p u r i f i e d e n z y m e w i t h a l a n y l - o r l y s y l - f i - n a p h t h y l a m i d e a t c o n c e n t r a t i o n s v a r y i n g f r o m 0 . 6 8 5 x 1 0 ‘4 M t o 6 . 8 5 X 1 0 '4 M in t h e p r e s e n c e o f t h e i n h i b i t o r . T h e e n z y m e w a s p r e i n c u b a t e d w i t h 0 . 0 0 1 - 0 . 0 1 p M p u r o m y c i n ( C a l b i o c h e m ) f o r 1 5 m i n a t 3 7 C b e f o r e t h e a d d i t i o n o f s u b s t r a t e .

Effect of metalsD u p l i c a t e s a m p l e s o f p u r i f i e d s h r i m p e n

z y m e w e r e u s e d . O n e w a s d i a l y z e d a g a i n s t 1 .0 l i t e r o f 0 .1 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , c o n t a i n i n g 4 . 0 m M m e r c a p t o e t h a n o l f o r 3 0 h r . T h e s e c o n d w a s d i a l y z e d a g a i n s t 0 .5 l i t e r o f 0 .1 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 0 , c o n t a i n i n g 4 . 0 m M m e r c a p t o e t h a n o l f o r 6 h r f o l

l o w e d b y d i a l y s i s a g a i n s t 1 .0 l i t e r o f t h e p h o s p h a t e b u f f e r , p H 7 . 0 , f o r 2 4 h r w i t h a t l e a s t t h r e e c h a n g e s o f t h e d i a l y s i s m e d i u m . 0 .1 m i o f e a c h e n z y m e s o l u t i o n w a s p r e i n c u b a t e d f o r

1 0 .0 m i n o r 1 .0 h r a t 3 7 ° C w i t h 0 .9 m l o f 0 .1 M p h o s p h a t e b u f f e r , p H 7 . 0 , c o n t a i n i n g 0 . 2 m M C o C l 2 , M g C h o r M n C l 2 . A f o u r t h s e t c o n t a i n e d n o m e t a l i o n , a n d a n u n d i a l y z e d s a m p l e s e r v e d

a s a c o n t r o l . A l a n y l - a n d l y s y l - / 3 - n a p h t h y l a m i d e s w e r e u s e d a s s u b s t r a t e s .

RESULTS & DISCUSSIONP a r t i a l p u r i f i c a t i o n o f s h r i m p m u s c l e a r y l a m i d a s e

D e t e r m i n a t i o n o f o p t i m a l e x t r a c t i o n m e d i u m . T h e a c t i v i t y o f t h e p r e p a r e d e x

t r a c t s a r e l i s t e d i n T a b l e 1 . A c t i v i t y w a s h i g h e s t u s i n g 0 . 1 M s o d i u m p h o s p h a t e b u f f e r a s t h e e x t r a c t i o n m e d i u m , t h e m e d i u m o f t h e h i g h e s t i o n i c s t r e n g t h . T h e

r e l a t i v e a c t i v i t y d i d n o t d e p e n d s o l e l y o n i o n i c s t r e n g t h , s i n c e t h e e x t r a c t p r e p a r e d i n d e i o n i z e d w a t e r h a d a h i g h e r a c t i v i t y t h a n w h e n p r e p a r e d i n 2 . 0 % ( 0 . 3 4 M ) N a C l .

A m m o n i u m s u l f a t e f r a c t i o n a t i o n . T h e

f r a c t i o n c o n t a i n i n g t h e h i g h e s t s p e c i f i c a c t i v i t y w a s t h e 5 0 —7 0 % f r a c t i o n . S p e c i f

i c a c t i v i t y v a l u e s o f 1 1 6 . 0 , 3 7 . 7 a n d 1 8 . 8 w e r e f o u n d u s i n g l y s y l - , a l a n y l - a n d l e u c y l - ( 3 - n a p h t h y l a m i d e s a s s u b s t r a t e s , r e s p e c t i v e l y .

Table 2—Purification of shrimp muscle arylamidase

FractionTotal activity

(units)3Recovery

(%)Specific activity

(units/mg protein)Purification

Crude extractlys-ß NA 83,430 100 .0 22.8 1 .0ala-ß NA 32,250 100 .0 8.8 1 .0leu-ß NA 11,800 100 .0 3.9 1 .0

50—70% Ammonium sulfate fractionlys-ß NA 42,600 51.2 116.0 5.1ala-ß NA 13,580 42.2 37.7 4.3leu-ß NA 7,075 60.0 18.8 4.8DEAE Cellulose chromatographylys-ß NA 17,765 21.3 312.0 13.7ala-ß NA 2,980 9.2 59.5 6.8leu-ß NA 880 7.5 19.1 4.9Hydroxylapatite chromatographylys-ß NA 20,280 24.4 5,860.0 257.0ala-ß NA 3,890 12 .0 1,113.0 126.8leu-ß NA 551 4.7 154.0 39.5a 1 u n it = 1 jug ß -na p hthy lam id e released''hr a t 37 °C

1 2 - J O U R N A L OF FOOD S CI EN C E-V o lu me 3 9 (1974)

F ig. 3 —E l u t io n o f t h e a c t i v e e n z y m e f r a c t io n o f t h e p H 8 . 6 D E A E c e l lu lo s e c o l u m n o n a h y d r o x y l a p a t i t e c o lu m n , p H 7 .0 . A v o lu m e o f

1 5 0 m l o f 0 . 0 5 M p h o s p h a t e b u f f e r , p H 7 .0 , w a s p a s s e d th r o u g h th e c o l u m n , a f t e r w h ic h a g r a d ie n t b e t w e e n 0 .1 a n d 0 .2 5 M p h o s p h a t e b u f f

er, p H 7 .0 w a s u s e d . T h e a b s o r b a n c e a t 5 6 0 n m w a s o b t a i n e d f r o m th e

r e a c t io n o f 0 .1 m l o f e a c h fr a c t io n a n d 0 . 9 m l p h o s p h a t e b u f f e r , p H 7 .0 , w i th 1 .0 m l o f a m in o a c id -p -n a p h th y ¡ a m id e fo r 1 h r a t 3 7 ° C.

D i e t h y l a m i n o e t h y l ( D E A E ) c e l l u l o s e c h r o m a t o g r a p h y . T h e e l u t i o n p a t t e r n s

f r o m D E A E c e l l u l o s e c h r o m a t o g r a p h y o f t h e 5 0 - 7 0 % a m m o n i u m s u l f a t e f r a c t i o n a t p H 8 . 2 a n d 8 . 6 a r e g i v e n i n F i g u r e s 1

a n d 2 , r e s p e c t i v e l y . W h e n t h e p H o f t h e c o l u m n a n d e l u t i o n b u f f e r w a s r a i s e d

f r o m 8 . 2 t o 8 . 6 , t w o p e a k s o f a c t i v i t y s h i f t e d i n t o o n e l a r g e p e a k , a n d a t e l u t i o n p H o f 8 . 2 , t h e r e l a t i v e a m o u n t o f a c t i v i t y i n e a c h a c t i v e p e a k v a r i e d f r o m c o l u m n t o c o l u m n . T h e e x p l a n a t i o n f o r t h i s p e a k b e h a v i o r i s n o t k n o w n , b u t w o u l d s u g g e s t s u b u n i t i n t e r a c t i o n a s B e h a i a n d C a r t e r ( 1 9 7 1 ) n o t e d a v a r i a n c e f r o m c o l u m n t o c o l u m n i n t h e r e l a t i v e a m o u n t s o f a c t i v i t y i n a r y l a m i d a s e p e a k s o f Sarcina lutea. T h e l a r g e a c t i v e p e a k f r o m t h e p H 8 . 6 c o l u m n w a s p o o l e d f o r f u r t h e r p u r i f i c a t i o n o f t h e e n z y m e .

H y d r o x y l a p a t i t e c h r o m a t o g r a p h y . T h ec h r o m a t o g r a p h i c s e p a r a t i o n o f t h e a c t i v e f r a c t i o n s f r o m D E A E c e l l u l o s e c h r o m a t o g r a p h y y i e l d e d t h e r e p r e s e n t a t i v e e l u t i o n p a t t e r n s h o w n i n F i g u r e 3 . A c t i v e f r a c t i o n s o f t h e l a r g e r p e a k w e r e p o o l e d a n d t e s t e d f o r h o m o g e n e i t y b y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . A s s h o w n i n F i g u r e 4 , f i v e p r o t e i n b a n d s a n d o n e a c t i v e b a n d w e r e e v i d e n t . W h e n t h e s a m p l e s w e r e p r e i n c u b a t e d i n a 4 . 5 M u r e a s o l u t i o n a n d r u n o n g e l s c o n t a i n i n g 4 . 5 M u r e a , t h e n u m b e r o f p r o t e i n b a n d s w a s r e d u c e d t o t h r e e a n d a r y l a m i d a s e a c t i v i t y w a s l o s t . T h i s s u g g e s t s t h a t t w o o f t h e f i v e p r o t e i n b a n d s i n t h e g e l m a y h a v e b e e n a g g r e g a t e s o f p a r t o r a l l o f t h e o t h e r t h r e e b a n d s , a n d t h a t t h e a c t i v e s p e c i e s r e q u i r e s t w o o r m o r e s u b u n i t s . L i t t l e a n d B e h a i ( 1 9 7 1 ) f o u n d a m o l e c u l a r w e i g h t o f

a p p r o x i m a t e l y 2 3 4 , 0 0 0 f o r l i v e r e n z y m e .

U s i n g 8 . 0 M u r e a , a v a l u e o f 1 6 7 , 0 0 0 w a s o b t a i n e d ; a n d t w o u n i t s o f 3 8 , 0 0 0 a n d 7 2 , 0 0 0 w e r e f o u n d w h e n 6 . 0 M g u a n i d i n e

w a s u s e d a n d p r o b a b l y a r e t h e s u b u n i t a n d i t s d i m e r , r e s p e c t i v e l y . T h e m o l e c u l a r w e i g h t f o u n d w i t h 8 . 0 M u r e a w a s a s -

I

s u m e d t o b e a p a r t i a l l y d i s s o c i a t e d e n z y m e . I n t h i s s t u d y t h e s h r i m p m u s c l e a r y l a m i d a s e w a s p o s s i b l y o n l y p a r t i a l l y d i s s o c i a t e d i n 4 . 5 M u r e a , a n d t h e t h r e e p r o t e i n b a n d s i n t h e u r e a g e l s m a y r e p r e s e n t a n o t y e t p u r e e n z y m e p r e p a r a t i o n o r i t m a y r e p r e s e n t a g g r e g a t e f o r m s o f a

p u r e e n z y m e .T h e s u m m a r y o f t h e r e s u l t s f o r e a c h

p u r i f i c a t i o n s t e p o f s h r i m p m u s c l e a r y l

a m i d a s e i s g i v e n i n T a b l e 2 . F r e e z i n g t h e e n z y m e a t a n y s t a g e o f p u r i f i c a t i o n r e s u l t e d i n a l o s s o f a c t i v i t y . W h e n t h e p a r

t i a l l y p u r i f i e d e n z y m e w a s s t o r e d a t 0 — 5 ° C , t h e e n z y m e l o s t a c t i v i t y s l o w l y , r e t a i n i n g 7 0 % o f i t s a c t i v i t y a f t e r 3 0 d a y s .

O p t i m a l p H o f t h e p a r t i a l l y p u r i f i e d e n z y m e

Using alanyl- and lysyl-|3-naphthylamides as substrates, the pH curve obtained for the purified enzyme is shown in Figure 5 . Activity dropped off sharply at pH 8 . 5 , the optimal pH for leucine aminopeptidase. High activity near neutrality indicates that the enzyme would probably be found in the muscle at or near its optimum pH value.S u b s t r a t e s p e c i f i c i t y

The natural substrate for arylamidase is not known, but a determination of the preferred amino acid-)3-naphthylamide substrate gives an indication of the nature of the in situ site of attack. The substrate specificity profile was obtained with ten amino acid-|3-naphthylamides (Fig. 6).

1 Î !O

P R O T E IN E N Z Y M E P R O TE INNO UREA A C T I V I T Y 4w5 M UREA

Fig. 4 —A c r y l a m i d e g e l e le c t r o p h o r e s i s o f t h e p a r t ia l l y p u r i f i e d e n z y m e .

PARTIALLY PURIFIED SHRIMP ARYLAMIDASE- 1 3

>

#>►-

o<III>H<IIIee

pH

F i g . 5 — E f f e c t o f p H o n t h e a c t i v i t y o f t h e e n z y m e . F i g . 6 — S u b s t r a t e s p e c i f i c i t y p r o f i l e o f t h e p a r t i a l l y p u r i f i e d e n z y m e .

F i g . 7 — E f f e c t o f p u r o m y c i n o n t h e a c t i v i t y o f t h e e n z y m e u s i n g a l a n y l -

¡ ¡ - n a p h t h y l a m i d e a s t h e s u b s t r a t e . C o n c e n t r a t i o n s o f p u r o m y c i n a r e

o---- o 0 . 0 pM; a ---- a 0 . 0 0 1 pM; o---- □ 0 . 0 0 5 pM; a n d □---- o 0 . 0 1 pM.

F i g . 8 — E f f e c t o f p u r o m y c i n o n t h e a c t i v i t y o f p a r t i a l l y p u r i f i e d s h r i m p

e n z y m e u s i n g l y s y l - f i - n a p h t h y l a m i d e a s s u b s t r a t e . C o n c e n t r a t i o n s o f

p u r o m y c i n a r e o------o 0 . 0 p M ; a ---- a 0 . 0 0 1 p M ; o— o 0 . 0 0 5 p M ; a n d

d ----o 0 . 0 1 p M .

T he rem o v a l o f th e m e th y l g ro u p fro m a lan y l-j3 -n ap h th y lam id es re su lte d in n e g ligible a c tiv ity w ith g ly cy l-j3 -n ap h th y lam id e as a su b s tra te . T h e a d d it io n to th e a lan y l- |3 -n ap h th y lam id e o f -C H 2 -C H 2 -N H 2 g ro u - p to m ak e ly sy l- |3 -n ap h th y lam id e re su lte d in an in c rease o f a c tiv ity . A c tiv ity was low w ith th e a -b ra n c h e d iso leucy l-|3 -naph- th y la m id e a n d th e n eg a tiv e ly ch arg ed asp arty l- |3 -n ap h th y lam id e .In h ib itio n by p u ro m y c in

A ry lam id ase , in c o n tra s t to leu c in e a m in o p e p tid a se , is in h ib i te d b y p u ro m y c in , an a n tib io tic w h ic h in h ib its p ro te in sy n th e s is a t th e r ib o so m a l stage

b y c o m p e tin g w ith a m in o acid tra n s fe r R N A (M ah ler a n d C o rd es , 1 9 6 6 ). P re v io u s r e p o r ts have sh o w n th e in h ib i t io n to be o f a c o m p e tit iv e n a tu re (S u szk iw a n d B rech e r, 1 9 7 0 ; M arks e t a l., 1 9 6 8 ; E llis a n d P e rry , 1 9 6 4 ; a n d M cD o n a ld e t a l., 1 9 6 8 ). T h e in h ib it io n cu rv es o f th e p a r tia l ly p u rif ie d e n z y m e u sin g a la n y l an d ly sy l-j3 -n ap h th y lam id es as su b s tra te s are g iven in F ig u re s 7 an d 8 , re sp ec tiv e ly . U sing a lan y l-/3 -n ap h th y lam id e as su b s tra te , p u ro m y c in c o m p e tit iv e ly in h ib i te d th e e n z y m e a n d w h e n lysy l-j3 -naphthy l- a m id e w as th e su b s tra te , in h ib i t io n te n d ed to w a rd c o m p e tit iv e b e h a v io r a lth o u g h

th e p a tte rn w as n o t w ell d e f in e d . In h ib it io n c o n s ta n ts c o u ld n o t b e assigned b e cau se th e c a lc u la te d K i v a lu es d ec reased w ith in c reas in g c o n c e n tra t io n s o f in h ib i to r . I t is p o ss ib le th a t o n e m o le c u le o f p u ro m y c in m ay a f fe c t m o re th a n o n e e n z y m e m o le c u le , c au sin g a ra p id d ecrease in a c tiv ity w ith in c reas in g in h ib i to r c o n c e n t r a t io n . P u ro m y c in c o n c e n tra t io n s cau sin g a 50% d e c re ase in a c t iv ity an d K m v a lu es o f th e e n z y m e w e re 2 .0 x 1 0 '9 M a n d 1.3 x lO ^ M , re sp ec tiv e ly , fo r th e a lan y l-)3 -n ap h th y lam id e a n d 4 .0 x 10~9 M a n d 1.5 x lO ^ M , re sp ec tiv e ly , for th e ly sy l-ß -n ap h th y lam id e .

V\-JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

Ï 0 0 300RELATIVE ACTIVITY (°/0)

F i g . 9 — R e l a t i v e a c t i v i t y o f t h e e n z y m e u p o n

t h e a d d i t i o n o f m e t a l s t o t h e a s s a y m e d i a w i t h

a n d w i t h o u t p r e v i o u s t r e a t m e n t w i t h E D T A .

A n u n d i a l y z e d s a m p l e o f e n z y m e s e r v e d a s t h e

c o n t r o l . A l a n y l - p - n a p h t h y l a m i d e w a s t h e s u b

s t r a t e .

E ffe c t o f m e ta ls

T h e re su lts o f th e a d d it io n o f d iv a len t m ag n e siu m , m an g an ese a n d c o b a lt to th e e n z y m e so lu t io n w ith a n d w ith o u t p rev io u s t r e a tm e n t w ith E D T A is g iven in F ig u re s 9 a n d 10 . S im ila r e f fe c ts w e re seen w h e th e r a la n y l- o r ly sy l- |3 -n ap h th y lam id es w e re u sed as su b s tra te s a f te r 10- an d 6 0 -m in p re in c u b a tio n p e rio d s . D ia lysis ag a in st so d iu m p h o s p h a te b u f fe r y ie ld e d a n in c re a se in a c t iv ity over th e u n d ia ly z e d c o n tro l w h e th e r th e 10 o r 6 0 m in p re in c u b a t io n tim e w as u se d . T h is c o u ld be d u e to th e re m o v a l o f a n in h ib i to ry ag en t o r t o a d ec rease in th e p h o sp h a te c o n c e n t r a t io n fro m th a t o f th e h y d ro x y la p a ti te a c tiv e f ra c t io n s ( 0 .1 5 M) to th a t o f th e d ia lysis m e d iu m (0 .1 M ). T h e e n z y m e seem ed to b e m o re liab le to lo ss o f ac tiv i ty in th e 6 0 -m in p re in c u b a tio n p e rio d a f te r t r e a tm e n t w ith th e c h e la to r . It a p p e a rs th a t th e s h o r te r p re in c u b a tio n tim e w as su ff ic ie n t fo r th e b in d in g o f th e m e ta l a n d e n z y m e .

T re a tm e n t w ith 0 .1 M E D T A d id n o t c o m p le te ly in a c tiv a te th e e n z y m e , in d ic a tin g th a t th e m e ta l m ay b e v e ry t ig h tly b o u n d , o r th a t it m ay n o t b e re q u ire d fo r

RELATIVE ACTIVITY (°/0 )

F i g . 1 0 — R e l a t i v e a c t i v i t y o f t h e e n z y m e u p o n

t h e a d d i t i o n o f m e t a l s t o t h e a s s a y m e d i a w i t h

a n d w i t h o u t p r e v i o u s t r e a t m e n t w i t h E D T A .

A n u n d i a l y z e d s a m p l e o f e n z y m e s e r v e d a s t h e

c o n t r o l . L y s y l - p - n a p h t h y l a m i d e w a s t h e s u b

s t r a t e .

a c tiv ity . F a ilu re o f th e a d d it io n o f m e ta ls a f te r E D T A t r e a tm e n t to a c tiv a te th e e n z y m e to levels n o te d fo r th e n o n -E D T A t r e a te d p re p a ra tio n s in d ic a te s th a t th e m e ta l m ay n o t b e ad d ed b a c k in su ff ic ie n t c o n c e n tr a t io n s fo r o p tim a l a c tiv a t io n , o r th e m o s t p re fe r re d m e ta l m ay n o t have b e e n te s te d . In th e cell th e m e ta l io n m ay ac t as a c o n tro l m ec h an ism o f th e re a c tio n o f sh rim p a ry la m id a se . W ith a h ig h c o n c e n tr a t io n o f a c tiv a tin g m e ta l, su c h as c o b a lt o r m an g a n ese , th e re a c tio n w o u ld p ro c e e d fa s te r th a n w h e n a m e ta l sh o w in g l i t t le e ffe c t su ch as m ag n esiu m w as p re se n t o r w h e n m e ta l c o n c e n tr a t io n w as n eg lig ib le . I t a p p e a rs th a t a m e ta l is n o t n ece ssa ry fo r a c tiv ity b u t e n h a n c e s o r in h ib i ts th e re a c tio n a c c o rd in g to th e in d iv id u a l m e ta l p re se n t.

CONCLUSIONP R O T E IN S p a r tia lly h y d ro ly z e d b y ca- th e p s in D a n d co llag en ase a re su sce p tib le to f u r th e r h y d ro ly s is b y c a th e p s in s a n d o th e r p e p tid a se s . Several s tu d ie s have su g g ested a p r o te o ly t ic ro le fo r a ry la m idase a t th e o lig o p e p tid e s tage o f h y d ro ly sis. T h e p re se n c e o f a ry la m id a se in sh r im p

m u sc le m ig h t b e a lin k in th e d e g ra d a tio n o f sh rim p m u sc le p ro te in . F u r th e r w o rk is n ece ssa ry to d e te rm in e th e e n z y m e ’s a b ili ty to d e g rad e n a tiv e s u b s tra te s ; h o w ever, th e p o te n t ia l o f a ry la m id a se as a n in f lu e n tia l f a c to r in th e s ta b il i ty o f u n c o o k e d m u sc le fo o d s d u r in g p ro c ess in g a n d s to r age is a p p a re n t.

REFERENCESBehai. F.J. and Carter. R.T. 1971. Naphthyl-

amidases of Sarcina lutea. Can J. Microbiol. 17: 39.

Cherkin, A., Martinez, F.E. and Dunn, M.S. 1953. Ar. expression for gradient elution. J. Am. Chem. Soc. 75: 1244.

Ellis, S. and Nuenke, J.M. 1967. Dipeptidyl arylamidase III of the pituitary. J. Biol. Chem. 242: 4623.

Ellis, S. and Perry, M. 1964. Inhibition of thiol-activated amino-peptidase by puromycin. Biochem. Biophys. Res. Comm. 15: 502.

Goldbarg, J.A. and Rutenburg, A.M. 1958. The colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects with cancer and other diseases. Cancer 11: 283.

Gomori, G. 1954. Enzymatic hydrolysis of acyl naphthylamines. Proc. Soc. Exptl. Biol. Med. 85: 570.

Gornall, A.G., Bardawill, C.J. and David, M.M. 1949. Determination of serum proteins by means of biuret reaction. J. Biol. Chem. 177: 751.

Little, G.H. and Behai, F.J. 1971. Human liver arylamidase: Molecular weight and subunit structure. Biochim. Biophys. Acta 243: 312.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 19 51. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265.

Mahler, H.R. and Cordes, E.H. 1966. “Biological Chemistry.” Harper and Row, Publishers, Inc., New York, N.Y.

Mäkinen, P. and Raekallio, J. 1968. Fractionation and characterization of arylamidases in regenerating wound tissue. Acta Chem. Scanc. 22: 597.

Marks, N., Datta, R.K. and Lajtha, A. 1968. Partial resolution of brain arylamidases and aminopeptidases. J. Biol. Chem. 243: 2882.

McDonald, J.K., Reilly, T.J., Zeitman, B.B. and Ellis, S. 1968. Dipeptidyl arylamidase II of the pituitary. J. Biol. Chem. 243: 2028.

Riley, P.S. and Behai, F.J. 1971. Amino acid-/3- naphthylamide hydrolysis by Pseudomonas aeruginosa arylamidase. J. Bacteriol. 108: 809.

Rüstow, B., Risse, S. and Hock, A. 1971. Einfluss der Qualität des Nahrungseiweises auf Hydrolasen des Verdauungstraktes. 5. Mitt. Einfluss des Aminosäuremusters auf ausgewählte Peptidasen und auf die Sauerstoffaufnahme (Q02) der Rattendunndarm- mucosa. Die Nahrung 15: 643.

Suszkiw, J.B. and Brecher, A.S. 1970. Brain aminoacyl arylamidase. Further purification of the soluble bovine enzyme and studies on substrate specificity and possible active site residues. Biochem. 9: 40008.

Sylvdn, B. and Lippi, U. 1965. The suggested lysomal localization of aminoacylnaphthyl- amide-splitting enzymes. Expt. Call, Res. 40: 145.

Tappel, A.L. 1968. In “Comprehensive Biochemistry.” Vol 23, Ed. Florkin, M. and Stotz, E.H. Elsevier Publishing Company, Amsterdam.

Ms received 7/18/73; revised 9/10/73; accepted 9/11/73.

R . L . H E i J R I C K S O N , J . L . M A R S D E N a n d R . D . M O R R I S O N

D e p a r t m e n t s o f A n i m a l S c i e n c e & I n d u s t r y a n d M a t h e m a t i c s & S t a t i s t i c s ,

O k l a h o m a A g r i c u l t u r a l E x p e r i m e n t S t a t i o n , O k l a h o m a S t a t e U n i v e r s i t y , S t i l l w a t e r , O K 7 4 0 7 4

AN EVALUATION OF A METHOD FOR MEASURING SHEAR FORCE FOR AN INDIVIDUAL MUSCLE FIBER

INTRODUCTIONT H E S H E A R F O R C E o f b o v in e m uscle tissu e has b e e n e x te n s iv e ly in v es tig a te d , b u t ra re ly a t th e f ib e r level. P re se n tly th e W arner-B ra tz le r in s tru m e n t is th e m o st w id e ly u se d p h y sica l m e th o d o f m e a su ring th e sh ea r fo rce o f m u sc le . H ow ever, th is m e th o d is c o n c e rn e d w ith c o o k ed p ieces o f m e a t , 1 in . in d ia m e te r , a n d its re la tio n sh ip to p h y s ic a l p ro p e r t ie s o f raw m u sc le is l im ite d (S h a rra h e t a l., 1 9 6 5 ; P oo l a n d K lose , 1 9 6 9 ). In a d d it io n , th e W arn er-B ra tz le r in s tru m e n t p ro v id es l it t le k n o w led g e a b o u t th e in f lu e n c e o f th e in d iv id u a l m u sc le tis su e c o m p o n e n t (e n d o - m y s iu m , sa rc o le m m a , f ib r il) o n te n d e r ness o r te x tu re . T h e re fo re , a n eed c learly ex is ts fo r a m e th o d th a t w o u ld re c o rd th e sh ear fo rc e o f a n in d iv id u a l m u sc le f ib e r.

S o m e w o rk o n th e p h y sica l p ro p e rt ie s o f th e m u sc le fa sc icu li a n d f ib e r has b e en re p o rte d (S ta n le y e t a l., 1 9 7 1 ; E in o an d S ta n le y , 1 9 7 3 a , b ) ; h o w e v e r , m o s t s tu d ie s have b e e n c o n c e rn e d w ith ten s ile s tre n g th . T h ere h a s b e e n n o o th e r investig a tio n c o n c e rn e d w ith th e sh ea r fo rce o f th e fascic le o r th e in d iv id u a l m u sc le f ib e r fo r th e d e te rm in a tio n o f m e a t te n d ern ess . T h e p u rp o se o f th is s tu d y w as to ev a lu a te a m e th o d w h e re b y th e sh ear fo rce o f th e in d iv id u a l m u sc le f ib e r is m easu red .

In o rd e r to d e te rm in e th e re p e a tib i l i ty an d p re c is io n o f th e m ic ro -se n sitiv e sh ear in s tru m e n t, a u n ifo rm ity s tu d y w as c o n d u c te d . U p o n c o m p le tio n o f th is w o rk , re sea rc h w as in it ia te d to d e te rm in e th e a b ili ty o f th e m ic ro se n sitiv e sh e a r in s t ru m e n t to m easu re a d iffe re n c e a m o n g th e sa r to r iu s m uscles ex c ised a f te r th re e d if fe re n t c o n d itio n in g p e rio d s .

MATERIALS & METHODSA M I C R O S E N S I T I V E I N S T R U M E N T w a s u t i l i z e d t o d e t e r m i n e t h e s h e a r f o r c e o f t h e i n d i v i d u a l m u s c l e f i b e r ( F i g . 1 ) . T h i s s h e a r i n s t r u m e n t w a s d e v e l o p e d a n d d e s c r i b e d b y H e n r i c k s o n e t a l . ( 1 9 6 7 ) a s a r e s e a r c h t o o l f o r m e a s u r i n g s h e a r f o r c e , a p h y s i c a l p r o p e r t y o f t h e m u s c l e f i b e r . T h e i n s t r u m e n t c o n s i s t e d o f a s h e a r g a u g e e q u i p p e d w i t h a t o r q u e d i a l w h i c h w a s e a s i l y r e a d f r o m t h e t o p o f t h e i n s t r u m e n t . T h e s h e a r g a u g e w a s s t r u n g w i t h a 1 / 1 0 0 in . d i a m w i r e w h i c h s u p p o r t e d a b l u n t e d g e d b l a d e f o r p a s s a g e t h r o u g h t h e f i b e r t o m a k e t h e s h e a r . T h e t o p e n d o f t h e w i r e w a s c o n n e c t e d t o t h e t o r q u e d i a l a n d t h e b o t t o m t o a t e n s i o n a r m .

T h e f i b e r w a s h e l d , b u t n o t t i g h t l y c l a m p e d , b e t w e e n a p l e x i g l a s s a n d a n a l u m i n u m p l a t e . A s h a l l o w V - c u t i n t h e a l u m i n u m p l a t e p r o v i d e d a g r o o v e t o p o s i t i o n t h e f i b e r . T h e t w o p l a t e s a r e t h e n a t t a c h e d b y a c l a m p t o a n a d j u s t a b l e s p e c i

m e n h o l d e r w h i c h s u p p o r t e d t h e f i b e r i n a v e r t i c a l p o s i t i o n . T h e h o l d e r a s s e m b l y a n d b l a d e w e r e p l a c e d u n d e r a p h y s i o l o g i c a l s a l i n e s o l u t i o n w h i l e t h e s h e a r w a s b e i n g m a d e . T h e t o r q u e r e q u i r e d t o s h e a r t h e f i b e r w a s c o n

v e r t e d t o u n i t s o f f o r c e b y a f o r m u l a d e s c r i b e d b y H e n r i c k s o n e t a l . ( 1 9 6 7 ) . S h e a r s t r e s s w a s d e t e r m i n e d b y d i v i d i n g t h e s h e a r f o r c e b y t h e

s q u a r e o f t h e d i a m e t e r ( H e n r i c k s o n e t a l . 1 9 6 7 ;

H e n r i c k s o n a n d M a r s d e n , 1 9 7 2 ) .I n a u n i f o r m i t y s t u d y , b o v i n e s a r t o r i u s m u s

c l e s w e r e e x c i s e d f r o m c h i l l e d c a r c a s s e s a n d a s e c t i o n o f e a c h m u s c l e w a s p l a c e d in 1 0 % f o r m a l i n s o l u t i o n . W h e n t h e m u s c l e s e c t i o n w a s t h o r o u g h l y f i x e d , a g r o u p o f f a s c i c u l i w a s r e m o v e d , s e p a r a t e d , a n d i t s f i b e r s h a r v e s t e d u s i n g a m o d i f i e d W a r i n g B l e n d o r . F r o m t h e c o m p o s

i t e g r o u p o f f i b e r s , 2 9 9 f i b e r s w e r e r a n d o m l y s e l e c t e d a n d d e t e r m i n a t i o n s w e r e m a d e o n e a c h f i b e r f o r d i a m e t e r , s h e a r f o r c e a n d s h e a r s t r e s s . I n a d d i t i o n , t h e f i b e r s w e r e a s s e s s e d f o r p e r c e n t

F i g . 1 — A m i c r o s e n s i t i v e s h e a r i n s t r u m e n t a n d c o n t a i n e r a s s e m b l y .

Volume 35 (1974)-JOURNAL OF FOOD SCIENCE- 15

1 6 - JOURNAL OF FOOD SCIENCE-Volume 39 f i974)

k i n k i n e s s b y t h e m e t h o d d e s c r i b e d b y C a g l e a n d H e n r i c k s o n ( 1 9 7 0 ) . T h e r e s u l t s w e r e a n a l y z e d s t a t i s t i c a l l y f o r c o n s i s t e n c y o f r e p l i c a t e d m e a s u r e m e n t . F i x a t i o n in f o r m a l i n is k n o w n t o a l t e r t h e m u s c l e t i s s u e p r o t e i n s a n d i n f l u e n c e t h e s h e a r f o r c e . C o n s e q u e n t l y , f u r t h e r r e s e a r c h

u t i l i z i n g r a w a n d c o o k e d f i b e r s i s n o w u n d e r

w a y .T o f u r t h e r t e s t t h e i n s t r u m e n t , m u s c l e s

f r o m 1 8 H e r e f o r d s t e e r c a r c a s s e s t h a t h a d b e e n c o n d i t i o n e d f o r 2 , 5 a n d 8 h r a t 1 6 ° C a n d t h e n c h i l l e d t o 2 ° C f o r 4 8 h r b e f o r e t h e s a r t o r i u s m u s c l e s w e r e e x c i s e d . I n e a c h o f t h e t h r e e t i m e p e r i o d s , t h e “ c o l d ” e x c i s e d m u s c l e s w e r e t r e a t e d t h e s a m e ; h o w e v e r , t h e “ h o t ” e x c i s e d m u s c l e s w e r e h e l d o n t h e s k e l e t o n 2 , 5 a n d 8 h r p o s t m o r t e m b e f o r e e x c i s i o n o f t h e m u s c l e s .

F a s c i c l e s e c t i o n s f r o m t h e s a r t o r i u s m u s c l e w e r e f i x e d i n 1 0 % f o r m a l i n , a n d i n d i v i d u a l m u s

c l e f i b e r s h a r v e s t e d u s i n g a W a r i n g B l e n d o r w h i c h w a s o p e r a t e d a t a s lo w s p e e d . 1 0 0 m e c h a n i c a l l y h a r v e s t e d f i b e r s f r o m e a c h m u s c l e w e r e e v a l u a t e d f o r d i a m e t e r , p e r c e n t k i n k i n e s s ,

s h e a r f o r c e a n d s h e a r s t r e s s .

RESULTS & DISCUSSIONU n ifo rm ity s tu d y

T h e re su lts o f th e u n ifo rm ity s tu d y d e m o n s tra te d th a t th e m ic ro se n sitiv e sh ea r in s t ru m e n t w as cap a b le o f m e a su ring th e sh ea r fo rc e o f an in d iv id u a l m u scle f ib e r. T h e m ea n sh ea r fo rc e fo r th e 2 9 9 m u sc le f ib e rs w as 5 .0 4 x 10_1g, w ith a s ta n d a rd d e v ia tio n o f 1 .90 (T ab le 1). T h e sh e a r fo rc e fo r th e in d iv id u a l f ib e rs ran g ed fro m 1 .22 — 1 0 .0 4 x 10_1g. T h e c o e ff ic ie n t o f v a r ia tio n fo r th e sh ea r fo rc e o f th e 2 9 9 fib e rs w as 37 .6 % , in d ic a tin g th a t th e re w as a large a m o u n t o f v a r ia tio n a sso c ia ted w ith th e m e a su re m e n t. H o w ever, th e g re a te r p a r t o f th is v a r ia tio n in

F i g . 2 - E f f e c t o f h o l d i n g t i m e a n d p r o c e s s i n g

m e t h o d o n f i b e r d i a m e t e r .

sh ea r fo rc e a m o n g m u sc le f ib e rs w as a t t r ib u te d to th e d iffe re n c e s in f ib e r d ia m e te r a n d d eg ree o f k in k in e ss . T h is w as s u b s ta n tia te d b y a m u ltip le lin e a r reg ress io n ana ly sis w h ic h sh o w ed th a t th e e ffe c ts o f f ib e r d ia m e te r a n d k in k in e ss o n sh ea r fo rc e w ere s ta tis t ic a lly s ig n ific a n t (P < 0 .0 0 1 ).

Table 1—Uniformity study: mean, standard deviation and range for fiber diameter, degree kinkiness, shear force and shear stress

Variable N Mean Std dev Range

D iam eter (p) 299 54.3 5 10.64 3 0 .0 0 -9 0 .0 0K in k in ess (%) 299 3 4.67 6 .9 8 1 6 .6 7 -6 6 .6 7Shear force X 10"1 g 299 5.04 1.90 1 .2 2 -1 0 .0 4Shear stress X 10‘4 g/M2 299 2 .30 1.05 0 .6 2 - 8 .2 0

Table 2—Fiber diameter, kinkiness, shear force, and shear stress of bovine sartorius muscle as influenced by holding time and treatment®

2-hr holding 5-hr holding 8-hr holdingHot Cold Hot Cold Hot Cold

Diameter( p )

59.9 ±15.1 47.6 +11.4 44.6 ±11.0 46.7 ± 10.6 51.9 ± 12.3 51.6 ± 12.2

Kinkiness(%)

60.8 ± 14.9 28.3 ± 6.7 34.7 ± 6.2 30.5 ± 7.5 35.7 ± 9.4 33.5 ± 7.6

Shear force X 10’1 g

7.86 ± 3.43 3.33 ± 1.04 2.75 ± 1.04 2.82 ± 0.98 3.48 ± 1.88 3.53 ± 1.40

Shear stress 2.9 ± 1.2a 2.1 ± 0.9 1.95 ± 1.0 1.8 ± 0.9 1.7 ± 0.8 1.83 ± 0.9X 10'4 g/u2

a ± sign indicates standard deviation

70|---------------------------------------------Sg^jHOT BONED

HOLDING TIME

F i g . 3 — E f f e c t o f h o l d i n g t i m e a n d p r o c e s s i n g

m e t h o d o n p e r c e n t k i n k i n e s s .

S h ea r s tre ss c a lc u la te d fo r th is g ro u p o f f ib e rs ra n g ed fro m 0 .6 2 —8 .2 0 x 10~4 g Iij2 , w ith a m e a n o f 2 .3 0 ± 1 .05 x 10"4 g/jU2 (T ab le 1). T h e m e a n s fo r f ib e r d ia m e te r a n d p e rc e n t k in k in e ss w ere5 4 .3 5 ± 1 0 .6 4 m an d 3 4 .6 7 ± 6 .9 8 % r e sp ec tiv e ly (T ab le 1).

E ffec t o f h o t a n d c o ld p ro cess in gAs sh o w n in F ig u re 2 , th e e f fe c t o f

h o ld in g tim e o n f ib e r d ia m e te r w as th e g re a te s t b e tw e e n th e “ h o t ” a n d “ c o ld ” ex c ised m u sc le s fo r th e 2 -h r p e r io d . T his d iffe re n c e w as s ta tis t ic a l ly s ig n ific a n t (P < 0 .0 1 ) . T h e d iffe re n c e s in f ib e r d ia m e te r b e tw e e n th e “ h o t ” a n d “ c o ld ” ex c ised m u sc les w as sm allest fo r th e 5- a n d 8 -h r h o ld in g p e r io d s , an d n e ith e r w as s ta tis tic a lly s ig n ific a n t. T h e re a so n fo r th e in c rease in f ib e r d ia m e te r in th e m u sc les c o n d it io n e d o n th e carcass fo r 2 h r b e fo re ex c is io n a n d c h illin g w as c lo se ly re la te d to th e d e v e lo p m e n t o f r ig o r m o r tis . T hese m u sc les w e re re m o v e d p r io r to th e o n se t o f rig o r, an d th u s w e re a llo w ed to c o n t r a c t f ree ly w h e n rig o r d id d e v e lo p . T h e re w as n o te n s io n p lac ed o n th e se m u sc le s o th e r th a n th a t o b ta in e d d u e to r e s tra in t o n th e carcass d u rin g th e d e v e lo p m e n t o f rig o r. T h e f ib e rs th e re fo re w ere a llo w ed to c o n tra c t to th e ir fu lle s t e x te n t o n c e th e m u sc le w as re m o v e d fro m th e sk e le to n re su ltin g in an in c re a sed f ib e r d ia m e te r .

A d iffe re n c e in p e rc e n t k in k in e ss b e tw e e n th e “ h o t ” a n d “ c o ld ” ex c ised sa rto r iu s m u sc le s fo r th e 2 -h r h o ld in g p e r io d w as s ta tis t ic a lly s ig n if ic a n t (P < 0 .0 1 ) , w h ile th e d iffe re n c e s fo r th e 5- a n d 8 -h r

MEASURING SHEAR FORCE OF MUSCLE FIBER-W

9.0|--------------------------------------------- ------£22 HOT BONED

2 HOUR 5 HOUR 8 HOUR HOLDING TIME


m e t h o d o n f i b e r s h e a r f o r c e .

ho ld ing periods were n o t (F ig . 3, Table2). As stated fo r f ib e r diam eter, the reason fo r the developm ent o f the severe kinkiness in the muscles held 2 h r be fore being excised was s im p ly tha t the muscles were removed before the onset o f rigor, and when rig o r m ortis developed, i t was allowed to proceed unrestric ted . In the case o f the muscles he ld 5 and 8 h r postm ortem before muscle excision, restrain-


m e t h o d o n f i b e r s h e a r s t r e s s .

m ent o f the muscles on the carcass during the onset and developm ent o f r ig o r m o rtis prevented some fib e r shortening.

The d iffe rence in shear force and shear stress between the “ h o t” and “ co ld ” excised muscles was s ta tis tica lly sign ificant o n ly fo r the 2-hr ho ld ing period (P < 0.01). In the 5- and 8-hr ho ld ing periods, the shear force and shear stress were com parable between the “ h o t” and “ co ld ”

excised muscles (F ig . 4 and 5, Table 2). The reason fo r the greater shear force requ ired fo r the fibers fro m muscles excised a fte r a 2-hr pos tm ortem ho ld ing period, m ust u ltim a te ly be re lated back to the developm ent o f rigo r. The data ind ica ted tha t as r ig o r m o rtis o f the muscle in creased, the force required to shear an in d iv idua l muscle f ib e r also increased.

REFERENCES

Cagle, E.D. and Henrickson, R.L. 1970. Influence of slicing warm porcine muscle on fiber diameter, kinkiness, and shear force. J. Food Sci. 35: 270.

Eino, M.F. and Stanley, D.W. 1973a. Catheptic activity, textured properties and surface ultrastructure of post-mortem beef muscle. J. Food Sci. 38: 45.

Eino, M.F. and Stanley, D.W. 1973b. Surface ultrastructure and tensile properties of cathepsin and collagenase treated muscle fibers. J. Food Sci. 38: 51.

Henrickson, R.L., Todd, F.C. and Dunn, J.J.1967. Mechanical properties of beef muscle fibers. Mimeograph series. Oklahoma Agri. Exp. Sta., Stillwater, Oklahoma.

Henrickson, R.L. and Marsden, J.L. 1972. Physical properties of the skeletal muscle fiber. Okla. Agri. Exp. Sta. Bull. 87.

Pool, M.F. and Klose, A.A. 1969. The relation of bone to sample dimensions in objective measurement of tenderness of poultry meat. J. Food Sci. 34: 524.

Sharrah, N., Kunze, M.S. and Pangborn, R.M. 1965. Beef tenderness: Comparison of sensory methods with Warner-Bratzler and L.E.E. Kramer shear presses. Food Technol. 19: 238.

Stanley, D.W., Pearson, G.P. and Coxworth,V.E. 1971. Evaluation of certain physical properties of meat using a universal testing machine. J. Food Sci. 36: 256.

Ms received 6/10/73; revised 9/28/73; accepted 9/30/73.____________________________

Journal Series Paper No. J2689, of the Oklahoma Agricultural Experiment Station.

S. E . M c C R A E a n d P . C . P A U L

F o o d & N u t r i t i o n D e p t . , U n i v e r s i t y o f N e b r a s k a , L i n c o l n , N E 6 8 5 0 3

RATE OF HEATING AS IT AFFECTS THE SOLUBILIZATION OF BEEF MUSCLE COLLAGEN

INTRODUCTION

TENDER NESS o f meat is a com plex sensation. Deatherage (19 63 ) states tha t tenderness o r toughness o f meat is a qu a lity representing the sum m ation o f properties o f the various p ro te in structures o f skeleta l muscles. However, the sensation o f tenderness o r toughness may also be in fluenced by the juiciness o f the meat, the w ater-ho ld ing capacity o f the prote ins, and the am ount and d is tr ib u tio n o f fa t (M atz, 1962). Cover et al. (1962), surmised th a t perhaps one reason fo r the lack o f understanding o f the causes o f tenderness or toughness is because tenderness is n o t a simple bu t a com ponent system.

Meat usually is classified as tender or less tender p rinc ip a lly on the basis o f connective tissue con tent. The exp lanation given fo r the su ita b ility o f a recom mended m ethod o f cooking is tha t the muscle fibers are toughened by extensive heating, w h ile the collagenous connective tissue requires a m oist atmosphere and long cooking to soften i t by conversion o f collagen to gelatin. Rate o f heat penetratio n has been shown to a ffec t the breakdown o f collagenous tissue. Cover and H oste tler (1960 ) have reported tha t the rate o f heat pene tra tion in to meat d if fers w ith the cooking m edium , and seems to a ffec t tenderness. These researchers stated tha t i t was qu ite evident th a t the tim e-tem perature com binations had an e ffec t on the tenderness o f the meat, p robab ly causing b o th physical and chemica l changes in the connective tissue and in muscle fibers.

The recent developm ent o f m icrowave ovens provides a d iffe re n t type o f energy fo r heating meat. This is considered a dry heat m ethod, since the atmosphere surround ing the meat is air (Rosen, 1972). M icrowaves act d ire c tly on the water molecules in the meat, producing heat by fr ic t io n fro m osc illa tion o f these m olecules (Takashima, 1969). The rap id rate o f heat p ro du c tion has un kno w n effects on the con tractile and connective tissue fibers. This study was designed to explore the in fluence o f various types and rates o f heating on the collagenous connective tissue, as assessed by the increase in ease o f e x trac tio n o f collagen fro m the meat tissue.

EXPERIMENTAL

MaterialsTwelve choice grade semitendinosus muscles

were purchased as required from a local supermarket. Neither the muscles nor the samples for this study was frozen.

Approximately 2 cm of m eat from each end of the muscle were discarded to eliminate the tapering ends. The fat layer which covered over a third o f the surface of the muscle was trimmed to approxim ately 0.7 cm thickness. Each muscle was divided into four portions about 6.5 cm in length, designated as the anterior end, the anterior center, the posterior center and the posterior end. Two of the roasts were left intact while the o ther two were cut in half to make two 2.5 cm thick steaks from each portion. The remaining 1.5 cm of each of these, plus a 1 cm slice from each of the two pieces not so divided, were ground for analysis raw. Cooking m ethods

Four rates o f heat penetration were produced by using four heating m ethods, microwave, oven broiling, braising and roasting. The two intact roasts were cooked by microwave or roasting m ethods while the steaks were oven broiled or braised. All samples were heated to an internal tem perature o f 70°C except one braising steak which was heated to 98°C and held at that tem perature for 30 min. Therm ocouples attached to a recorder were used to obtain tim e-temperature records.

The cuts representing the four positions within each muscle were distributed among the heating treatm ents in a Latin-square design, with four positions, four heating m ethods and four muscles from different animals. This design was replicated three times. Analysis o f variance (AV), Duncan’s m ultiple range test (DMRT), and correlation coefficients were utilized in assessing the significance o f the results. The four m ethods using the 70°C internal tem perature were compared in the major statistical analysis. The comparison of braising to 70°C and to 98°C plus 30 min holding was done as a separate analysis, since the longer cooking time did no t fit in to the Latin-square design.

The roasts cooked most rapidly (microwave) were placed, fat side up, on glass rods (serving as a rack) in pyrex pans. The therm ocouple was inserted to record the initial tem perature. After removal o f the therm ocouple, the meat was heated in the microwave oven for 1 min, then removed and the tem perature recorded. This procedure was repeated until the internal tem perature o f the m eat reached 55°C. The meat sample was then removed, and covered. The tem perature continued to rise until 70° C. The meat sample o f this size and shape took up to 10 min to rise to 70°C after taking it from the oven. A num ber of studies have found a consid

erable internal tem perature rise in roasts after removal from the microw'ave oven. Decareau (1967) points out that this must be allowed for when comparing m eat heated by microwave with m eat heated by other m ethods.

For oven broiling (Paul et al., 1956), the two steaks for each sample were cooked at an oven tem perature o f 204°C to 70°C internal tem perature, and showed no tem perature rise after removal from the oven.

The braising m ethod used was that described by Paul and Bean (1956), with the omission of initial browning, as recom mended by Rogers (1969). The two steaks were placed in separate pans. One of the steaks was heated to an internal tem perature of 70°C while the other remained in the oven until the m eat tem perature reached 98°C plus an additional 30 min. The 70°C sample provided data for com parison with the other heat penetration rates, while the 98°C plus 30 min treatm ent more nearly resembled braising as done for consum ption.

The cuts heated most slowly, by roasting in a conventional oven, were heated on racks in open pans at an oven tem perature o f 1633C, to an internal tem perature o f 70°C. They were placed on the rack with the external fat cover uppermost. Since the roast was so small, there was no tem perature rise after the meat was removed from the oven.Sampling and analysis

The heated m eat was trim m ed of all ex ternal fat, and allowed to cool to room tem perature before sampling for chemical analysis and for shear force and penetration measurements. The fluid and solids remaining in the pan were transferred quantitatively to a stoppered graduated cylinder, using two petroleum ether (b.p. 30 -6 0 °C ) rinses to remove all the fat from the pan. The cylinder contents, term ed cooking liquid, were then handled as described bv Paul et al. (1973).

Penetration and Warner-Bratzler shear force values were measured on the lean m eat samples according tc m ethods described by Paul et al.(1970). Four penetrom eter readings and six shear force values were obtained for each heating treatm ent. Sections were reserved for h istological exam ination. All the remaining lean meat, including the samples used for tenderness measurement, was ground and sampled for chemical analysis. Total solids, fat content, total nitrogen, total hydroxyproline and solubilized hydroxyproline were determ ined as described by Paul e t al. (1973). The hydroxyproline data were corrected for the hydroxyproline from elastin.

Samples for m icroscopic exam ination were fixed in formalin, em bedded in paraffin, sectioned 10m thick and stained with W eighert’s triple connective tissue stain.

18- J O U R N A L O F F O O D S C IE N C E - V o lu m e 3 9 (1 9 7 4 )

SOLUBILIZATION OF BEEF MUSCLE COLLAGEN- 1 9

F i g . 1 - T i m e - i n t e r n a l t e m p e r a t u r e c u r v e s f o r h e a t i n g b e e f m u s c l e .

RESULTS & DISCUSSIONTHE T IM E -T E M P E R A T U R E curves calculated fro m the po ten tiom e te r records are shown in F igure 1. M icrowave heating gave the m ost rap id heat pene tra tion , fo llowed by oven b ro iling , braising and roasting, in order o f decrease in rate. A V and D M R T showed tha t the differences in rate were h igh ly s ign ificant, w hether calculated by m inutes per u n it weight or m inutes per u n it depth to the center o f the cut.

Table 1 lists the rates o f heat penetratio n calculated fo r each 10-degree in te rval. The slowest heating rates generally occurred in the 6 0 —70°C in te rva l, w ith the 50—60° in te rva l next. These are the tem perature ranges during w h ich part o f the energy is though t to be u tilize d fo r denaturation o f prote ins and fo r evaporation o f water. The slow rate fo r the 10—20°C in te rva l in braising probably was due to the extra energy demand fo r heating the added water.

The to ta l cooking loss d id no t d iffe r s ign ificantly among electron ic cookery, braising o r roasting (Table 2). However, it was s ign ifican tly less in oven b ro iling , probably due to the re la tive ly short cooking tim e.

Table 1—Mean values fo r rates o f heat penetra tio n

Heat penetra tion rate, m in /°C

interval("C | M icrow ave

Ovenb ro ilin g Braising Roasting

10-20 0.25 0.30 0.60 0.902 0 -3 0 0.25 0.30 0.45 0.753 0 -4 0 0.15 0.30 0.30 0.754 0 -5 0 0.10 0.30 0.45 0.905 0 -6 0 0.20 0.45 0.45 1.206 0 -7 0 0.35 0.45 0.60 1.80

When the to ta l cooking loss was divided in to its com ponents, d rip and evaporation, m ore differences among the rates o f heating were apparent. The evapo ra tion losses did no t d iffe r s ign ifican tly between b ro ilin g and roasting, bu t the vo la tile loss fro m the m icrowave was sign ific a n tly less. This could be a ttr ibu ted , in part, to the greatly decreased cooking tim e o f the meat samples and the surround ing lo w oven tem perature in the m icrowave oven. Braising had the lowest evaporation losses since the covered conta iner reduced evaporation.

M icrowave heating showed a s ign ificant increase in d rip loss over conventio n a l roasting and oven b ro iling . Ruyack and Paul (19 72 ) suggested tha t th is m ight be caused by increased ease o f release o f water due to osc illa tion o f the water molecules in the a lte rnating e lectric fie ld .

Braising produced the highest d rip loss, a re fle c tio n o f the added w ater and o f reduced evaporation in a covered container. The highest percent non fa t solids retained in the meat and low er percent lost in to the liq u id in m icrowave and braising tend to support these conclusions. Oven- b ro iled samples had the lowest d rip loss, perhaps due to short cooking tim e plus increased evaporation fro m the high oven tem perature. The high externa l tem peratures fo r roasting and oven b ro ilin g were p robab ly re flected in the high fa t con tent o f the cooking liq u id , again due to in creased evaporation o f water.

In com m on w ith o the r studies, the n itrogen con ten t o f the heated meat (d ry , fat-free basis) was no t in fluenced by o ther variables. The fa t con ten t o f the lean meat also was no t in fluenced s ign ifican tly by heating m ethod. However, the fa t con tent o f the cooking liq u id in creased w ith increase in no n fa t solids in the liq u id , except fo r oven b ro iling . The exposure o f m ore fa t as m arb ling to the d irect heat cou ld account fo r the s ig n ifican tly higher fa t con tent in the b ro iling liq u id than in the roasting liq u id .

Table 3 lists the values fo r the to ta l h y d ro x y p ro lin e con ten t o f the solid samples, the cooking liq u id , the am ount o f h yd ro x y p ro lin e fro m the collagen extracted w ith water at 40°C fro m the solid, and the percent o f the h y d ro x y p ro line present in the raw meat w h ich was so lub ilized by heating.

The m icrowave energy solub ilized more collagen fro m the meat samples than did conven tiona l heat energy. These higher soluble h y d ro x y p ro lin e values suggest tha t the m icrowave energy influences the collagen so lub iliza tion in a d iffe re n t way than does the heat energy. A fu rth e r study using the tw o types o f energies to

Table 2—Means, F-values fro m analysis o f variance and s ign ifican t d iffe rences among means fo r cook ing losses and solids co n te n t o f p roducts

Measure

Heating m ethods

M icrow aveOven

b ro iling Braising Roasting F-value

% to ta l cooking Mean 28.32 23.71 29.58 28.20 9 .7 8 * *loss D M R T a a b a a

% d r ip loss Mean 12.41 3.31 20.02 5.62 1 9 6 .8 8 **D M R T b d a c

% evaporation Mean 15.95 20.41 9.57 22.58 3 9 .2 2 * *D M R T b a c a

N on fa t solids in Mean 32.18 30.04 32.19 30.44 2 3 .6 5 **heated meat, g/100g D M R T a b a b

N onfa t solids in Mean 13.71 29.40 8.91 30.33 6 8 .4 5 * *liq u id , g/100g D M R T b a c a

Fat in liq u id . Mean 14.35 36.07 5.64 29.05 4 2 .2 6 * *g/100g D M R T c a d b

aDMRT—Duncan's Multiple Range Test; means having same letter are not significantly dif-ferent at p < 0.05.

•Significant at p < 0.05 •Significant at p < 0.01

20-JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

produce the same rate o f heat pene tra tion w ou ld be needed to compare the percent collagen so lub ilized by each heat source. I t is apparent fro m th is study tha t the rate o f heat pene tra tion alters the solub iliz a tio n o f the collagen less than does the manner in w h ich the energy is supplied to produce the heating effect, since there were no sign ificant differences among d iffe re n t heating rates produced by the three conventional methods.

The shear force averages (Table 3) were no t affected by the heating m ethod. Paul et al. (1973 ) also found th is to be true in the semitendinosus muscle when it

was heated to d iffe re n t in te rna l tem peratures. The results o f these tw o studies suggest tha t the tenderness o f the semitendinosus muscle is no t in fluenced by m oist o r d ry heat methods, at least th rough the in te rna l tem perature range o f 58—82°C.

M icrowave and oven b ro ilin g gave a p roduct s ig n ifica n tly softer, as measured by the penetrom eter, than the braised meat. There was no sign ificant d ifference between the softness o f the braised and roasted meat.

The high percent h y d ro xyp ro lin e solub ilized by the m icrowave energy and the

re la tive ly high pene tra tion value seem to indica te tha t m icrowave heating has a d if ferent action on the con trac tile pro te ins than does conven tiona l heating. The muscle fibers rem ain soft even w ith the large am ount o f h y d ro x y p ro lin e so lub ilized. This cou ld be due to the m anner in w h ich the H-bonds are re form ed a fte r the co llagenous tissue is broken dow n and the collagen is released, o r to the d irec t action o f the m icrowave rays on the contrac tile fibers.

The data on the 70°C in te rna l tem pe rature samples showed h igh ly s ign ifican t variations among the 12 animals, fo r percent fa t (d ry w e igh t) and to ta l h y d ro x y p ro line in the cooked meat and cook ing liq u id , fo r percent non fa t solids in the cooked meat, and fo r percent collagen (h y d ro x y p ro lin e ) so lub ilized by heating. Com parison o f the fa t con ten t o f raw and cooked meat ind ica ted tha t high fa t conten t o f raw meat tended to be associated w ith high values fo r cooked mea:, and tha t low values also were found fo r raw and cooked samples fro m the same an imal. This suggests tha t the m ain source o f va ria tion was the in it ia l fa t con ten t o f the raw meat. H y d ro x y p ro lin e data shewed a s im ilar tendency, high values in raw tended to be associated w ith high values in cooked. However, none o f the corre lat io n coe ffic ien ts was s ign ificant. I t is c u rre n tly recognized tha t the degree o f in te rchain crosslinking in fluences m a te ria lly the extent o f heat so lub iliza tion o f co llagen.

The fo u r d iffe re n t parts o f the muscle accounted fo r s ign ificant va ria tio n in the percent non fa t solids in the cooked meat and the percent fa t (d ry w e igh t) in the cooking liq u id , and fo r h igh ly s ign ifican t variations in the percent m oistu re and percent fa t (d ry w e igh t) in the cooked meat, h y d ro xyp ro lin e in the cook ing liq u id , and depth o f pene tra tion . The poste r io r end o f the muscle had higher m oisture con tent, low er fa t con ten t, and low er depth o f pene tra tion than any o f the o ther three parts. The tw o end cuts (an te rio r, poste rio r) had m ore h y d ro x y p ro line in the cooking liq u id than did the tw o center cuts.

When com paring the steaks braised to an end po in t o f 70°C w ith those cooked to 98°C and held an add itiona l 30 m in (Table 4), i t was found th a t to ta l and evaporation losses were greatly increased by the longer tim e o f heating, w h ile the am ount rem aining as d rip decreasec. The m oisture rem aining in the meat decreased, and the non fa t solids o f the cooked meat and cooking liq u id and fa t in the cooking liq u id increased w ith longer cooking. These results correspond w ith the know n effects o f higher tem pe ratures and longer cooking tim es in causing decrease in w ater b ind ing capacity and increased evaporation o f water.

The percent o f extractab le h y c ro x y -

Table 3—Means, F-values from analysis of variance and significant differences among meansfor chemical and physical measures ___________________________ ___________________

Heating methods

OvenMeasure Microwave broiling Braising Roasting F-value

H yd ro xyp ro lin e , Mean 0.48 0.47 0.48 0.47 0.43g/100g meat D M R T a a a a a

H yd ro xyp ro lin e , Mean 0.09 0.05 0.10 0.05 8 8 .8 9 **g/100g cook- D M R T b a C aing liq u id

Extractab le Mean 0.0357 0.0290 0.0277 0.0338 3 .4 *h yd ro xyp ro lin e , D M R T a b b a,b

g/100g meat% h y d ro xyp ro lin e Mean 7.82 5.63 6.37 6.79 4 .6 9 *

in raw meat D M R T a b b bso lub ilized by cook ing

Shear force, Mean 4.23 4.40 4.58 4.49 0.93

kg D M R T a a a aPenetration Mean 5.82 5.91 5.13 5.38 4 .6 3 *mm D M R T a a b a,b

a DMRT— Duncan's Multiple Range Test; means having same letter are not significantly differ-ent at p < 0.05.

* Significant at p < 0.05 * * Significant at p < 0.01

Table 4—Means and F-values for comparison of braising to 70°C or to 98°C plus 1/2 hr holding

MeasureBraise 70°C,

Mean

Braise 98°C + 1/2 hr.

Mean F-value

% to ta l loss 29.58 44 .38 2 1 6 .7 9 **% d r ip loss 20.02 15.24 11.0 1 * *% evaporation 9.57 29.14 1 4 8 .1 9 **Cooked meat

% m o isture In lean 62.28 55.69 1 5 3 .9 8 **% fa t-fre e solids, meat 32 .19 38.59 2 4 6 .6 0 **% fa t-free solids, cook ing liq u id 8.91 18.75 2 9 .5 2 **% fa t , d ry basis, meat 14.57 12.84 1.44ns% fa t, d ry basis, cook ing liq u id 5.54 20.39 1 8 .8 7 **

% h y d ro x y p ro lin e in raw meatso lub ilized by cooking 6.37 27.26 6 7 .7 8 * *

Shear force, kg 4.58 3.78 4.03nsPenetra tion, mm 5.13 3.34 3 4 .7 2 **• - p < 0.01ns —not significant

SOLUB1 LIZA TION OF BEEF MUSCLE COLLAGEN—2"\

'p ro lin e increased, bu t the depth o f penetra tio n decreased w ith longer heating, ind ica ting tha t a lthough collagen so lub iliza tion increased m a te ria lly , the meat was becoming m ore resistant to penetra tion . The shear fo rce required was s ligh tly low er fo r the longer tim e , bu t the d iffe rence was not sign ificant.

The typ ica l patterns o f heat-induced change observed in collagenous connective tissue, con trac tile fibers and endo- m ysia l re ticu lu m , in sections stained w ith W eighert’s, have been described in some detail by Paul (1963, 1966). In term edia te rates o f heating by oven b ro ilin g and braising to 70°C caused the least changes in collagenous fibers, w ith the slowest m ethod, roasting, second, braising to 98°C plus 1/2 h r nex t, and the fastest

> m ethod, m icrowave heating, producing the most a lte ra tion . These observations perhaps go along w ith the decreasing rate o f heat pene tra tion among oven b ro iling , braising to 70°C , and roasting, and the greatest increase in extractab le collagen in m icrowave heating and braising to 98°C plus 1/2 hr. The endom ysial re ticu lum showed some fray ing and breakup w ith a ll the conventional heating m ethods, bu t remained essentially in tac t w ith m icro

wave. The con tra c tile fibers were cracked and broken a fte r oven b ro ilin g , rcasting, o r braising to 70°C , w ith the la tte r tw o showing some granu la tion o f the muscle fibers themselves. The samples heated by m icrowave showed granu la tion bu t l i t t le cracking in these fibers. The samples braised to 98°C plus 1/2 h r were considerably shrunken w h ich tended to cbscure any breakup o f the muscle fibers w h ich m ight have occurred.

REFERENCES

Cover, S. and Hostetler, R.L. 1960. An examination of some theories about beef tenderness by using new methods. Texas Agri. Exp. Sta. Bui. 947, College Station, Texas.

Cover, S., Ritchey, S.J. and Hostetler, R.L. 1962. Tenderness of beef. 1. The connective-tissue component of tenderness. J. Food Sci. 27: 469.

Deatherage, F.E. 1963. The effect of water and inorganic salts on tenderness. Proc., Meat Tenderness Symposium, p. 45. Campbell Soup Co., Camden, N.J.

Decareau, R.V. 1967. Utilization of microwave cookery in meat processing. Proc. 20th Ann. Recip. Meat Conf., p. 216. National Live Stock Meat Board, Chicago, 111.

Matz, S.A. 1962. “Food Texture.” Avi Publ. Co., Westport, Conn.

Paul, P.C. 1963. Influence of methods of cooking on meat tenderness. Proc., Meat Tenderness Symposium, p. 225. Campbell Soup Co.. Camden, N.J.

Paul, P.C. 1966. Storage- and heat-induced changes in the microscopic appearance of rabbit muscle. J. Food Sci. 30: 960.

Paul, P.C., and Bean, M. 1956. Method for braising beef round steaks. Food Res. 21: 75.

Paul, P.C., Bean, M. and Bratzler, L.J. 1956. Effect of cold storage and method of cooking on commercial grade cow beef. Mich. Agr. Exp. Sta. Bull. 256, East Lansing, Mich.

Paul, P.C., Mandigo, R.W. and Arthaud, V.H.1970. Textural and histologic differences among three muscles in the same cut of beef. J. Food Sci. 35: 505.

Paul, P.C., McCrae, S.E. and Hofferber, L.M.1973. Heat-induced changes in beef muscle collagen. J. Food Sci. 38: 66.

Rogers, P.J. 1969. Standardization of methods of heating and sampling meat. Proc. 22nd Ann. Recip. Meat Conf., p. 166. National Live Stock Meat Board, Chicago, 111.

Rosen, C.G. 1972. Effects of microwaves on food related materials. Food Technol., 26(7): 36.

Ruyack, D.F. and Paul, P.C. 1972. Conventional and microwave heating of beef: Use of plastic wrap. Home Econ. Res. J. 1: 98.

Takashima, S. 1969. Dielectric properties of proteins. 1. Dielectric relaxation. In: “Physical Principles and Techniques of Protein Chemistry,” Part A, Ed. Leach, S.J., p. 291. Academic Press, New York.

Ms received 6/16/73; revised 8/21/73; accepted 8/24/73.

Presented at the 33rd Annual Meeting of the Institute of Food Technologists in Miami Beach.

Paper No. 3647, Journal Series, Nebraska Agricultural Experiment Station, Lincoln. This research was supported by Project No. 91-009, Nebraska Agricultural Experiment Station.

OXYGEN

INTRODUCTION

OXYGEN uptake in excised postrigor muscle could result from tissue respiration, heme pigment oxygenation, dissolution into tissue fluids of low oxygen tension, lipid oxidation, and/or bacterial demands.

Postmortem respiratory oxygen consumption would require that the muscle contain active enzyme systems and residual stores of respiratory substrates and intermediates. Active respiratory enzymes in postmortem muscle have been demonstrated by several investigators (Andrews et al., 1952; Grant, 1955; Bodwell et al.,1965) and substantial quantities of respiratory intermediates and substrates have been observed after various postmortem storage times (Bodwell et al., 1965a; Atkinson et al., 1969; Kasten- schmidt, 1970; Newbold and Scopes,1971). Furthermore, studies by Cheah and Cheah (1971) have indicated that bovine muscle mitochondria remain intact and active up to 144 hr postmortem, as long as the tissue pH is maintained at or above 5.5. The existence of intact mitochondria in bovine muscle after several days of storage has been supported by recent investigations at Rutgers University (Franke, personal communication). Significant respiratory oxygen consumption by postrigor bovine muscle has been reported previously by Urbin and Wilson (1961) and Bendall and Taylor(1972).

Urbin and Wilson (1961) also indicated that substantial quantities of oxygen were consumed in postrigor muscle resulting from oxygenation of myoglobin and dissolution into tissue fluids, especially during the first 15 hr of oxygen consumption.

Total oxygen uptake in excised sections of postrigor muscle would then result predominantly from tissue respiration, heme pigment oxygenation, and saturation of low oxygen tension tissue fluids. However, lipid oxidation (Tappel et al., 1961; Kwoh, 1971) and bacterial demands (Rahn and Richardson, 1940; Greig and Hoogerheide, 1941) could also consume measurable amounts of oxygen.

This study was designed to measure manometrically the time-course of oxygen uptake in postrigor bovine muscle

D . P. D e V O R E .

B a tte l le , C o lu m b u s L a b o ra to r ie s , 5 0 5 K in g A ve. C o lu m b u s , O H 4 3 2 0 1

M . S O L B E R G

D e p t, o f F o o d S cience, R u tg e rs — The S ta te U n iv e rs ity o f N e w Jersey, N e w B ru n s w ic k , N J 0 8 9 0 3

UPTAKE IN POSTRIGOR BOVINE MUSCLE

and to determine the magnitude of oxygen consumption attributable to respiration, heme pigment oxygenation, tissue fluid saturation, and lipid oxidation. Oxygen consumption resulting from bacterial demands was maintained at negligible levels by using aseptic techniques to limit the bacterial populations.

An understanding of the primary oxygen requiring systems in postrigor muscle may result in the development of improved storage parameters. The resulting data should also allow a more comprehensive assessment of the respiratory processes in postrigor muscle and possibly indicate the in situ functions of tissue myoglobin.

EXPERIMENTALO X Y G E N U P T A K E o r c a r b o n d io x id e e v o lu t i o n w a s m e a s u r e d m a n o m e t r i c a l l y u s in g a d i f f e r e n t i a ] r e s p i r o m e te r ( G i ls o n M o d e l G R - 2 0 , G ils o n M e d ic a l E l e c t r o n i c s , M a d is o n , W ise .) w h ic h w a s e q u ip p e d w i th N o . 5 a ll-g la s s v o lu - o m e te r s . A ll m e a s u r e m e n t s w e re m a d e a t 5 ° C u n d e r a n o x y g e n h e a d s p a c e m a in t a in e d a t o n e a tm o s p h e r e o f p r e s s u re . I n o x y g e n u p ta k e m e a s u r e m e n t s , f l u t e d f i l t e r p a p e r , s a t u r a t e d w i th 1 0 % K O H , w a s p la c e d in t h e s a m p le c e l ls t o a b s o r b c a r b o n d io x id e .

F r e s h t o p r o u n d o f b e e f f r o m 5 - 7 d a y s p o s t m o r t e m w a s p u r c h a s e d lo c a l ly . T h e s u r f a c e w a s w a s h e d w i th 9 5 % e t h a n o l a n d a s e p t ic a l ly c le a n e d o f s u r f a c e f a t . T h e s e m im e m b r a n o s u s m u s c le w a s r e m o v e d a n d p la c e d in a p r e s t e r i l i z e d , c o n t r o l l e d a tm o s p h e r e c h a m b e r ( S t a n d a r d S a f e ty E q u i p m e n t , P a l a t i n e , 111.). T h e c h a m b e r w a s c o n t i n u o u s l y f lu s h e d w i th h i -p u r e n i t r o g e n d u r in g s a m p le p r e p a r a t i o n p r o c e d u r e s . A ll p r o c e d u r e s w e re p e r f o r m e d a t 5 ° C a n d a s e p t ic t e c h n iq u e s w e re e m p lo y e d . T i s s u e s lic e s 1 3 m m t h i c k w i th a s u r f a c e a r e a o f 1 2 .5 c m 2 w e r e o b t a i n e d b y s l ic in g m u s c l e s e c t io n s p e r p e n d ic u l a r t o t h e d i r e c t i o n o f t h e m u s c l e f ib e r s . T is s u e d is c s w e re s u b s e q u e n t ly p la c e d in th e g la ss s a m p le c e l ls , 15 m m in d e p t h a n d w i th a d i a m e te r o f 4 . 0 c m . T h e g r o u n d g la ss s u r f a c e s o f t h e s a m p le c e l ls w e re s e a le d u s in g s to p c o c k g re a s e a n d t h e c e l l o v e r s e a le d w i th m o d e l in g c la y . T h e s a m p le s w e re p la c e d in t h e d i f f e r e n t i a l r e s p i r o m e te r a n d e q u i l i b r a t e d f o r 3 0 m in . D u r in g t h e e q u i l i b r a t i o n , t h e h e a d s p a c e s y s te m , e x c lu s iv e o f t h e s a m p le c e l l , w a s f lu s h e d w i th o x y g e n . M e a s u r e m e n t s o f o x y g e n u p t a k e w e re th e n in i t i a t e d . A ll t im e s r e p o r t e d d u r in g th e s t u d y a re b a s e d o n a z e r o t im e a t th i s p o i n t .

P e r f u s io n t e c h n iq u e s w e re d e v e l o p e d t o in t r o d u c e r e a g e n t s p a s s iv e ly i n t o t h e i n t e r i o r o f t h e m u s c le . P e r f u s io n w a s a c c o m p l i s h e d b y in

s e r t in g a 6 -h o le s t i t c h p u m p n e e d l e i n t o a n e a s i ly d e t e c t e d a r t e r y . P e r f u s a t e w a s p u m p e d i n t o t h e m u s c l e u s in g a f i l a m a t ic a u t o m a t i c v ia l f i l le r ( N a t io n a l I n s t r u m e n t C o m p a n y . B a l t i m o r e , M d .) . H e m o s ta t s w e re u s e d t o c lo s e b lo o d v e ss e ls in w h ic h u n o b s t r u c t e d r e a g e n t f lo w w a s o b s e r v e d .

T h e d i lu t i o n o f t i s s u e h e m e p ig m e n t c o n c e n t r a t i o n w a s p e r f o r m e d b y t o t a l i m m e r s io n o f t h e s a m p le d is c s in a c o n t i n u o u s f l o w o f K re b s - R in g e r - p h o s p h a t e i s o to n ic s o lu t io n (p H 5 .6 ) a t 5 ° C a n d a t a f lo w r a t e o f 5 0 m l p e r h r . T h e d i l u t i o n s y s te m w a s o p e r a t e d f o r 2 4 h r in a n i t r o g e n a tm o s p h e r e .

O x y g e n u p t a k e in c o n t r o l a n d t r e a t e d p o s t r i g o r b o v in e m u s c l e

O x y g e n u p t a k e in c o n t r o l a n d in a n t im y c in A p e r f u s e d ( r e s p i r a t i o n in h i b i t e d ) m u s c l e s lic e s w a s m e a s u r e d to d e t e r m i n e th e n e t o x y g e n c o n s u m p t io n r e s u l t in g f r o m t i s s u e r e s p i r a t i o n . In t h e p r e l im in a r y e x p e r im e n t s , b o t h o x y g e n c o n s u m p t io n a n d c a r b o n d io x id e e v o lu t io n w e r e d e t e r m in e d in f o u r s a m p le s e a c h u p to 1 2 0 h r o f e x p o s u r e t o o x y g e n . I n th e s u c c e e d in g e x p e r im e n ts , o n ly o x y g e n u p t a k e w a s d e t e r m i n e d a n d th e e x p o s u r e t im e w a s r e d u c e d t o 3 0 h r ; 12 c o n t r o l s a m p le s a n d 1 0 t r e a t e d s a m p le s w e re e x a m in e d . S a m p le s e c t io n s w e re o b t a i n e d f r o m t h e e x c i s e d s e m i m e m b r a n o s u s m u s c l e a n d w e re p e r f u s e d w i th e i t h e r 2 0 0 m l o f K re b s - R in g e r - p h o s p h a te i s o to n ic s o lu t io n (p H 5 .6 ) a lo n e o r w i th 2 0 0 m l o f t h e i s o to n ic s o l u t i o n c o n t a in in g 2 0 m g o f a n t i m y c i n A in 2 0 m l o f e t h a n o l . A f te r p e r f u s io n , s a m p le s l ic e s w e r e p r e p a r e d a n d p la c e d in t h e s a m p le c e l ls , w h ic h w e re t h e n a t t a c h e d to t h e d i f f e r e n t i a l r e s p i r o m e t e r f o r s u b s e q u e n t g as e x c h a n g e m e a s u r e m e n ts .

T h e e f f e c t o f l ip id o x i d a t i o n o n o x y g e n c o n s u m p t io n in p o s t r i g o r s e m i m e m b r a n o s u s m u s c l e w a s d e t e r m in e d b y c o m p a r in g t h e t im e c o u r s e o f o x y g e n u p t a k e in c o n t r o l a n d r e s p i r a t i o n - i n h i b i t e d , h e m e o x id iz e d m u s c l e s l ic e s p e r f u s e d e i t h e r w i th i s o to n ic s o lu t io n o n ly o r i s o to n ic s o lu t io n c o n ta in in g 3 .2 x 1 0 '3 M a - t o c o p h e r o l a n d 1 .5 X 1 0 '3 M a s c o r b a t e . O x y g e n u p t a k e w a s m e a s u r e d in f o u r s a m p le s o f e a c h t r e a t m e n t p r e p a r a t i o n .

T h e e f f e c t o f h e m e p ig m e n t c o n c e n t r a t i o n ( r e d u c e d s t a t e ) o n o x y g e n u p t a k e in n o r m a l a n d in r e s p i r a t i o n - in h ib i t e d p o s t r i g o r m u s c l e w a s d e t e r m in e d b y c o m p a r in g th e t im e c o u r s e o f o x y g e n u p t a k e in m u s c l e s lic e s w i th v a r y in g c o n t e n t s o f h e m e p ig m e n t . H e m e p ig m e n t c o n c e n t r a t i o n w a s r e d u c e d u s in g th e d i l u t i o n p r o c e ss d e s c r ib e d p r e v io u s ly . In a n a d d i t i o n a l s e t o f e x p e r im e n t s , o x y g e n u p t a k e w a s m e a s u r e d in r e s p i r a t i o n - in h ib i t e d m u s c l e c o n t a i n i n g n o r m a l ( r e d u c e d s t a t e ) a n d o x id i z e d h e m e p ig m e n ts . M u sc le s e c t io n s w e re p e r f u s e d w i t h 2 0 0 m l o f K r e b s - R m g e r - p h o s p h a te i s o t o n i c s o l u t i o n (p H 5 .6 ) c o n ta in in g 2 0 m g o f a n t im y c in A ir. 2 0 m l o f 9 5 % e th a n o l . H e m e p ig m e n ts w e re o x id i z e d


O X Y G E N U P T A K E I N P O S T R I G O R B O V I N E M U S C L E - 2 3

b y in c lu d in g 2 g o f p o t a s s iu m f e i r i c y a n id e in t h e p e r f u s io n s o lu t io n . R e f l e c t a n c e s p e c t r a w e re r e c o r d e d s p e c t r o p h o t o m e t r i c a l l y ( B e c k m a n D K -2 s p e c t r o p h o t o m e t e r , B e c k m a n I n s t r u m e n t C o m p a n y . F u l l e r t o n , C a lif .

A n a ly t ic a l D e te r m in a t io n s

M u sc le s a m p le s in a ll e x p e r im e n t s , e x c e p t th e p r e l im in a r y r u n s , w e re a n a ly z e d f o r p H , h e m e p ig m e n t c o n c e n t r a t i o n , b a c t e r i a l c o n t a m in a n t c o n t e n t , m o is tu r e c o n t e n t , a n d c y t o c h r o m e c r e d u c t a s e a c t i v i t y . A n a ly s e s w e re p e r f o r m e d b e f o r e b e g in n in g o x y g e n u p ta k e m e a s u r e m e n ts a n d a t t h e c o m p le t io n o f th e ru n s . T h e p H o f m u s c le s a m p le s w a s m e a s u re d u s in g a n e x p a n d e d s c a le p H m e te r ( B e c k m a n E x p a n d o m a t ic , B e c k m a n I n s t r u m e n t C o ., F u l l e r to n , C a l if .) , e q u ip p e d w i th a c o m b in a t io n s u r fa c e e le c t r o d e . p H m e a s u r e m e n t s o f p e r f u s io n e f f lu e n t s a n d p ig m e n t d i l u t i o n s o lu t io n s w e re m a d e u s in g a d ig i ta l p H m e t e r ( R a d i o m e te r p H m e te r , R a d i o m e te r I n s t r u m e n t s , C o p e n h a g e n , D e n m a r k ) . T o t a l h e m a t in w a s d e t e r m in e d s p e c t r o p h o t o m e t r i c a l l y u s in g th e m e th o d o f H o rn s e y ( 1 9 5 6 ) . A b s o r b a n c e o f th e t is s u e e x t r a c t s w a s m e a s u r e d a t 6 4 0 n m (B e c k m a n D B G s p e c t r o m e te r , B e c k m a n I n s t r u m e n t C o ., F u l l e r to n , C a l if .) . H e m a t in c o n t e n t w a s c a l c u la te d b y m u l t i p l y in g th e a b s o r b a n c e a t 6 4 0 n m b y 6 8 0 . H e m e p ig m e n t c o n c e n t r a t i o n in m g /g w a s o b t a i n e d b y m u l t i p l y in g th e t o t a l h e m a t in c o n t e n t b y 0 .0 2 6 . T h e m o i s tu r e c o n t e n t o f s a m p le p r e p a r a t i o n s w a s d e t e r m in e d u s in g th e A O A C o v e n -d ry in g m e t h o d (A O A C ,1 9 7 0 ) . M ic r o b io lo g ic a l a n a ly s is w a s p e r f o r m e d u s in g th e m e t h o d d e s c r ib e d b y S o lb e rg a n d P r o c to r ( 1 9 6 0 ) . V ir i le c o lo n ie s w e re o b s e rv e d b y th e p r o d u c t i o n o f a r e d c o lo r r e s u l t in g f r o m th e b a c t e r ia l r e d u c t io n o f a t e t r a z o l iu m c h lo r id e s o lu t io n .

T h e a c t iv i ty o f s u c c in a te o r N A D H c y t o c h r o m e c r e d u c t a s e w a s d e t e r m in e d s p e c t r o - m e tr ic a l ly u s in g a m o d i f i c a t i o n o f t h e m e th o d d e s c r ib e d b y T a p p e l ( 1 9 6 0 ) a n d K a n iu g a e t a l.( 1 9 6 8 ) . A c t iv i ty w a s m e a s u r e d in t i s s u e h o m o g e n a t e s w i th a n d w i t h o u t e x o g e n o u s s u b s t r a te . D i lu te t i s s u e h o m o g e n a te s w e re o b t a i n e d b y a d d in g f in e ly m in c e d m u s c le t o t h r e e t im e s th e s a m p le w e ig h t o f c o ld 0 .9 % KC1. A f t e r m ix in g in a t i s s u e h o m o g e n iz e r f o r 1 - 2 m in , 1 .0 m l o f t h e s u p e r n a t a n t s o lu t io n w a s d i lu t e d w i th 5 0 m l o f c o ld 0 .9 % KC1 t o g iv e t h e d i l u t e t is s u e h o m o g e n a te . E n z y m e a c t iv i t y w a s a s s a y e d s p e c t r o p h o t o m e t r i c a l l y b y m e a s u r in g th e i n

c re a s e o f a b s o r b a n c e o f r e d u c e d c y to c h r o m e c a t 5 5 0 n m . T h e r e a c t io n m ix tu r e o f 3 m l c o n t a in e d 2 0 m M s u c c in a te , 1 m g o f c y t o c h r o m e c, 0 .1 M p h o s p h a te b u f f e r ( p H 7 .4 ) , 2 m M E D T A , 1 m M K C N , 0 .1 m l o f d i s t i l l e d w a te r , a n d 0 .1 m l o f d i l u t e h o m o g e n a te c o n ta in in g 0 . 1 4 - 0 . 3 3 m g o f p r o t e in . I n m e a s u r e m e n ts o f a c t iv i ty w i t h o u t e x o g e n o u s s u b s t r a t e , s u c c in a te w a s o m i t t e d f r o m t h e r e a c t io n m ix tu r e . T h e p r o t e in c o n t e n t o f t h e d i l u t e t is s u e h o m o g e n a te w as d e t e r m in e d b y th e B iu r e t m e t h o d (B a ile y , 1 9 6 7 ) .

RESULTSTissue respiration—preliminary experiments

In the preliminary experiments, the rate of oxygen uptake and carbon dioxide evolution was rapid up to 4 hr. Then it decreased to an intermediate rate lasting to approximately 50 hr before falling off to a slower, steady-state rate lasting more than 120 hr. The cumulative volume of carbon dioxide evolution was consistently larger than the cumulative volume of oxygen uptake. Consequently, the respiratory quotient (R.Q.) values were greater than1 . 0 during the entire recording period. However, during the latter steady-state period, the rate of oxygen uptake was greater than the rate of carbon dioxide evolution resulting in an apparent R.Q'. of less than 1.0. The calculated R.Q. values of 1 . 0 or greater indicated that active respiration was occurring in the postrigor muscle samples. Furthermore, the substrate for respiration was most likely a carbohydrate rather than a fat or fatty acid. The reduction in R.Q. value after 50 hr indicated a possible depletion of carbohydrate substrate stores. Oxygen consumption and carbon dioxide evolución in antimycin A perfused muscle systems were reduced significantly compared to the control systems. The difference between the time-course of oxygen uptake in control and inhibited muscle theoretically represents the oxygen consumption

resulting from respiration. This uptake accounted for approximately 80% of the total oxygen consumption. The corresponding differential for the carbon dioxide curves indicated that approximately 75% of the carbon dioxide evolution resulted from respiration. Respiration inhibition was assumed to be 1 0 0 % since Potter and Reif (1952) observed nearly total inhibition of succinoxidase activity in muscle containing antimycin A at a level of 2 Mg/g of tissue. In this study, the antimycin A content in the perfused muscle sections was at least this great.

Tissue respirationThe time-course of oxygen uptake in

control of postrigor muscle and in antimycin A perfused muscle slices is shown in Figure 1. The rate of oxygen uptake remained constant in the treated muscle up to 30 hr. Respiration inhibition was biochemically determined by measuring the specific activity of succinate/NADH cytochrome c reductase. The results are shown in Table 1 together with the data from several other analytical determinations. The specific activity of cytochrome c reductase was essentially zero in the antimycin A perfused tissue confirming the observed inhibited respiration based on oxygen consumption measurements. Uptake in the antimycin A perfused muscle sample represented the oxygen utilization resulting from heme pigment oxygenation, saturation of tissue fluids, and possibly lipid oxidation. This nonres- piratory oxygen consumption rate remained constant and accounted for approximately 50% of the total oxygen uptake after 30 hr.

The difference in uptake between the control sample and the antimycin A perfused sample then represents the oxygen utilization attributable to respiration. The rate of uptake from respiration was dramatically reduced after 10 hr. The in situ activity of cytochrome c reductase

Table 1—Physical and chemical characteristics o f the perfusion effluents end of control and antim ycin A perfused postrigor bovine semimembranosus muscle

Contro la Antim ycin A perfused

to(In itia l)

t f(Final)

to(In itia l)

t f(Final)

Perfusioneffluent

pH 5.3 5.3 5.4 5.5 5.7Total heme (mg/g) 9.2 - 9.2 - -Moisture content (%) 75.8 - 75.5 - -0 2 Depth o f penetration (mm) - 6 - 8 -Specific activity

(pmoles cytochrome c reduced/mg/min)With succinate 0.890 0.458 0.014 0.0 0.0W ithout succinate 0.416 0.340 0.014 0.0 0.0

A v e r a g e o f 1 0 — 1 6 s a m p l e s

2 4 - J O U R N A L O F F O O D S C I E N C E - V o l u m e 3 9 ( 1 9 7 4 )

also decreased during the recording period. This indicated that the decrease in postrigor respiration may have resulted from enzyme degradation. However, subsequent addition of exogenous substrate to the 30-hr homogenate resulted in a significant increase in enzyme activity. The observed rate decline may have also been a function of substrate depletions. It is difficult, from these data, to determine whether substrate limitation, enzyme degradation, or both, specifically contributed to the observed rate decline.

Effect of lipid oxidation on oxygen uptake in postrigor bovine muscle

Tne effect of lipid oxidation on oxygen uptake in postrigor muscle was investigated by comparing the time-course of oxygen uptake in normal and in treated muscle perfused with isotonic solution or isotonic solution containing «-tocopherol and sodium ascorbate. The results shown in Figure 2 are similar for both antioxidant and nonantioxidant containing sam

ples regardless of other treatments used throughout all periods of the test. The chemical and physical characteristics of comparative samples, which are shown in Table 2, were also similar, and oxygen uptake resulting from lipid oxidation was insignificant in this experiment.

It is noted in Table 2 that there were variations in the activity of cytochrome c reductase in the isotonic solution perfused muscle samples with and without antioxidant. At the end of the uptake recordings, samples with antioxidant

Table 2—Chemical and physical characteristics o f postrigor bovine semimembranosus muscle perfused w ith : (1) isotonic solution, (2) isotonic solution plus antioxidant, (3) antimycin A plus K 3Fe(CN)6 and (4) antimycin A plus K 3Fe(CN)6 plus antioxidant

Characteristics

S.A. of cytochrome c reductase

pH

pigmentcontent,

mg/gIn itia l3

Watercontent,

%Finalb

(pmoles cyt. c recuced/mg/min)

Added substrate No added substrate

Sample description In itia l3 Final*3 In itia l3 Final13 In itia l3 Finalb

Isotonic Sol perfused0 5.3 5.3 10.3 77.2 0.820 0.600 0.640 0.400Isotonic sol

+ antioxidant0 5.3 5.2 10.7 76.7 0.880 0.300 0.690 0.250Antimycin A +

K3 Fe(CN)6c 5.3 5.2 8.2 74.1 0.000 0.000 0.000 0.000Antimycin A +

K, Fe(CN)6 + antioxidant0 5.4 5.3 7.6 76.2 0.000 0.000 0.000 0.000

a P r i o r t o e x p o s u r e t o o x y g e n

h A f t e r e x p o s u r e t o o x y g e n f o r 3 0 h r

c A v e r a g e v a l u e s f o r 1 0 — 1 6 s a m p l e s

Fig. 1—The tim e -co u rse o f o x y g e n u p ta k e in u n tre a te d (o -o -o ) a n d a n t im y c in A p e rfu s e d (a -a -a ) p o s tr ig o r b o v in e m u sc le a n d th e tim e -

c o u rse o f o x y g e n u p ta k e du e to re s p ira t io n (n -n -u ) in p o s tr ig o r b o v in e m usc le . E ach p o in t re p rese n ts th e average o f 10— 16 sam ples.

Fig. 2 —O xyg e n u p ta k e in p o s tr ig o r b o v in e m u sc le p e r fu s e d w ith (1 ) is o to n ic s o lu t io n (o -o -o ), (2 ) is o to n ic s o lu t io n c o n ta in in g a n t io x id a n t c o m p le x (a -a -a ) , (3) a n t im y c in A is o to n ic s o lu t io n c o n ta in in g fe r r i- c y a n id e ( • - • - • ) a n d 14) a n t im y c in A is o to n ic s o lu t io n c o n ta in in g fe r r i- c y a n id e p lu s a n t io x id a n t c o m p le x (*-*-*■). Each p o in t re p rese n ts th e average o f 10— 16 sam ples.

O X Y G E N U P T A K E I N P O S T R I G O R B O V I N E M U S C L E - 25

showed decreased enzyme activity compared to the samples without antioxidant. The addition of exogenous substrate to the antioxidant containing system did not significantly increase the enzyme activity. It is extremely difficult to determine why the activity of cytochrome c reductase was apparently destroyed by the antioxidants during the time-course of the experiment.

Effect of heme pigment concentration on oxygen uptake

The time-course of oxygen uptake in isotonic solution perfused muscle with normal and diluted heme pigments is shown in Figure 3. Uptake followed the typical three-phase course. There was a significant reduction in the rate of oxygen uptake in the treated samples in each phase. In the first hour and again at 10

hr, there was a net reduction in uptake of 50%. At 30 hr, a 42% reduction was observed. However, the reduced oxygen uptake was not completely attributable to the dilution of heme pigments since this dilution could only account for 1 0 % of the change based on a direct stoichiometric relationship.

A comparison of the chemical characteristics of the control and treated samples (Table 3) shows that the in situ activity of cytochrome c reductase was also lowered in the samples treated to dilute the heme pigment concentration. Subsequent examination of the soak effluents from the dilution process indicated that approximately 25% of the enzyme activity was removed. Thus, oxygen uptake would be reduced correspondingly. The effect of the differential in enzyme activity was eliminated by normalizing the

data in both the control and treatment samples to an equivalent enzyme activity. The result of normalization is shown in Figure 4. The variation in oxygen uptake attributable to the dilution of heme pigments was decreased. However, the differences observed were still 8 times greater than expected for the change in heme pigment concentration noted. Based on the analytically determined heme pigment concentration, it was calculated that a net difference of 2.4 pi of oxygen per cm2

was expected from the variation in heme pigment concentration observed between the control and treated samples after 30 hr. Experimentally, the net adjusted difference after 30 hr was 20 /rl/cm . The experimental results indicate that the oxygen-utilizing capacity of in situ heme pigment is greater than that due to a simple stoichiometric interaction between

Fig . 3 —The tim e -co u rse o f o x y g e n u p ta k e in p o s tr ig o r b o v in e m u sc le F ig . 4 —O xyg e n u p ta k e in p o s tr ig o r b o v in e m u sc le w ith n o rm a l ( o - o - o )

w ith n o rm a l ( o - o - o ) a n d d i lu te d (&-&-&) he m e p ig m e n t c o n te n t. Each a n d d i lu te d (A-A-Aj h e m e p ig m e n t c o n te n ts a n d n o rm a liz e d e n z y m e

p o in t re p rese n ts th e average o f 10— 16 sam ples. a c t iv ity . Each p o in t re p rese n ts th e average o f 10— 16 sam ples.

Fig. 5 —The tim e -co u rse o f o x y g e n u p ta k e in a n t im y c in A p e r fu s e d Fig. 6 —The tim e -co u rse o f o x y g e n u p ta k e in p o s tr ig o r b o v in e m usc le p o s tr ig o r b o v in e m usc le w ith n o rm a l to-o'" 1) a n d d i lu te d (a -a -a ; he m e p e rfu s e d w ith a n t im y c in A is o to n ic s o lu t io n ( o - o - o ) a n d w ith a n t im y c in

p ig m e n t c o n te n ts . Each p o in t re p rese n ts th e average o f 1 0 —12 sam ples. A is o to n ic s o lu t io n c o n ta in in g 2% p o ta s s iu m fe r r ic y a n id e (A-A-A). Eachp o in t re p resen ts th e average o f 8 — 12 sam ples.


the oxygen molecule and the heme pigment molecule. The additional oxygen utilized by the heme pigments may be a function of a facilitated oxygen transport through the tissue by the heme pigments. Although not experimentally determined, it is assumed that myoglobin was predominant tissue heme pigment. Since myoglobin-facilitated oxygen flux likely involves a concurrent interaction with respiratory enzymes, it was of interest to determine the effect of the heme pigment (myoglobin) dilution on oxygen uptake in respiration-inhibited muscle samples. As shown in Figure 5, the rate of oxygen uptake in both control and soaked samples was nearly linear up to 30 hr as observed in the previous experiments. The activity of cytochrome c reductase was essentially zero as shown in Table 4. The curves then represent the oxygen uptake

resulting from interaction with heme pigments and dissolution into tissue fluids of low oxygen tension. The total predicted oxygen uptake attributable to heme pigment variations observed was calculated to be 3.9 /il of oxygen per cm2 after 30 hr. Experimentally, the difference was21.3 /il of oxygen per cm2. The resultant differential is similar to that noted in the samples with normal respiratory activity indicating that the heme pigments’ interaction with oxygen was apparently not affected by tissue respiration.

Oxygen uptake in respiration- inhibited postrigor muscle normal (reduced state) and chemically oxidized heme pigments

The time-course of oxygen uptake in respiration-inhibited muscle slices with normal heme pigment (reduced state) and

Table 3—Physical and chemical characteristics of postrigor bovine semimembranosus muscle perfused w ith isotonic solution, pH 5.6 and then either untreated or treated to lower the heme pigment concentration

Normal heme Loweredconcentration3 heme concentration*5

Characteristic In itia l Final In itia l Final Perfusate Soak

pH 5.4 5.4 5 2 5.3 5.7 5.7Water, % 75.9 - 79 2 - - -Heme pigment, mg/g Penetration depth

9.4 — 60 — 1.4 2.3

of 02, mm Specific activity (jumoles cyt. c. reduced per mg/min)

5.0 6.0

with added substrate 0.85 0.41 0.41 0.25 0.00 0.21No added substrate 0.35 0.30 0 26 0.22 0.00 0.10

3 A v e r a g e v a l u e s f o r 1 6 s a m p l e s

b A v e r a g e v a l u e s f o r 1 0 s a m p l e s

with chemically oxidized heme pigments is shown in Figure 6 . The rate of oxygen uptake in each sample remained constant up to 30 hr. Reflectance spectra of oxygen-exposed samples of control and ferricyanide-treated muscle slices are shown in Figure 7. Myoglobin in the control samples was predominantly in the oxygenated state while the treated samples showed a predominance of the oxidized form of the muscle pigment. The difference in oxygen uptake between the samples theoretically represents the effect of the exposed heme pigments on oxygen utilization. A total net difference of 10.5 /d of oxygen per cm2 was predicted between the control and oxidized heme pigment samples. Experimentally, the net difference after 30 hr was 5.1 /il/cm2 . The observed differential was partially attributed to the incomplete oxidation of heme pigment in the sample interior. The difference in chemically reactive heme pigment concentration was less than that predicted. These results deviate from the results of the previous experiment in which the effect of heme pigments on oxygen utilization by heme pigments was 8 times larger than predicted. The enhanced oxygen utilization by the heme pigments in the previous experiments, was possibly associated with modifications in the muscle characteristics, mediated by the pigment-dilution process. Each set of oxygen uptake data was statistically evaluated to determine variability between like samples and to determine whether significant differences existed between control and treated samples. In all cases, the intra- and interexperimental variability between like samples was insignificant. Significant differences in oxygen uptake were, however, observed between

Table 4—Physical and chemical characteristics o f postrigor bovine semimembranosus muscle perfused w ith antim ycin A isotonic solution, pH 5.6, and then either untreated or treated to lower the heme pigment concentration

Normal Reduced hemeheme concentration3 concentration^

Characteristic In itia l Final In itia l Final Perfusate Soak

pH 5.4 5.6 5.2 5.3 5.7 5.7Water, % 75.4 - 79.2 — - —Heme pigment, mg/g Penetration depth

9.2 — 5.9 - 1.2 2.4

of oxygen, mm Specific activity (/imole cyt. c. reduced per mg/min)

6.0 8.0

with added substrate 0.014 0.014 0.009 0.000 0.000 0.006No added substrate 0.014 0.010 0.000 0.000 0.000 0.002

3 A v e r a g e v a l u e s f o r 1 2 s a m p l e s

b A v e r a g e v a l u e s f o r 1 0 s a m p l e s

Fig. 7 —R e fle c ta n c e s p e c tra o f o x y g e n -e x p o s e d p o s tr ig o r b o v in e m u s c le sam ples p e r fu s e d w ith a n t im y c in A t ) a n d w ith a n t im y c in A c o n

ta in in g 2% p o ta s s iu m fe r r ic y a n id e (— ).

O X Y G E N U P T A K E I N P O S T R I G O R B O V I N E M U S C L E - 2 1

control and treated systems in those experiments in which the effect of tissue respiration and heme pigment concentration were examined.

DISCUSSION

THE EXPERIMENTAL RESULTS presented show that oxygen uptake in postrigor bovine semimembranosus muscle results from tissue respiration, reaction with heme pigments, and dissolution into tissue fluids.

Oxygen uptake as a result of lipid oxidation was not detected since the addition of antioxidant solutions to muscle sections did not influence oxygen uptake. Tappel et al. (1961), using the same antioxidant complex, reported a protective index of 1 1 0 for heme-catalyzed linoleic acid oxidation. Evidence of significant lipid oxidation in postrigor beef has been reported by Liu and Watts (1970), Greene (1969) and Kwoh (1971). Both heme and nonheme iron were implicated as catalysts of lipid oxidation. However, these studies were all performed using ground beef. Liu and Watts (1970) suggested that in an intact muscle system, lipid oxidation would be minimized since the interaction of polyunsaturated fatty acid with heme proteins would be limited by compartmentation and structural integrity. The failure to detect oxygenutilizing lipid oxidation processes in this present study perhaps confirms this suggestion since intact muscle systems were investigated.

Oxygen uptake by contaminating bacterial populations was also found insignificant. The maximum bacterial count observed was 20,000 cells/g. According to Greig and Hoogerheide (1941), one cell of Pseudomonas fluorescens requires 9.7x 1(T8 l±\ of oxygen per hour. A 20-g tissue sample containing 2 0 , 0 0 0 organisms per g would then utilize 1 . 1 /rl of oxygen after 30 hr. This quantity was insignificant since muscle samples utilized more than 1 . 0 Ail in 1 0 min.

The uptake of oxygen by heme pigment (myoglobin) oxygenation and tissue water saturation was linear up to 30 hr of measurement and accounted for approximately 50% of the total uptake after 30 hr. Valid information concerning the function of myoglobin in the intact tissue system was not obtained. Attempts to passively dilute the heme pigment concentration in intact samples apparently resulted in the elution of other soluble materials from the cellular fluid. Significant quantities of heme pigment and significant cytochrome c reductase activity were measured in the soak solution, as shown in Table 3, indicating that there may have been a partial disruption of cellular structure in the postrigor muscle, as suggested by Bendall (1972).

In a subsequent experiment, oxygen uptake in respiration-inhibited, heme- oxidized tissue was only slightly less than the uptake in a similar sample with normal (reduced state) heme pigments. The effect of the heme pigments on oxygen utilization was less than that predicted on the basis of a 1 : 1 molar interaction of oxygen with heme pigments.

Myoglobin facilitation of oxygen transport would be expected to increase the penetration depth of oxygen in proportion to the concentration of the heme pigments. This result was not observed. The depth of oxygen penetration was greater in the diluted heme pigment samples, as shown in Tables 3 and 4. This likely resulted from the observed reduction in the concentration of oxygenutilizing systems (heme pigment and cytochrome c reductase activity) thereby allowing more oxygen to penetrate the muscle due to diffusive processes.

Additional experimentation is required to more thoroughly examine the function of myoglobin in the intact and respiring muscle system.

The existence of active postrigor respiration was confirmed by this study. Oxygen consumption was significantly reduced in antimycin A perfused muscle sections. Antimycin A is thought to specifically bind to a nonheme component of the electron transport chain, in the region of cytochrome b, resulting in an efficient and selective inhibition of electron transfer (Kaniuga et al., 1969; Rieske and Gupta, 1972). Effective inhibition of electron transport by antimycin A was demonstrated biochemically by the negligible activity of cytochrome c reductase in the perfused muscle sections.

Active postmortem respiration has been reported by several other investigators including Urbin and Wilson (1961), Cheah and Cheah (1971), Ashmore et al.(1971) , Ashmore et al. (1972), Bendall(1972) and Bendall and Taylor (1972). In addition, Bendall (1972) observed active oxidative phosphorylation in postmortem muscle. Cheah and Cheah (1971) observed similar activity in mitochondrial isolates from ox neck-muscle after 144 hr postmortem, as long as the pH of the tissue was maintained at or above 5.5. The importance of postmortem pH in maintaining mitochondrial functionality was supported by Ashmore et al. (1971).

The decline in oxygen uptake rate observed in this study was noted in previous investigations (Brooks, 1929; Urbin and Wilson, 1961; Bendall, 1972; Bendall and Taylor, 1972). Urbin and Wilson (1961) observed a rate change at 15 hr and related the reduction in oxygen uptake rate to diffusion limitations. Uptake rates of approximately 1 . 0 jul/hr/g were reported up to 15 hr. After 15 hr, the rate decreased to 0.7 /ul/hr/g. Brooks (1929) proposed that postrigor muscle indefinitely

retained a residue of respiratory activity. When freshly excised muscle was exposed to air, a steady state was reached with the depth of oxygen penetration determined by relative rates of diffusion and oxygen uptake. Initial uptake rates of approximately 4.8 jUl/cc/hr were reported by Brooks. The rate of oxygen uptake decreased to 1 . 2 ftl/cc/hr in latter stages of measurement. In the present study, uptake rates of 4.4 ¿rl/cnr/hr were recorded in initial uptake stages. After 10 hr, the rate was reduced to 2.4 |Ul/cm2 /hr. The results indicated that the decline in oxygen uptake rate resulted primarily from a decay in the rate of respiratory oxygen consumption. Uptake from heme pigment oxygenation and tissue fluid saturation remained constant during the storage exposure periods. The reduction in respiration rate appeared to result from substrate depletions or enzyme degradations.

These results support the findings of Bendall and Taylor (1972) who showed that the main factor determining the postrigor oxygen consumption rate was attributed to a deterioration of mitochondrial function at the low postrigor pH. In the present experiments, the oxygen consumption rate decline appeared to result from substrate depletions, enzyme degradations, or a combination of these factors. Experimental results regarding the rate-limiting factors involved in postrigor respiration will be reported subsequently.

REFERENCESAndrews, M.M., Guthneck, B.T., McBride, B.H.

and Schweigert, B.S. 1952. Stability of certain respiratory and glycolytic enzyme systems in animal tissue. J. Biol. Chem. 194: 715.

Ashmore, C.R., Doerr, L., Foster, G. and Car- roll, F. 1971. Respiration of m itochondria isolated from dark-cutting beef. J. Anim. Sci. 33: 574.

Ashmore, C.R., Parker, W. and Doerr, L. 1972. Respiration of m itochondria isolated from dark-cutting beef: Postm ortem changes. J. Anim. Sci. 34: 46.

AOAC. 1970. “ Official Method of Analysis,” 11th ed., p. 392. Assoc. Offic. Agri. Chemists, Washington, D.C.

Atkinson, J.L., Follett, M.J. and Ratcliff, P.W.1969. Postm ortem changes in oxygen uptake and NAD content of lamb muscularis semimembranosus. Nature 223: 1372.

Bailey, J.L. 1967. “ Techniques in Protein Chemistry,” p. 341. Elsevier Publishing Co., New York.

Bendall, J.R . 1972. Consum ption of oxygen by the muscles of beef animals and related species and its effect on the colour of meat.I . Oxygen consum ption in pre-rigor muscle.J. Sci. Food Agric. 23: 61.

Bendall, J.R . and Taylor, A.A. 1972. Consumption of oxygen by the muscles of beef animals and related species. 2. Consum ption of oxygen by Post-rigor muscle. J. Sci. Food Agric. 23: 707.

Bodwell, C.E., Pearson, A.M. and Fennell, R.A. 1965. Postm ortem changes in muscle. 1. Chemical changes in beef. J. Food Sci. 30: 766.

Bodwell, C.E., Pearson, A.M. and Fennell, R.A. 1965a. Postm ortem changes in muscle. 3. Histochemical observations in beef and pork. J. Food Sci. 30: 944.

Brooks, J. 1929. Postm ortem form ation of m ethaemoglobin in red muscle. Biochem. J. 23: 1391.

2 8 - J O U R N A L O F F O O D S C I E N C E - V o / u m e 3 9 ( 1 9 7 4 )

Cheah, K.S. and Cheah, A.M. 1971. Postm ortem changes in structure and function of ox m itochondria. 1. Electron microscopic and polarographic investigation. J. Bioenergetics 2: 85.

Grant, N.W. 1955. The respiration enzymes of m eat. 1. Identification of active enzymes. Food Res. 20: 250.

Greene, B.E. 1969. Lipid oxidation and pigm ent changes in red beef. J. Food Sci. 34:10.

Greig, M.E. and Hoogerheide, J.C. 1941. The correlation of bacterial growth with oxygen consum ption. J. Bacteriol. 41: 549.

Hornsey, H.C. 1956. The colour of cooked cured pork. 1. Estim ation of the nitric oxide-haem pigments. J. Sci. Food Agric. 7: 534.

Kaniuga, Z., Bryle, J. and Slater, E.C. 1968. Mechanism of interaction between antimycin and the respiratory chain. FEBS Symposium 19: 285.

Kastenschmidt, L.L. 1970. The metabolism of

muscle as a food. In “ The Physiology and Biochemistry of Muscle as a Food ,” Ed. Briskey, E.J., Cassens, R.G. and Marsh, B.B., Vol 2, p. 735. The University of Wisconsin Press, Madison.

Kwoh, T.L. 1971. Catalysts of lipid peroxidation in meats. J. Am. Chem. Soc. 48: 523.

Liu, H. and Watts, B.M. 1970. Catalysis of lipid oxidation in meats. 3. Catalysis of oxidative rancidity in meats. J. Food Sci. 35: 596.

Newbold, R.P. and Scopes, R.K. 1971. Postm ortem glycolysis in ox skeletal muscle: Effect of adding nicotinamide-adenine dinucleotide to diluted mince preparation. J. Food Sci. 36: 215.

Potter, V.R. and Reif, A.E. 1952. Inhibition of an electron transport com ponent by anti- m ycin A. J. Biol. Chem. 194: 287.

Rahn, O. and Richardson, G.L. 1940. Oxygen demand and oxygen supply. J. Bacteriol. 41: 225.

Rieske, J.S. and Gupta, V.D. 1972. On the sigmoidal relationship between inhibition of

respiration and antim ycin tite r. FEBS Letters 20: 316.

Solberg, M. and Proctor, B.E. 1960. A technique utilizing 2,3,5-triphenyltetrazolium chloride for recognition of bacterial colonies in the presence of large num bers of food particles. Food Technol. 14: 343.

Tappel, A.L. 1960. Inhibition of electron transport by antim ycin A, alkylhydroxynaph- thoquinones and metal coordination com plexes. Biochem. Pharmacol. 3: 289.

Tappel, A.L., Brown, W.D., Zalkin, J. and Maier, V.P. 1961. Unsaturated lipid peroxidation by hem atin com pounds and its in hibition by vitamin E. J. Am. Oil Chem. Soc. 38: 5.

Urbin, M.C. and Wilson, G.D. 1961. The postmortem requirem ents of bovine tissue. J. Food Sci. 26: 314.

Ms received 6 /28 /73 ; revised 8 /24 /73 ; accepted 8/28/73.

Ft. L . W E S T ,' P. W. M O E L L E R , B. A . L IN K a n d W. A . L A N D M A N N

D e o a rtm e n ts o f A n im a l S cience a n d B io c h e m is try & B io p h ys ics

Texas A g r ic u ltu ra l E x p e r im e n t S ta tio n , Texas A & M U n iv e rs ity , C o llege S ta tio n , T X 7 7 8 4 3

LOSS OF CALCIUM ACCUM ULATING ABILITY IN THE SARCOPLASM IC RETICULUM FOLLOW ING DEGRADATION BY CATHEPSINS

INTRODUCTION

MANY of the quality attributes of meat are established during the postmortem period when muscle is transformed into meat. The postmortem changes have been shown to be associated with the state of muscle contraction at the onset of rigor mortis (Herring et al., 1965; Marsh and Leet, 1966). The loss of calcium- accumulating ability of the sarcoplasmic reticular membranes causes the onset of rigor mortis (Greaser et ah, 1969; Schmidt et ah, 1970). Goll et al. (1971) have theorized that the loss of the Ca2+- accumulating ability of the sarcoplasmic reticulum (and rigor onset) could have three possible underlying causes: ( 1 ) Uncoupling of the Ca2+-pump by proteolysis; (2) Postmortem pH decline; and(3) Postmortem loss of ATP. The above authors concluded that while decreased ATP concentration and pH decline definitely cause a loss of Ca2+-accumulating ability, a very limited proteolysis of sarcoplasmic reticular membranes could account for more of the observations (rigor onset at high pH and varying ATP concentrations) on postmortem loss of Ca2+-accumulating ability. A brief tryptic digestion was shown by the above authors to produce effects very similar to those produced by postmortem storage of muscle.

The present study was initiated to determine whether cathepsins could reduce the Ca2+-accumulating ability of the sarcoplasmic reticulum in postmortem muscle. Since cathepsins are intracellular proteolytic enzymes, these enzymes could very well be a factor in altering the Ca2+-accumulating ability of the sarcoplasmic reticulum postmortem with the subsequent onset of rigor mortis.

Bovine spleen cathepsins have been purified and characterized to a greater extent than bovine muscle cathepsins. The cathepsins of the spleen can be more readily extracted and are more abundant than those from muscle. Due to the difficulty in obtaining cathepsins from bo-

1 Present address: Dept, of Animal Science, University of Florida, Gainesville, FL 32601

vine muscle in a purified form and since the activities of the cathepsins from both sources are similar, spleen cathepsins were used in this study.

EXPERIMENTALT H E S T E R N O M A N D IB U L A R IS m u s c le w a s r e m o v e d f r o m o n e s id e o f a b o v in e c a r c a s s i m m e d ia t e ly a f t e r s l a u g h te r . T h e m u s c l e w a s t r i m m e d o f a l l e x t e r n a l f a t a n d c o n n e c t iv e t i s s u e a n d p a c k e d in ic e f o r im m e d ia t e e x t r a c t i o n a n d p r e p a r a t i o n o f t h e s a r c o p la s m ic r e t i c u lu m b y a m o d i f i c a t i o n o f t h e m e th o d s o u t l in e d b y G re a s e r e t a l. ( 1 9 6 9 ) . T h e m u s c le t i s s u e w a s h o m o g e n iz e d in 4 v o l o f ic e c o ld 0 .1 M K C1, 5 m M h i s t i d in e b u f f e r ( p H 7 .2 ) in a W a r in g B le n d o r in 6 b u r s t s o f 1 5 s e c e a c h . T h e h o m o g e n a te w a s c e n t r i f u g e d f o r 2 0 m in a t1 ,0 0 0 X G . A f t e r f i l t r a t i o n th r o u g h c i e e s e c l o t h t o r e m o v e f lo a t in g l i p id , t h e s u p e r n a t a n t w a s c e n t r i f i g e d f o r 2 0 m in a t 8 ,0 0 0 X G to r e m o v e m i t o c h o n d r i a a n d s m a l l m y o f i b r i l l a r c o m p o n e n ts . T h e s u p e r n a t a n t w a s c e n t r i f u g e d f o r 1 h r a t 3 0 ,0 0 0 X G to o b t a i n a s a r c o p la s m ic r e t i c u lu m p e l le t . T h e p r e c i p i t a t e w a s r e s u s p e n d e d in 3 m l o f b u f f e r w i th a g la ss h o m o g e n iz e r . T h e p r o t e i n c o n c e n t r a t i o n o f t h e s a r c o p la s m ic r e t i c u lu m w a s d e t e r m in e d b y t h e b i u r e t p r o c e d u r e ( G o m a l l e t a l . , 1 9 4 9 ) u s in g b o v in e s e r u m a l b u m in in 5 m M h i s t i d in e b u f f e r a s t h e s t a n d a r d .

C a t h e p t i c e n z y m e s w e re p r e p a r e c f r o m b o v in e s p le e n b y t h e m e t h o d o f O t t o ( 1 9 7 1 ) . T h e 4 0 - 7 0 % s a t u r a t e d a m m o n iu m s u l f a te p r e c ip i t a t e w a s d i s s o lv e d , d ia ly z e d a n d s e p a r a t e d c n a S e p h a d e x G - 1 0 0 c o lu m n in 0 .0 5 M c i t r a t e b u f f e r a t p H 5 .0 . T h e p e a k c o n ta in in g c a t h e p s in B1 a c t iv i ty w a s r e c h r o m a to g r a p h e d o n a S e p h a d e x G -7 5 c o lu m n u n d e r th e s a m e c o n d i t i o n s . T h e c r u d e c a t h e p s in f r a c t i o n o b t a i n e d b y d ia l y s is o f t h e 4 0 - 7 0 % s a t u r a t e d a m m o n iu m s u lf a t e p r e c ip i t a t e w a s a ls o e x a m in e d , a n d is d e s ig n a te d a s c a t h e p s in 4 0 - 7 0 % f r a c t io n .

T h e 4 0 - 7 0 % f r a c t io n h y d r o ly z e d th e f o l lo w in g s u b s t r a t e s : 2 3 .9 M m oles c a r b o b e n z c x y - g l u t a m y l t y r o s i n e / m g / h r a t p H 5 .0 ( c a th e p s in A ) ; 3 3 .9 g im o le s b e n z o y la r g in in e - p - n i t r o a n i - l i d e / m g / h r a t p H 6 .5 ( c a th e p s in B l ) ; 1 8 .6 M m oles b e n z o y l a r g in in e a m i d e / m g / h r a t p H 5 .0 ( c a th e p s in B l a n d B 2 ) ; 5 7 .9 M m oles g ly c y lp h e n - y la la n in e a m i d e / m g / h r a t p H 5 .0 ( c a th e p s in C ). T h e i s o la te d c a t h e p s in B l c le a v e d 3 8 4 .6 /c m a le s b e n z o y l a r g in in e - p - n i t r o a n i l i d e /m g /h r a t p H 5 .5 . A c t iv i t ie s r e l a t e d t o c a t h e p s in s A , B 2 a n d C c o u ld n o t b e d e t e c t e d in t h e c a t h e p s in B l p e a k . T r y p s in , 2 x c r y s t a l l i z e d , p u r c h a s e d f r o m S ig m a C h e m ic a l C o ., S t . L o u i s , M o ., w a s s p e c i f i e d t o c o n ta in a p p r o x i m a t e l y 1 0 ,0 0 0 u n i t s o f b e n z o y l a r g in in e e t h y l e s t e r a c t i v i t y p e r m g .

T h e t r e a t m e n t s o f s a r c o p la s m ic r e t i c u lu m

u t i l i z e d in t h i s s t u d y w e r e a s f o l l o w s : (1 ) T r y p s in d ig e s t io n f o r 3 m in a t 2 5 ° C ; ( 2 ) C a th e p s i n B l d ig e s t io n f o r 2 h r a t 2 5 ° C ; (3 ) C a t h e p s in 4 0 - 7 0 % f r a c t i o n d ig e s t io n f o r 2 h r a t 2 5 ° C ; ( 4 ) C a t h e p s in B l d ig e s t io n f o r 1 8 h r a t 2 ° C ; a n d (5 ) C a th e p s in 4 0 —7 0 % f r a c t io n d ig e s t i o n f o r 1 8 h r a t 2 ° C . A ll d ig e s t io n s w e re c o n d u c t e d a t a n e n z y m e c o n c e n t r a t i o n w h ic h w a s o n e - t e n t h t h a t o f t h e c o n c e n t r a t i o n o f s a r c o p la s m ic r e t i c u l u m . C o n t r o l s p r e p a r e d w i t h o u t e n z y m e w e r e i n c u b a t e d f o r 2 h r a t 2 5 ° C a n d f o r 1 8 h r a t 2 ° C .

Sarcoplasm ic re ticu lu m p repara tio n s were digested w ith ca thepsins or trypsin a t pH 7 .2 . T he in cu b a tio n m edium consisted o f 0 .1 2 m g/m l sarcoplasm ic re ticu lu m , 1 2 Mg/ml en zym e and 1 2 .5 Mmoles/ml (3-m ercaptoethyl- am ine. In th e trypsin d igestion (3-m ercaptoeth- y lam ine was rep laced w ith an equal volum e o f w ater. A fter th e in cu b a tio n periods, enzym atic d igestion was m in im ized by p lacing th e tubes in ice an d im m edia te ly determ in in g th e calcium - accum ulating ab ility .

F o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f th e C a 2 + - a c c u m u la t in g a b i l i t y , t h e p r o c e d u r e s e t f o r t h b y G re a s e r e t a l . ( 1 9 6 9 ) w a s u t i l i z e d . T h e r e a c t io n m i x t u r e c o n s i s t e d o f 0 .1 M K C 1, 5 m M h i s t i d in e , 5 m M A T P , 5 m M p o ta s s iu m o x a la t e , 5 m M M g C l2 a n d 0 .2 m M C a C l2 w i th 4 S C a. T h e f in a l v o lu m e o f 5 m l c o n t a i n e d a t o t a l o f 0 .1 8 m g o f s a r c o p la s m ic r e t i c u l u m p r o t e i n . T h e in c u b a t i o n o f 2 5 ° C w a s t e r m i n a t e d a f t e r 15 m in b y f i l t r a t i o n t h r o u g h a 0.45m M ill ip o re ® f i l t e r . T h e f i l t r a t e w a s a d d e d t o 1 0 m l o f 6 m M E D T A . 2 m l o f th i s s o lu t io n w e re t r a n s f e r r e d to a s c in t i l l a t i o n s o lu t io n . T h e a m o u n t o f 4 5 C a n o t b o u n d w a s d e t e r m in e d b y l i q u id s c in t i l l a t i o n . A n a l i q u o t o f t h e 2 h r 2 5 ° C c o n t r o l w a s s to p p e d i m m e d ia t e ly a f t e r 4 S C a a d d i t i o n f o r u s e a s a b la n k .

RESULTS & DISCUSSION

THE STERNOMANDIBULARIS muscle was found to yield approximately 0.24% crude sarcoplasmic reticular membranes on a wet weight basis. Although some workers (Greaser et al., 1969; Eason,1969) have used sucrose density gradients for the final separation of the sarcoplasmic reticulum, negative staining and electron microscopy (Fig. 1) revealed that the extraction procedure utilized in the present study was sufficient since mitochondrial and myofibrillar contamination was minimal.

The effects of enzyme and temperature treatments on the calcium-accumulating ability of the sarcoplasmic reticulum

Volume 39 (19741-JOURNAL OF FOOD SCIENCE-29


Fig . 1—E le c tro n m ic ro g ra p h o f s a rco p la sm ic re t ic u lu m p re p a ra tio n s n e g a tiv e ly s ta in e d w ith 1% p o ta s s iu m p h o s p h o tu n g s ta te , p H 7 .0 17 0 ,0 0 0 X ) .

Table 1—Effect o f enzyme and temperature treatments on the calcium-accumulation ab ility o f the sarcoplasmic reticulum (SR)

Treatment3

mM Ca2 + bound/mg

protein % Lossb

Trypsin digestion (3 min (s> 25°C) 1.02 522 hr @ 25°C

SR Control 2.10 0SR + B1 fraction 0.96 55SR + 40—70 cathepsin fraction 0.59 72

18 hr @ 2°CSR Control 0.57 73SR + B1 fraction 0.00 100cSR + 40—70 cathepsin fraction 0.00 100c

3 R e a c t i o n c o n d i t i o n s o u t l i n e d i n E x p e r i m e n t a l . V a l u e s a r e m e a n s o f

d u p l i c a t e s .

b C o n t r o l f o r 2 h r a t 2 5 ° C t r e a t m e n t u s e d a s 1 0 0 % a c c u m u l a t i o n . c C o u n t s w e r e h i g h e r t h a n b l a n k .

are presented in Table 1. The control for the 2 hr at 25°C treatment was utilized to indicate 100% level of binding. This level of 2.1 /rmoles Ca2+ bound/mg protein can be compared to a value of 2 . 2 /umoles Ca2+ bound/mg protein at 0 hr postmortem as reported by Goll et al. (1971). Tryptic digestion resulted in a 52% reduction in Ca2+-accumulating ability. Ca- thepsin Bl, which has trypsin-like activity, resulted in a 55% reduction at 25°C

for 2 hr comparable to the effect of trypsin at 3 min. The 40—70% cathepsin fraction caused an even greater loss (72%) of Ca2+-accumulating ability at 25°C for 2 hr. This may be the result of the synergistic action of both exo- and endopepti- dases present in the partially purified 40—70% fraction.

Incubation of the control at 2°C for 18 hr resulted in a 73% loss of Ca2+- accumulating ability. The enzyme treat

ments at this time and temperature showed a complete loss ( 1 0 0 %) of the accumulating ability. Actually, the counts for these treatments were higher than the blank (2 hr, 25°C control), indicating that although the reaction was stopped immediately in the blank, some Ca2+ had been bound.

Since the onset of rigor has been shown to be triggered by the loss of the Ca2+-sequestering ability of the sarcoplasmic reticulum (Greaser et al., 1969; Schmidt et al., 1970), catheptic enzymes may be responsible for the loss of Ca2+- accumulation of the sarcoplasmic reticulum even in the presence of ATP and at an elevated pH. Thus, although Ca2+- accumulating ability may also be lost by ATP depletion and/or pH drop, proteolytic activity may accelerate this process (Goll et al.. 1971).

Treatment of the sarcoplasmic reticulum preparation with bovine spleen cathepsins in the presence of a sulfhydryl activator indicates that cathepsins could, under optimal conditions, play a role in the onset of rigor. One must recognize some limitations. Intact sarcoplasmic reticulum may not be as readily attacked immediately postmortem. The activation of cathepsins by naturally occurring sulfhydryl reducing compounds such as glutathione may be limited. Still it is probable that the anaerobic conditions and reduced pH in postmortem muscle may maintain the proteolytic activity of cathepsins over extended periods of time. The cathepsins occurring in muscle have at least the activities corresponding to the spleen cathepsins, regardless of the compositional and structural differences (Otto, 1971). Nonetheless, it remains to be demonstrated whether muscle cathepsins can also affect the onset of rigor.

Since it is not known whether calcium is transported into the lumen of the reticulum vesicles and deposited as free ions or bound to some structure in the lumen (Ebashi and Endo, 1968), the exact nature of the catheptic effect cannot be stated. It can be postulated that cathepsins degrade the membrane (and/or lumen structures) thereby releasing the calcium into the sarcoplasm. This reaction could presumably take place prior to ATP depletion or postmortem pH decline. The cathepsins, therefore, may play a significant role in the early stages of the rigor process.

REFERENCESEason, B.A. 1969. Purification and properties

of skeletal muscle microsomes. Ph.D. thesis, Iowa State University, Ames, Iowa.

Ebashi, S. and Endo, M. 1968. Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol. 18: 123.

Goll, D.E., Strom er, M.H., Robson, R.M., Temple, J., Eason, B.A. and Busch, W.A.1971. Tryptic digestion of muscle com ponents simulates many of the changes caused by postm ortem storage. J. Anim. Sci. 33: 963.

LOSS OF Ca ACCUMULATING A B IL IT Y -3 \

Gornall, A.G., Bardawill, C.J. and David, M.M. 1949. Determ ination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177: 751.

Greaser, M.L., Cassens, R.G., Briskey, E.J. and Hoekstra, W.G. 1969. Postm ortem changes in subcellular fractions from norm al and pale, soft, exudative porcine muscle. 1. Calcium accumulation and adenosine triphosphatase activities. J. Food Sci. 34: 120.

Herring, H.K., Cassens, R.G. and Briskey, E.J. 1965. Further studies on bovine muscle

tenderness as influenced by carcass position, sarcomere length and fiber diam eter. J. Food Sci. 30: 1049.

Marsh, B.B. and Leet, N.G. 1966. Studies in m eat tenderness. 3. The effects of cold shortening on tenderness. J. Food Sci. 31: 450.

O tto, K. 1971. Cathepsins B1 ana B2. In “ Tissue Proteinases,” Ed. Barret, A.J. and Dingle, J.T ., p. 1. American Elsevier Publishing Co., Inc., New York, N.Y.

Schmidt, G.R., Cassens, R.G. and Briskey, E.J.

1970. Relationship of calcium uptake by the sarcoplasmic reticulum to tension developm ent and rigor m ortis in striated muscle. J. Food Sci. 35: 574.

Ms received 8 /8 /73; revised 10/2 /73; accepted 10/8/73.

Technical Article No. TA10675, Texas Agricultural Experim ent Station, College Station, Texas.

This work was supported in part by funds from the King Ranch Chair, Project S-1550.

T. Ft. D U T S O N , ' A . M . P E A R S O N a n d R . A . M E R K E L 4

D e p t, o f F o o d S c ie n c e & H u m a n N u t r i t i o n

a n d G. C. S P IN K

C o lle g e o f O s te o p a th ic M e d ic in e , M ic h ig a n S ta te U n iv e r s i t y , E a s t L a n s in g , M l 4 8 8 2 4

U LTRA STRU CTU RA L POSTMORTEM CHANGES IN NORMAL AND LOW QUALITY PORCINE MUSCLE FIBERS

INTRODUCTION

SEVERAL STUDIES have been concerned with the biochemical and histochemical differences of fiber types in pig skeletal muscle (Beecher et ah, 1965, 1968; Moody and Cassens, 1968; Cooper et ah, 1969; Schmidt et ah, 1970). These studies have shown that pig muscle is composed of red fibers (those high in mitochondrial oxidative enzymes), white fibers (those high in glycolytic enzymes) and intermediate fibers (those having properties between the red and white fibers). Cassens and Cooper (1971) have reviewed the properties of different fiber types and found the biochemical and histochemical characteristics of porcine muscle to be similar to that of other mammalian muscles. Gauthier (1970) stated that different fiber types (red, white and intermediate) can be distinguished in rat skeletal muscle on the basis of ultrastructural differences. Grinyer and George (1969) have also outlined the ultrastructural differences between the small red fibers and large white fibers in pigeon pectoralis muscle. Gauthier (1970) has pointed out that failure to recognize or understand ultrastructural differences of the various fiber types has often resulted in research workers attributing the cytological characteristics of different fiber types to experimental or pathological conditions.

Henderson et al. (1970) and Cassens et al. (1963) have studied some of the postmortem ultrastructural changes that take place in intact porcine muscle. However, they did not discuss the differences in ultrastructure or the postmortem changes in different fiber types. Thus, this investigation was carried out to determine if ultrastructural differences could be detected for various fiber types and to determine the nature of postmortem changes occurring in porcine muscle.

MATERIALS & METHODS

F IV E N O R M A L and five low -quality pigs were selected from each o f the Hampshire and Y o rk shire breeds on the basis o f a com bin ation o f 4 5

1 Present Address: Dept, of Animal Science, Texas A&M University, College Station, TX 77843

min postm ortem pH values (K o ch e t a l., 1 9 7 0 ) and percent transm ission values o f a sarcoplasm ic protein ex tract at 24 hr postm ortem as ou tlined by Hart ( 1 9 6 2 ) . T he 10 low -quality pigs had m ean 4 5 min m uscle pH values o f 6 .0 5 ± 0 .4 3 and average percent transm ission values o f5 7 .5 + 1 2 .1 5 com pared to corresponding values o f 6 .7 2 ± 0 .0 9 and 1 0 .6 ± 3 .8 3 for the 10 normal anim als. Sam ples from the 20 pigs selected (10 norm al and 10 low -quality) were removed from the second and third lum bar vertebrae area o f the right longissimus m uscle at 15 min postm ortem . Since K och et al. ( 1 9 7 0 ) dem onstrated that drastic postm ortem changes o c curred in m uscle sampled im m ediately fo llow ing death, the initial sam ple was rem oved at 15 min follow ing death. A pproxim ately 4 5 min after death, all carcasses were placed in a 2 °C chill room and held until a fter the 24 hr postm ortem sam ples were rem oved from the le ft longissimus m uscle in the region o f the 1 3th thoracic vertebra.

All samples were cut in to strips 3 mm in cross section by I cm in length before subsequent fixation . F ixatio n was im m ediately carried out in 1 .25% glutaraldehyde (Fisher Sc ien tific C o., P ittsburgh, Pa.; Biological grade 50% ) so lution buffered w ith 0 .0 0 7 M NaH2 P 0 4 and 0 .0 4 1 M N a, H P 0 4 . T he fixing solution had a pH o f 7 .4 and contained 0 .0 4 3 M NaCl. Sam ples were then washed with two changes o f a pH 7 .4 solution containing 0 .01 3M NaH2 P O „, 0 .0 8 1 M Na2 H P 0 4 and 0 .0 8 6 M NaCl. A fter washing, the samples were postfixed in 1% osmium tetroxid e for 1 hr as described by S jostrand (1 9 6 7 ) and dehydrated in ethan ol. F ixatio n and dehydration were accom plished at room tem perature. A fter dehydration, the sam ples were washed in two changes o f propylene oxid e, em bedded in Epon 8 1 2 (L u ft , 1 9 6 1 ) and sectioned on an L K B 4 8 0 1 A ultram icrotom e. Sectio n s were m ounted on uncoated copper grids and stained in uranyl acetate (S jostran d , 1 9 6 7 ) and lead citrate (R ey n old s, 1 9 6 3 ) . E lectron m icrographs were obtained with a Philips EM 3 0 0 transm ission electron m icroscope operated at an accelerating voltage o f 8 0 kv and recorded on K odak 70-rr m , fine-grain positive film .

F iber types were identified in porcine muscle according to num ber o f m itochondria and relative width and density o f the Z line. Tw o distinct fiber types were noted in this study and were characterized as red fibers (th ose having a large num ber o f subsarcolem m al and interm yo- fibrillar m itochon dria and having wide d istinct Z lines) and w hite fibers (th ose having relatively few m itochondria and very thin , less dense Z lines). Although fibers interm ediate betw een these two fiber types were observed, there was considerable variation in their ultrastructure. Three fiber types in porcine longissimus m uscle (a fast w hite m uscle) were described by B eecher et al. ( 1 9 6 5 ) , which agrees with D ubow itz

(1 9 7 0 ) , w ho found three fiber types in fast w hite m uscles.

T he num ber o f m itochon dria was used in this study to id en tify fiber types since m ito chondrial staining by su ccin ic dehydrogenase histochem istry has been used by o th er au th ors (Stein and Padykula, 1 9 6 2 ; N ystrom , 1 9 6 8 ; D ubow itz, 1 9 7 0 ) to identify fiber types in light m icroscopy and also because m itochon d rial num bers was used by G authier ( 1 9 6 9 , 1 9 7 0 ) to iden tify fiber types o f rat m uscle at an ultrastructural level. W idth and density o f the Z line were also used by G authier ( 1 9 6 9 , 1 9 7 0 ) as an indicator o f fiber types.


SINCE DIFFERENCES in the ultrastructure of fiber types were not found between the two breeds, they were pooled to include 1 0 normal and 1 0 low-quality pigs. Figure 1 shows a longitudinal section through adjacent red and white fibers. These fibers are typical of their respective types at 15 min postmortem as shown by the differences in the width of their Z lines and in the arrangement, structure and number of mitochondria. The characteristic differences shown between the two fiber types were used for classification purposes in this study and will be discussed later herein. Although fibers that are intermediate between the typical red and white types were frequently observed, only red and white fibers are considered in this study.Normal muscle—15 min postmortem

Myofibrils. Myofibrils from a typical red fiber are presented in Figure 2. The red fibers have very wide, dense Z lines of approximately 1200X in width. Figure 3 shows an electron micrograph of myofibrils from a white muscle fiber, which is characterized by narrow, less dense Z lines of approximately 625X in width. Gauthier (1970) has reported the Z line width of rat diaphragm muscle to be 634A for red fibers and 339^ for white fibers. It is apparent that there are large differences in the width of Z lines between myofibrils of red and white fibers in both rat diaphragm muscle and porcine longissimus muscle. Although actual measurements of Z line width are quite different between the rat diaphragm and pig longissimus muscle, it is not clear from this study whether the variation is due to species or to muscle differences.

32- J O U R N A L O F F O O D S C I E N C E - V o l u m e 3 9 ( 1 9 7 4 )

U L T R A S T R U C T U R E O F F IB E R T Y P E S - 3 3

Although sarcomere length was not measured in this study, some variation existed which appeared to reflect differences in the amount of contraction within different fibers. No attempt was made to control the degree of post-mortem contraction. However, the state of contraction was not different between fiber types, and did not appear to be related to postmortem time of sampling.

Mitochondria. Electron micrographs of representative mitochondria from red and white fibers are presented in Figures 4 and 5, respectively. The mitochondria from the red fibers (Fig. 4) were very large, showed very closely packed cristae and were dense in appearance. Red fibers contained large numbers of mitochondria, which were found in groups just beneath the sarcolemma, in interfibrillar rows or

in pairs at the Z line. The mitochondria from white fibers (Fig. 5) were less dense in appearance and had less closely packed cristae than those from red fibers. The mitochondria in white fibers were also smaller in size than those found in red fibers. Most of the mitochondria in white fibers were located between the myofibrils at the Z line with a limited number observed near the sarcolemma. Mitochon-

F ig . 1 - E le c t r o n m ic r o g r a p h s h o w in g a d ja c e n t r e d a n d w h ite m u s c le

f ib e rs f r o m n o r m a l m u s c le a t 1 5 m in p o s tm o r te m . T h e c h a r a c te r is t ic

u l t r a s t r u c t u r e o f e a ch f ib e r t y p e is e v id e n t . F tF = r e d f ib e r ; W F = w h ite

f ib e r ; L = l i p i d d r o p le ts ; Z = Z l i n e ; M = m i t o c h o n d r ia ; S = s a rc o le m m a .

10,900X.

F ig . 2 —E le c t r o n m ic r o g r a p h s h o w in g c h a r a c te r is t ic m y o f i b r i l s f r o m a

n o r m a l r e d m u s c le f ib e r a t 1 5 m in p o s t m o r t e m . A = A b a n d ; 1 = 1 b a n d ;

Z = Z lin e . 1 6 ,3 0 0 X .

F ig . 3 —E le c t r o n m ic r o g r a p h s h o w in g c h a r a c te r is t ic m y o f i b r i ls f r o m a

n o r m a l w h ite m u s c le f ib e r a t 1 5 m in p o s tm o r te m . A = A b a n d ; 1 = 1

b a n d ; Z = Z l in e . 1 6 .3 0 0 X .

F ig . 4 —E le c t r o n m ic r o g r a p h o f c h a r a c te r is t ic m i t o c h o n d r ia o f n o r m a l

r e d f ib e r s a t 15 m in p o s tm o r te m . M = m it o c h o n d r ia . 2 9 , 3 0 0 X .

3 4 - J O U R N A L O F F O O D S C I E N C E - V o l u m e 3 9 (1 9 7 4 )

dria were completely absent in many sections of white muscle fibers. Muir (1970) has also reported a very low mitochondrial content for white fibers from pig muscle as compared to red fibers. The ultrastructural differences in mitochondria between fiber types of the pig appear to be essentially the same as those observed in rat muscle by Gauthier (1969, 1970). However, less mitochondria were found in pig muscle.

Sarcoplasmic reticulum and transverse tubular system. The sarcoplasmic reticulum (SR) and transverse tubular system (TTS) in pig muscle appear to be similar to that of other mammalian species (Porter and Palade, 1957; Peachey, 1970). Differences do exist, however, between red and white type muscle fibers. A longitudinal section showing characteristic SR and TTS from red fibers is presented in Figure 6 . Figure 7 is a longitudinal sec

tion through a white muscle fiber showing the SR and TTS between two myofibrils. A comparison of Figures 6 and 7 shows that the SR of white fibers has larger more open longitudinal tubules, and in the region of the H zone, contains more open sac-like structures with the appearance of some fenestrations. The longitudinal tubules of red fibers are narrower and more tortuous in the area of the H zone with fewer connections or

F ig . 5 —E le c t r o n m ic r o g r a p h o f c h a r a c te r is t ic m it o c h o n d r ia o f n o r m a l

w h ite f ib e r s a t 1 5 m in p o s tm o r te m . M = m it o c h o n d r ia . 2 9 . 3 0 0 X

F ig . 6 —E le c t r o n m ic r o g r a p h s h o w in g th e s a rc o p la s m ic r e t i c u lu m a n d

t ra n s v e rs e t u b u la r s y s te m c h a r a c te r is t ic o f n o r m a l r e d f ib e r s a t 1 5 m in

p o s tm o r te m . L T = lo n g i t u d in a l t u b u le s ; T C = te r m in a l c is te r n a e ; T T =

tra n s v e rs e tu b u le s ; G = g ly c o g e n g r a n u le s ; A = A b a n d ; / = / b a n d ; Z = Z

l in e . 4 I . 2 0 0 X .

F ig . 7 —E le c t r o n m ic r o g r a p h s h o w in g s a rc o p la s m ic r e t i c u lu m a n d t ra n s

ve rse t u b u la r s y s te m c h a r a c te r is t ic o f n o r m a l w h ite m u s c le f ib e rs a t 1 5

m in p o s t m o r t e m . L T — lo n g i t u d in a l t u b u le s ; T T = t ra n s v e rs e tu b u le s ;

T C = t e r m in a l c is te r n a e ; F = fe n e s t ra t io n s ; G = g ly c o g e n g ra n u le s ; I = /

b a n d ; A = A b a n d ; Z = Z l in e . 4 1 , 2 0 0 X .

F ig . 8 - E l e c t r o n m ic r o g r a p h s h o w in g a r e p r e s e n ta t iv e r e d f ib e r f r o m

n o r m a l m u s c le a t 2 4 h r p o s tm o r te m . M = m i t o c h o n d r ia ; Z = Z U n e ; V =

v e s ic u la r s t ru c tu r e s . 1 2 ,0 0 0 X .

U L T R A S T R U C T U R E O F F IBE R T Y P E S - 3 5

fenestrations. Terminal cisternae of red fibers also appear more dense across the entire structure, whereas, in white fibers the dense area is primarily next to the transverse tubule. Gauthier (1969, 1970) has reported that the SR of red fibers from rat muscle was in the form of tortuous tubules in the H zone and that the SR from white fibers appeared more as flattened sacs.

Normal muscle—24 hr postmortemMyofibrils. Electron micrographs of

representative red and white fibers at 24

hr postmortem are presented in Figures 8

and 9, respectively. The width of the Z lines is essentially the same for each fiber type as at 15 min postmortem; however, some Z line material was absent in white fibers at 24 hr postmortem. The arrows in Figure 9 mark areas where Z lines are disrupted in white fibers. The Z lines from red fibers at 24 hr postmortem showed very little or no disruption. Henderson et al. (1970) found very little disruption in the ultrastructure of porcine muscle stored at 2°C for 24 hr, but did find some disruption of the Z line. After storage at

25 or 37°C for 24 hr postmortem, however, they found more extensive disruption of the Z line. Cassens et al. (1963) found no evidence of alteration in the myofibrils from normal pig muscle during 24 hr postmortem holding, but Greaser et al. (1969a) showed a loss of some Z line material in isolated myofibrillar fractions of normal pig muscle at 24 hr postmortem. Thus, results of this study showing that the Z lines are more disrupted in white than in red fibers of porcine muscle at 24 hr postmortem helps to explain the discrepancies reported in previous re-

F ig . 9 —E le c t r o n m ic r o g r a p h o f a re p re s e n ta t iv e w h it e m u s c le f ib e r f r o m

n o r m a l m u s c le a t 2 4 h r p o s tm o r te m . M = m it o c h o n d r ia ; Z = Z l in e ;

A r r o w s m a rk p o in t s o f Z l in e d is r u p t io n . 1 2 .0 0 0 X .

F ig . 1 0 —E le c t r o n m ic r o g r a p h s h o w in g c h a r a c te r is t ic m y o f i b r i ls o f a r e d

f ib e r f r o m lo w - q u a l i t y m u s c le a t 2 4 h r p o s tm o r te m . Z = Z l i n e ; A = A

b a n d ; 1 = 1 b a n d . 1 3 .6 0 0 X .

F ig . 11 —E le c t r o n m ic r o g r a p h s h o w in g c h a r a c te r is t ic m y o f i b r i ls o f a

w h ite f ib e r f r o m lo w - q u a l i t y m u s c le a t 2 4 h r p o s tm o r te m . Z = Z l in e ; V

= v e s ic u la r r e m n a n ts o f s a rc o p la s m ic r e t ic u lu m . A r r o w s in d ic a t e a reas

s h o w in g Z l in e d is r u p t io n . 1 3 .6 0 0 X .

F ig . 1 2 —E le c t r o n m ic r o g r a p h o f a c h a r a c te r is t ic w h ite f ib e r f r o m lo w -

q u a l i t y m u s c le a t 1 5 m in p o s tm o r te m . M = m i t o c h o n d r ia ; Z = Z lin e .

1 4 .0 0 0 X .

3 6 - J O U R N A L OF F O O D S C / E N C E - V o l u m e 3 9 (1 9 7 4 )

F ig . 1 3 - E le c t r o n m ic r o g r a p h o f a c h a r a c te r is t ic r e d f ib e r f r o m lo w -

q u a l i t y m u s c le a t 1 5 m in p o s tm o r te m . Z = Z l in e ; M = m it o c h o n d r ia .

1 4 ,0 0 0 X .

search and suggests that the differences were due to the observations being made on different fiber types.

Mitochondria. The appearance of mitochondria from normal red and white porcine muscle at 24 hr postmortem are shown in Figures 8 and 9, respectively. The cristae of red fiber mitochondria were still very distinct and closely packed at 24 hr postmortem, but there was less dense material between the cristae. White fiber mitochondria seem to be more disrupted at 24 hr postmortem than those of red fibers, and although cristae are evident, they are not clearly distinct in mitochondria from white fibers. Most of the dense background material is absent from white fiber mitochondria at 24 hr postmortem and some of the white fiber mitochondria appear as open vesicles.

Sarcoplasmic reticulum and transverse tubular system. The appearance of SR and TTS of red and white fibers from 24 hr postmortem muscle can be seen in Figures 8 and 9, respectively. By 24 hr postmortem, no triads or transverse tubules are apparent in either of the fiber types. The SR is no longer recognizable, and all that remains are vesicular type structures. In a small number of red fibers, structures that appeared similar to longitudinal tubules of the SR are found; but in most of the red fibers no SR was observed. Although Greaser et al. (1969a, b) reported no marked change in the isolated heavy sarcoplasmic reticular fraction of muscle at 24 hr postmortem, they did show that a marked decrease in the calcium accumulating ability of the SR had occurred. The marked structural alterations of the SR in intact muscle found in this study may be a factor in the lowered calcium accumulating ability of SR as reported by Greaser at al. (1969a, b).Low-quality muscle

Myofibrils. No differences in the myofibrils were found between low-quality and normal muscle for any of the fiber types at 15 min postmortem. However, at 24 hr postmortem there was more disruption in the myofibrils of low-quality muscle, particularly for white type fibers as can be seen by comparing Figures 10 and11. A comparison of these two figures shows there is little change in the red fibers, but extensive Z line disruption and vesicular remnants of the SR are apparent in the white fibers at 24 hr postmortem.

Mitochondria, sarcoplasmic reticulum and transverse tubular system. Mitochondria from low-quality red fibers at 15 min postmortem showed very little difference from mitochondria of normal red fibers at the same postmortem times. However, mitochondria from low-quality white muscle fibers had an open-structured appearance and show considerable disruption at 15 min postmortem, which is shown in Figure 12. Comparison of Fig

ure 12 with Figures 5 and 9 shows that mitochondria from low-quality white muscle fibers at 15 min postmortem corresponded closely to normal white fibers at 24 hr postmortem. There was considerable variation in the appearance of mitochondria from low-quality muscle at 15 min postmortem, which may reflect a difference in the rate of postmortem change between fibers of the same type. The variation is further substantiated by the observation that the white fibers from low- quality muscle having the most disrupted mitochondria at 1 5 min showed extensive disruption of the SR and had very few glycogen granules, while low-quality white fibers with relatively intact mitochondria showed less disruption of the SR and had larger amounts of glycogen. These observations also indicate that disruption of the ultrastructure of these three components (mitochondria, SR and glycogen) occurs simultaneously in the same fibers.

Figure 13 shows the ultrastructural appearance of a low-quality red fiber at 15 min postmortem. Comparisons of this Figure with Figures 4, 7 and 8 demonstrate that the myofibrils and mitochondria are essentially the same as for normal red fibers at 15 min postmortem. However, there seems to be more disruption of the SR and a reduction in the amount of glycogen granules.

The ultrastructure of all fiber types from low-quality muscle at 15 min postmortem was essentially the same in appearance as normal muscle at 24 hr postmortem, except that myofibrils from low-quality muscle showed a larger amount of disruption, especially in the white fibers.

REFERENCES

Beecher, G.R., Cassens, R.G., Hoekstra, W.G. and Briskey, E.J. 1965. Red and white fiber content and associated post-m ortem properties of seven porcine muscles. J. Food Sci. 30: 969.

Beecher, G.R., Kastenschmidt, L.L., Cassens, R.G., Hoekstra, W.G. and Briskey, E.J.1968. A comparison of the light and dark portions of a striated muscle. J. Food Sci. 33: 84.

Cassens, R.G., Briskey, E.J. and Hoekstra, W.G. 1963. Electron microscopy of postm ortem changes in porcine muscle. J. Food Sci 28: 680.

Cassens, R.G. and Cooper, C.C. 1971. Red and white muscle. Advan. Food Res. 19: 1.

Cooper, C.C.. Cassens, R.G. and Briskey, E.J.1969. Capillary distribution and fiber characteristics in skeletal muscle of stress-susceptible animals. J. Food Sci. 34: 299.

Dubowitz, V. 197 0. Differentiation of fiber types in skeletal muscle. In “ The Physiology and Biochemistry of Muscle as a Food,” Vol 2, p. 87. Ed. Briskey, E.J., Cassens, R.G. and Marsh, B.B. University of Wisconsin Press, Madison, Wis.

Gauthier, G.R. 1969. On the relationship of ultrastructural and cytochem ical features to color in mammalian skeletal muscle. Z. Zell- forsch. 95: 462.

Gauthier, G.R. 1970. The u ltrastructure of three fiber types in mammalian skeletal muscle. In “ The Physiology and Biochemistry of Muscle as a Food,” Vol 2, p. 103. Ed. Briskey, E.J., Cassens, R.G. and Marsh, B.B. University of Wisconsin Press, Madison, Wis.

Greaser, M.L.. Cassens, R.G., Briskey, E.J. and Hoekstra, W.G. 1969a. Postm ortem changes in subcellular fractions from norm al and pale, soft, exudative porcine muscle. 2. Electron microscopy. J. Food Sci. 34: 125.

Greaser, M.L., Cassens, R.G., Briskey, E.J. and Hoekstra, W.G. 1969b. Postm ortem changes in subcellular fractions from norm al and pale, soft, exudative porcine muscle. 1. Calcium accumulation and adenosine triphosphatase activity. J. Food Sci. 34: 120.

Grinyer, I. and George, J.C. 1969. Some observations on the u ltrastructure of the hum mingbird pectoral muscle. Can. J. Zool. 47: 771.

Hart, P.C. 1962. The transmission value, a m ethod fcr meat quality evaluation. Research Institute for Animal Husbandry “ Schoonocrd.” Zeist, The Netherlands (mimeograph).

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Henderson, D.W., Goll, D.E. and Strom er, M.H.1970. A comparison of shortening and Z line degradation in postm ortem bovine, porcine, and rabbit muscle. Am. J. Anat. 128: 117.

Koch, D.E., Merkel, R.A. and Purchas, B.J. 1970. The effect of postm ortem m yotom y on glycolysis and ultim ate qualitative characteristics of porcine longissimus muscles. J. Agr. Food Chem. 18: 1073.

Luft, J.H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409.

Moody, W.G. and Cassens, R.G. 1968. Histo- chemical differentiation of red and white muscle fibers. J. Anim. Sci. 27: 961.

Muir, A.R. 1970. Normal and regenerative skeletal muscle fibers in Pietrain pigs. J. Comp. Path. 80: 137.

Nystrom, B. 1968. Histochemistry of developing cat muscles. Acta Neurol. Scand. 44: 405.

Peachey, L.D. 1970. Form of the sarcoplasmic reticulum and T system of striated muscle. In “ The Physiology and Biochemistry of Muscle as a Food ,” Vol 2, p. 273. Ed. Briskey, E .J., Cassens, R.G. and Marsh, B.B. University of Wisconsin Press, Madison, Wis.

Porter, K.R. and Palade, G.E. 1957. Studies on endoplasmic reticulum . 3. Its form and distribution in striated muscle cells. J, Biophys. Biochem. Cytol. 3: 299.

Reynolds, E.S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17: 208.

Romanul, F.C.A. 1964. Enzymes in muscles. 1. Histochemical studies of enzymes in individual muscle fibers. Amer. J. Physiol. 213: 295.

Schmidt, G.R., Cassens, R.G. and Briskey, E.J. 1970. Changes in tension and certain m etabolites during the developm ent of rigor

m ortis in selected red and white skeletal muscles. J. Food Sci. 35: 571.

Sjostrand, F.S. 1967. “ Electron Microscopy of Cells and Tissues,” p. 145. Academic Press, New York.

Stein, J.M. and Padykula, H.A. 1962. Histochemical classification of individual skeletal muscle fibers of the rat. Amer. J. Anat. 110: 103.

Ms received 6 /28/73; revised 9 /16 /73 ; accepted9/19/73.

Michigan Agricultural Experim ent Station Journal Article No. 6441.

This investigation was supported in part byU.S. Public Health Service Grant No. FD00097 from the Food & Drug Administration.

Appreciation is expressed to Mr. Gary Gann for assistance in reproducing the electron micrographs presented in this paper.

J A M E S R . O 'H A R A , M . S. C H IN , B . D A I N I U S a n d J O H N H . K I L B U C K

__________S ta n g e C o ., 3 4 2 N . W e s te rn A y e , , C h ic a g o , I L 6 0 6 1 2

DETERM INATION OF BENZO(a)PYRENE IN SMOKE CONDENSATES BY HIGH PRESSURE RAPID LIQUID-LIQUID CHROM ATOGRAPHY

INTRODUCTION

IN RECENT YEARS, a number of investigators have determined the amount of po lycyclic aromat ic hydrocarbons (PAH’s) in such nonfood items as: rubber and tire dust (Snyder, 1971; Smith et al.,1968), cigarette smoke (Oakley, 1970), and ambient air (Moore et al., 1967; Lao et al., 1973). Since a number of PAH’s such as benzo(a)pyrene, dibenz(a,h)an- thracene are carcinogens, their presence in food products has been the subject of intense analytic investigations with particular emphasis on smoked foods (Howard et al., 1966a, b, c; White et al., 1971). The temperature at which smoke flavors or liquid smokes are produced approaches the ignition tempera

ture of hardwood sawdust, which is also conducive to PAH formation (Toth and Blass, 1972). Therefore, a quick reliable analytic method to determine PAH content of smoked flavors and smoked foods is of current importance to the food industry (Fazio et al., 1973). To date, the procedures cited involve a lengthy sample preparation followed by estimation of PAH content by ultraviolet and fluorescence spectrophotometry.

Recent advances in the development of adsorbents for liquid-liquid or liquid- solid chromatography render a method of separating and quantitating PAH’s (Karger et al., 1973; Ives and Guiffida,1972). This report illustrates the applicability of LLC to the analysis of benzo(a)- pyrene in smoke condensates.

EXPERIMENTAL

Materials

Reagents. Iso o ctan e , reagent grade; phosphoric acid, 85% , reagent grade; d im ethyl su lfox id e, reagent grade; benzene, reagent grade; F lorisil, 6 0 - 1 0 0 m esh. Prepared according to Howard et a.. (1 9 6 6 a ) w ith 3% w ater added. Pre-test by placing 10 Mg o f ben zo(a)p y ren e in 100 ml o f isooctane on a co lu m n contain ing 60g o f F lorisil at the bo tto m and 50g o f anh ydrous N a2 S 0 4 on the top. A fter discarding all o f the isooctane that was passed thru the co lum n, elute ben zo(a)p yren e w ith 2 5 0 m l o f ben zene follow ed by 5 0 ml o f ch lo ro fo rm . A fter removing the solvent, add 0 .2 ml o f a solution contain ing 0 .02g o f 1 ,2 ,4 ,5 -d iben zan th aracen e in 1 0 0 ml o f ch loroform and swirl. B en zo(a)p y- rene is qu antitatively determ ined by L L C . R e covery should exceed 90% .

F ig . 1 —P r e p a ra t iv e t h in - la y e r c h r o m a to g ra p h o f a q u e o u s s m o k e n u m b e r F ig . 2 —P re p a ra t iv e t h in - la y e r c h r o m a to g ra p h o f re s in o u s s m o k e c o n d e n -

1. M id d le s p o t in lo w e r r o w re p re s e n ts 0 .0 5 g g o f b e n z o (a )p y r e n e . s a te n u m b e r 2 . M id d le s p o t in lo w e r r o w re p re s e n ts 0 . 0 5 g g o f b e n -

z o fa lp y r e n e .

3 8 - J O U R N A L O F F O O D S C I E N C E - V o l u m e 3 9 (1 9 7 4 )

A N A L Y S I S OF B E N Z O (a )P Y R E N E IN S M O K E F L A V O R S - 3 9

Developing so lution, anhydrous ethanol- toluene-w ater ( 1 7 :5 :2 v /v/v).

Apparatus

Column ch ro m a to g ra p h y -a 38 mm i.d. x 3 0 0 mm long colum n, equipped with fritted disc.

Thin-layer ch ro m a to g ra p h y -20 cm x 20 cm plates were coated w ith a 250m layer o f 20% acetylated cellulose (Brin km an n In strum en t In c ., W estbury, N .Y .).

Liquid-liquid ch rom atog rap h y -N ester/F au st 121 0 Liquid Chrom atograph equipped with a 2 5 4 mm ultraviolet d etector. A lm 3 1 6 stainless steel colum n having an i.d. o f 2 .1 mm was packed with V Y D A C Reverse Phase (T h e Sep aration G roup, Hesparia, C alif.).

U ltraviolet la m p -lo n g wavelength, 36 6 0 A .U V sp e ctro p h o to m etry -B eck m a n A CTA III

U V Visible Sp ectrop h otom eter.

Procedure

Aqueous soluble sm oke flavors. A pre ¡4 tered lOOOg sample o f aqueous sm oke was placed in a 2 liter separatory funnel and e x tracted five tim es w ith 2 0 0 m l each o f isooctan e. T he com bined isooctane ex tra cts were placed in a 1 liter distilling flask and vacuum concentrated to about 2 0 m l. Q uantitative transfer o f the co n cen trate to a 2 5 0 m l separatory funnel was facilitated w ith three 20 ml portions o f isooctane. T he e x tra c t was consecutively washed w ith 10 0 m l each o f the follow ing: 5% NaOH (w /v) five tim es, w ater tw ice, 2N sulfuric acid tw ice and finally w ater tw ice. F or each washing the separatory funnel was vigorously shaken fo r 2 min and allow ed to separate for at least five m inutes before discarding the aqueous layer.

Resinous sm oke condensates. A 200g sam ple o f the condensate was dissolved in 1.5 liters o f 20% NaOH (w/v) using 3 0 0 m l p ortions o f the alkali solution to transfer the condensate in to a 2 liter separatory funnel as outlined by W hite e t al. ( 1 9 7 1 ) . An alternative m ethod of dissolving the condensate was em ployed using1.5 liters o f 70% acetic acid (v/v) and heating to 80° to dissolve the sample. W hen the solution was cooled it was likewise placed in a 2 liter funnel. T he alkali or acidic so lution was then extracted five tim es with 2 0 0 m l each o f isooctan e. T he com bined isooctane ex tracts were vacuum concen trated to approxim ately 5 0 ml and qu antitatively transferred to a 2 5 0 ml separatory funnel with an additional 5 0 m l o f isooctane. The ex tract was consecutively washed w ith 100 m l each o f the follow ing: 5% NaOH five tim es, w ater tw ice, 2N sulfuric acid tw ice and finally w ater tw ice.

V egetable oil soluble sm oke flavors. The PAH’s were extracted from the flavor w ith isooctane and washed with 85% H 3P 0 4 , 5% NaOH, and subjected to liquid-liquid partition as outlined by Howard et al. ( 1 9 6 6 c ) .

Chrom atographic clean-up. The previously washed ex tract o f w ater soluble, resinous co n densate or oil soluble sm oke flavor was further cleaned up by colum n chrom atography. A co lumn containing 60g o f F lorisil on the bo tto m and 50g o f anhydrous Na2 S 0 4 on top was prewet w ith 100 ml o f isooctane. T he e x tra c t was placed on the colum n and the isooctane was allowed to drain com pletely. A fter discarding the isooctan e, the PAH ’s were eluted with 2 5 0 ml o f benzene follow ed by 5 0 ml o f ch lo roform .

The volum e o f the com bined eluates was reduced to approxim ately 0 .5 m l under vacuum in a 60° oil bath , follow ed by room tem pera-

T IM E (M IN )

F ig . 3 —L i q u id - l iq u id c h r o m a to g r a p h o f vege

ta b le o i l s o lu b le s m o k e c o n ta in in g 0 . 3 p p b b e n -

z o fa /p y re n e . N u m b e r 1 is b e n z o 'a tp y r e n e ;

n u m b e r 2 is 1,2 ,4 ,5 - d ib e n z p y r e n e .

0 .6-

F ig . 4 —L i q u id - l iq u id c h r o m a to g ra p h o f a q u e o u s

s m o k e c o n t a in in g 5 p p b b e n z o (a )p y r e n e . N u m

b e r 1 is b e n z o fa fp y r e n e ; n u m b e r 2 is 1 ,2 ,5 ,6 -

d ib e n z a n th ra c e n e .

F ig . 5 —U l t r a v io le t a b s o r p t io n s p e c tra o f b e n z o (a )p y r e n e . B r o k e n l in e ,

u n k n o w n ; s o l id l in e , re fe re n c e .

40—JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

A B CI

_ i________ i_________i---------- _i_-----“ T o 2 0 5 0

TIME (MIN)

F ig . 6 —L iq u id - l iq u id c h ro m a to g ra p h o f f lu o re s

c e n t spo ts e lu te d fro m th e to p o f th e res inous con d e n sa te T L C p la te .

ture drying w ith nitrogen. A fter dissolving the residue in 0 .2 m l o f benzene, the ex tract was spotted on one o f two 20% acety lated cellulose T L C plates. Tw o 0 .2 ml portions o f benzene were used to rinse the flask and were spotted on the second plate. E ach plate was also spotted w ith 5 ,ul o f a solution containing 0 .0 0 lg benzo(a)pyren e in 100 ml o f benzene to obtain a referen ce R f value. T he flu orescen t spots, w hich were d etected under U V light, were scraped and covered with 10 ml o f m ethanol. The PAH ’s were eluted from acetylated cellulose w ith five 10 m l portions o f warm m ethanol, and separated on a buchner funnel equipped with a num ber 4 0 W hatm an filter paper. T he filtra te was quantitatively transferred to a 1 0 0 m l flask and vacuum concentrated to approxim ately 1 ml in a 55° oil bath . Evaporation o f the m ethanol was com pleted at room tem perature under a stream o f nitrogen. A fter chilling the flask in a refrigerator for 15 m in, 0 .2 ml o f internal standard solution (0 .0 2 g o f either 1 ,2 ,5 ,6-d iben zanth racene or1 ,2 ,4 ,5-d iben zp yren e in 1 0 0 ml o f ch loroform ) was added and swirled. A 10 /ctl portion was withdraw n from the flask and in jected on the colum n.

T he lm colum n packed w ith V Y D A C reverse phase was allowed to equilibrate in the m ethanol-w ater ( 6 5 :3 5 v/v) m obile phase overnight. Optim um separations were found to occu r under the follow ing instrum ent conditions: tem perature 5 5 ° , pressure 12 0 0 psi, a tten u ation 0 .2 , flow rate 0 .7 5 m l/m in.

Fig . 7 —U ltra v io le t a b s o rp t io n spe c tra o f c o m p o u n d B -P hen an th ren e . B ro k e n lin e , u n k n o w n ; s o lid l in e , re fe rence .

RESULTS & DISCUSSIONT H E U S E o f an in te rn a l s t a n d a rd o n LLC a f fo rd s a h igh ly re p ro d u c ib le a n d re liable m e t h o d to q u a n t i t a t e b e n z o ( a ) p y r e n e .

A 10 /fl s am p le o f a s o lu t io n t h a t c o n ta in s O.Olg o f in te rn a l s t a n d a r d a n d O.Olg o f b e n z o ( a ) p y r e n e in 100 m l o f c h lo r o f o r m can be u se d to ca lcu la te th e re la tive re sp o n se .

_ B e n z o (a )p y re n e r e sp o n se In te rn a l s t a n d a rd re sp o n se

Since 0 .2 m l o f a s o lu t io n c o n ta in in g 0 .0 2 g o f t h e in te rn a l s t a n d a rd (4 x 10“5g) p e r 100 m l c h lo r o f o r m was used to dissolve th e re s id u e , t h e b e n z o ( a ) p y r e n e c o n te n t c an be c a lcu la ted :

[B e n z o (a )p y re n e b e n z o ( a ) p y r e n e _ re sp o n se ] (4 0 /ag)

(/ig) ~~ (K ) ( In te rn a ls t a n d a r d re sp o n se )

T he d ev e lo p ed T L C p la te s o f f e r a q u ick and easily d iscern ib le s tep to d e te r m in e if a n y ap p rec ia b le a m o u n t o f b e n z o (a )p y - re n e is p re sen t in t h e sm o k e e x t r a c t (Fig. 1 a n d 2). In th ese f igures , b e n z o ( a ) p y r e n e is lo ca te d in th e lo w er f lu o re s ce n t ro w

w i th t h e m id d le sp o t c o n ta in in g 0 .0 5 /ig o f s t a n d a r c . Since a sp o t c o n ta in in g as l i t t le as 0 .031 /ig o f b e n z o ( a ) p y r e n e is visible u n d e r u l t rav io le t i l lu m in a t io n , d e t e c t io n can be c o n f i rm e d b u t n o t q u a n t i t a t e d visually.

T h e sensi t iv i ty l im it o f t h e LLC u l t r a v io le t d e t e c t o r fo r b e n z o ( a ) p y r e n e is 0 .0 1 5 /ig. If 10 /il o r 1 /20 o f t h e f inal s o lu t io n is p laced o n t h e c o lu m n , t h e n 0.3 /ig o f b e n z o ( a ) p y r e n e w o u ld be d e t e c t e d f ro m a lOOOg sa m p le (a ssu m in g 100% re co v e ry ) w h ic h is e q u iv a le n t t o 0.3 p p b (Fig. 3). A re p re se n ta t iv e c h r o m a t o g ram o f an a q u e o u s s m o k e t h a t h a s b e e n fo r t i f ied w i th 5 .0 p p b b e n z o ( a ) p y r e n e is in c lu d e d in F ig u re 4.

Peaks n u m b e r 1 in b o t h (Fig. 3 a n d 4 ) w ere c o n f i rm e d as b e n z o ( a ) p y r e n e by c o m p a r in g t h e U V s p e c t r u m o f t h e e lu te d p e a k w i th t h a t o f a p u re s t a n d a rd (Fig .5). B e n z o (a )p y re n e was re m o v e d f ro m m e th a n o l - w a te r m o b i le ph ase b y e x t r a c t ing th re e t im e s w i th i so o c ta n e t o give a f inal v o lu m e e q u a l t o t h e v o lu m e o f t h e e lu te d peak . A re fe re n c e so lu t io n was o b ta in e d by a s im ila r e x t r a c t i o n o f an e q u a l v o lu m e o f t h e m o b i le phase . B o th w e re c o n c e n t r a t e d to 1 m l w i th n i t ro g e n a n d t r a n s fe r r e d to a cell o f 1 cm p a th len g th .

ANALYSIS OF BENZO(a)PYRENE IN SMOKE FLAVORS- 4 1

R ecovery o f b e n z o ( a ) p y r e n e a d d e d at a level o f 10 p p b was 7 4 - 5 8 % fo r a q u e o u s sm o k e , 6 0 —81 % in re s in o u s sm o k e c o n d en sa tes , 6 8 —84% in oil so lub le c o n den sa te s as d e te r m in e d b y LLC.

In an e f fo r t t o i d e n t i fy t h e u p p e r ro w o f f lu o rescen t sp o t s in F ig u re s 1 a n d 2, several T L C p la te s s p o t t e d w i th res in o u s sm o k e c o n d e n s a te w e re d e v e lo p ed and th e area e lu te d as p rev ious ly o u t l in e d . The l iqu id - l iqu id c h r o m a to g r a p h o b ta in e d c o n ta in e d 4 peak s , h o w e v e r t h e y w ere n o t c o m p le te ly reso lved u n d e r t h e i n s t ru

m e n t c o n d i t i o n s used f o r t h e b e n z o (a )p y - ren e analysis . W hen a m o b i le ph ase o f m e th a n o l - w a te r ( 5 0 : 5 0 v /v) was passed t h r u th e c o lu m n a t a ra te o f 0 .6 5 m l /m in , su f f ic ie n t s e p a ra t io n o c c u r re d t o co llect t h e e lu te d p e ak s (Fig . 6) . O f th e f o u r p e ak s , th r e e (B, C a n d D) have b e e n id e n t if ied b y th e i r r e t e n t i o n t im e s an d th e i r U V sp ec tra . In F igure 7 , p e a k B is i d e n t i f ied as p h e n a n t h r e n e by c o m p a r in g its U V sp e c t r u m w i th t h a t o f a re fe ren ce p h e n a n t h r e n e s o lu t io n (1 .6 7 /ig p e r m l o f i so o c ta n e ) . T h e U V sp e c tra o f p e a k C in

F ig u re 8 is a p p a r e n t ly a c o m p o s i t e o f p y re n e an d f lu o r a n th e n e w h ic h has been verif ied by th e va r iab le re fe re n ce t e c h n iq u e . In F ig u re 9, th e U V s p e c t r u m o f p e a k D is c o m p a r e d w i th a n d id en t i f ie d as t r ip h e n y le n e (0 .6 7 /ug p e r m l o f i so o c t a n e ) . A t t e m p t s t o i d e n t i fy p e a k A were u n su ccess fu l a l t h o u g h i ts U V sp e c t r u m is s im ila r to several a lky l su b s t i t u te d b e n zenes. A l th o u g h th ese c o m p o u n d s have b e e n p rev io u s ly id en t i f ie d in sm o k e d f o o d s b y H o w a rd et al. ( 1 9 6 6 a ) and W hite e t al. ( 1 9 7 1 ) , t h e use o f L LC o ffe rs t h e in v es t ig a to r a m e t h o d o f q u a n t i t a t i n g th e se P A H ’s.

It is n o t e w o r t h y t h a t b e n z o ( a ) p y r e n e a n d b e n z o ( e ) p y r e n e can be s e p a ra ted and q u a n t i t a t e d in d iv id u a lly w h ic h has b e en a d i f f ic u l ty in t h e past . It is also n o t e w o r t h y t h a t t h e ca rc in o g e n s b e n z o (a )a n - t h r a c e n e a n d d ib e n z ( a ,h ) a n th r a c e n e w ere n o t d e te c t e d in t h e sm o k e f lavors te s ted .

REFERENCESF a z io , T . , W h ite , R .H . and H o w a rd , J .H . 1973.

C o lla b o ra tive s tu d y o f the m u ltic o m p o n e n t m e th o d fo r p o lyc y c lic a ro m a tic h y d ro c a rbons in foods. J. Assoc. O ff ic . A n a l. C hem . 56: 68.

H ow ard , J .W ., Teague, R .T ., W h ite , R .H . and F ry , B .E . 1966a. E x tra c tio n and e s tim a tio n o f p o lyc y c lic a rom a tic hyd rocarb ons insm oked foods. 1. G enera l m e th o d . J. Assoc.O ff ic . A n a l. C hem . 49 : 595.

H o w a rd , J .W ., W h ite , R .H ., F r y , B .E . and T u ric - ch i, E .W . 19 66b . E x tra c tio n and e s tim a tio n o f p o lyc y c lic a rom a tic hyd roc arb o ns insm oked foods. 2. B enzo(a )p y rene . J. Assoc. O ff ic . A n a l. C hem . 49 : 611 .

H ow ard , J .W ., T u r ic c h i, E .W ., W h ite , R .H . and F a z io , T . 1966c. E x tra c tio n and e s tim a tio n o f p o lyc y c lic a rom a tic hyd roc arb o ns invegetable o ils . J. Assoc. O ff ic . A n a l. Chem . 49 : 1236.

Ives, N .F . and G u iff id a , L . 1972 . L iq u id c h ro m atog rap hy o f p o lyc y c lic a ro m a tic h y d ro carbons. J. Assoc. O ff ic . A n a l. C hem . 5 5 (4 ): 757 .

Karger, B .L ., M a rt in , M ., Loheac , J. and G u io - chon , G. 19 73 . S ep a ra tion o f p o ly a ro m a tic hyd roc arb o ns by liq u id -so lid ch rom a tog ra p hy using 2 ,4 ,7 -tr in it ro f lu o re n o n e im preg nated C oras il I . co lum ns. A n a l. Chem . 4 5 (3 ): 496 .

L a o , R .C ., Th om a s, R .S ., O ja , H . and D ub o is , L . 1973 . A p p lic a tio n o f a gas c h ro m a to g ra p h -m ass spectrom eter-data processor c o m b in a tio n to the analysis o f the p o ly cyc lic a rom a tic hyd ro c a rb o n c o n te n t o f a irborne p o llu ta n ts . Presented a t E ig h th In te r n a t io n a l S ym p o s iu m o f Advances in C h rom a to g rap h y, A p r il 16—19 , 1973 at T o ro n to , Canada.

M o o re , G .E ., Thom as, R .S . and M o n km a n , R .L .1967 . Th e ro u t in e d e te rm in a tio n o f p o ly cyc lic hyd roc a rb o ns in a irb o rne p o llu ta n ts . J. C h rom a to g r. 29: 45 6 .

O ak ley , E .T ., Jo hnso n , L .F . and S tah r, H .M .1970 . R ap id m e thod fo r the d e te rm in a tio n o f benzo(a )p y rene in c igarette sm oke. To b . Sci. 16: 19.

S m ith , G .C ., N au , C .A . and Law rence , C .H .1968. S ep a ra tion and id e n t if ic a t io n o f p o ly cyclic hyd roc arb o ns in ru b b er dus t. A m . In d . H yg . Assoc. J. 29 : 242.

S nyd e r, M .S . 19 71 . A m e th o d fo r th e separat io n , id e n t if ic a t io n and q u a n tita t io n o f3 ,4-benzpyrene f ro m ru b b e r t ire dust. Ph .D . thesis, U n ive rs ity o f O k lahom a.

T o th , L . and Blass, W . 1972 . E f fe c t o f sm oking techn o log y on the c o n te n t o f carcinogenic hyd rocarb ons in sm oked m eat products. F le is c h w ir ts c h a ft, 5 2 (7 ): 1419.

W h ite , R .H ., H o w a rd , J.W . and Burnes, C.J.19 71 . D e te rm in a tio n o f p o lyc y c lic arom atic hyd roc arb o ns in liq u id sm oke fla vo rs . J. Agr. F o o d C hem . 1 9 (1 ): 143.

Ms received 6 /2 8 /7 3 ; revised 9 /1 6 /7 3 ; accepted 9 /1 9 /7 3 .

Fig . 8 —U ltra v io le t a b s o rp t io n spe c tra o f c o m p o u n d C—a m ix tu re o f p y re n e a n d flu o ra n th e n e . B ro k e n Une, u n k n o w n ; s o lid Une, p y re n e ; d a s h -d o t Une, flu o ra n th e n e .

Fig. 9 —U ltra v io le t a b s o rp t io n spe c tra o f c o m p o u n d D —tr ip h e n y le n e . B ro k e n Une, u n k n o w n ; s o lid

Une, tr ip h e n y le n e .

S. H . R I C H E R T ' , C . V . M O R R 1 2 a n d C . M . C O O N E Y

D e p t , o f F o o d S c i e n c e & N u t r i t i o n , U n i v e r s i t y o f M i n n e s o t a , S t . P a u l , M N 5 5 1 0 1

EFFECT OF HEAT AND OTHER FACTORS UPON FOAMING PROPERTIES OF WHEY PROTEIN CONCENTRATES

INTRODUCTIONS E V E R A L C O M M E R C IA L a n d sem ic o m m e rc ia l p rocesses have b e e n develo p e d t o f r a c t io n a t e w h e y a n d p rep are w h e y p r o t e in c o n c e n t r a t e s (see M orr et al., 1 9 7 3 ) . W hey p r o t e in c o n c e n t r a te s (WPC), p re p a re d b y th ese p rocesses w i th care t o m in im iz e p r o t e in d é n a tu r a t io n , a re o f c o n s id e ra b le in te re s t t o t h e in d u s t r y b e cau se o f th e i r p o t e n t i a l value as fu n c t io n a l f o o d ing red ien ts . P re l im in ary in ves t iga t ions in d ic a te d t h a t t h e fo a m in g p ro p e r t i e s o f all WPC te s te d w ere in fer io r t o egg w h i te p r o te in a n d c ase in a te (M orr e t al., 19 7 3 ) . H ow eve r , t h e l a t t e r s tu d ies w e re c o n d u c t e d u n d e r l im i te d e x p e r im e n ta l c o n d i t i o n s w i th n o a t t e m p t s t o o p t i m iz e p r o c e d u r e s t o d e te r m in e fo a m in g p ro p e r t i e s o f WPC.

T h is s tu d y was u n d e r t a k e n to inves tiga te t h e e f fec ts o f h ea t t r e a tm e n ts , pH levels, c a lc iu m c o n c e n t r a t io n , re d o x p o te n t i a l a n d so d iu m lau ry l su lfa te u p o n fo a m in g p ro p e r t i e s o f WPC. T h e resu l ts o f th ese s tu d ie s sh o u ld p rov ide th e basis fo r o p t im iz in g fo a m in g c o n d i t i o n s t o o b ta in m a x i m u m o v e r ru n a n d fo a m s ta b i l i ty fo r w h ip p e d WPC d ispers ions .

EXPERIMENTALWhey protein con cen trate

Whey protein co n cen trate (W PC), prepared by Sephadex gel filtra tio n fractio n ation o f partially d elactosed whey con cen trate (F o o d In g red ien ts D iv., S tau ffer Chem ical C o ., R och ester, M inn.), was used for the m ajor portion o f this study. T he com position o f the product is given in T ab le 1. O ther sources o f WPC w ere: e lectrod ialyzed , delactosed whey (F o rem o st F oo d s C o ., San F ran cisco , C alif.), u ltrafiltra tion (A b co r, In c., Cam bridge, M ass.), and polyphosphate precip itate (Borden In c ., E lg in , 111.) (R ich e rt, 1 9 7 2 ). P rotein co n ten t was determ ined as to ta l m icro-K jeldahl N x 6 .3 8 . U ndenatured p rotein , rem aining in the supern atan t obtained by adjusting the pH o f a 1% protein WPC in w ater dispersion to 4 .6 and centrifuging at 1 ,0 0 0 X G for 10 m in, was also determ ined by m icro-K jeld ahl. M oisture, ash and tota l lipids were determ ined by procedures adapted from Official Methods o f Analysis (A O A C , 1 9 7 0 ) , as previously described (M orr e t a l„ 1 9 7 3 ) . Calcium was by the E D T A titration procedure o f Jen n ess (1 9 5 3 ) and phosphorus

1 P resen t address: U n iv e rs ity o f W isconsin — R iv e r Fa lls , R iv e r Fa lls , W I 54022

2 P resen t address: R a ls to n P u r in a C om p any ,C heckerboard Square, S t. L o u is , M O 6 3 188

Table 1—Composition of Sephadex gelfiltration WPC

% dry basis

Protein 41.8Undenatured protein3 87.1Lipid 1.97Ash 13.6Calcium 1.01Phosphorus 1.32Moisture 4.97a Percent o f to ta l p ro te ins

was by the m olybd ate procedure o f Sum ner (1 9 4 4 ) .Sam ple preparation and treatm en t

Su ffic ien t WPC was dispersed in distilled w ater w ith gentle stirring, to m ake 1 kg o f a 4% (w /w ) protein so lution. V arying am ounts o f 0 .25M sodium oxalate were added to precip ita te the desired am ount o f calcium , held 10 m in , and centrifuged a t 1 ,0 0 0 x G for 10 min to sedim ent Ca oxalate . T he solution was transferred to a vacuum flask and evacuated several tim es w ith a w ater aspirator to remove dissolved air. 0 .1M sodium sulfide (N a2 S) was added to adjust the redox poten tia l to the desired level, as determ ined w ith a platinum

electrod e. T h e pH, m easured by glass e lectro d e, was adjusted to the desired level w ith IN HC1 or NaOH. F in ally , the desired volum e o f a 10% sodium lauryl su lfate so lution was added.

T he entire sample was transferred to a sta in less steel beaker and heated at tem peratu res o f 5 0 - 8 5 ° C for 30 min by passing steam through a coiled 0 .2 5 in. stainless steel tubing im m ersed in the sam ple. Sam ples were easily heated to < 8 5 °C w ithin 1 m in by sim ultaneously co n trolling the steam flow through the co ;l and providing rapid sample m ixing w ith a m agn etic stirrer. W hen the desired tem perature was achieved, the beaker was transferred to a therm ostatically contro lled w ater bath , held for 30 m in, and cooled to am bient tem perature w ithin 3 - 4 m in by imm ersing the beaker in an ice bath . A com p lete tim e-tem perature profile was obtained fo r each heating exp erim en t using a cop p er-corstan tan therm ocouple im m ersed in the sample and con n ected to a Sargent S R L recorder (F ig . 1).

Protein denaturation/aggregation

Whey protein d énaturation was determ ined as the percent reduction o f to ta l N obtained by adjusting the pH o f a 5 :1 d ilution to pH 4 .6 , then centrifuging at 1 ,0 0 0 x G fo r 10 m in. Protein aggregation was determ ined by the same procedure, exce p t that the pH was n o t adjusted to 4 .6 . D én aturation was studied by determ ining the increase in viscosity produced by heating. V iscosity m easurem ents were m ade at 20° C w ith a B roo k field L V F v iscom eter

TIME (min)

F i g . 1 - T i m e t e m p e r a t u r e p r o f i l e f o r a t y p i c a l h e a t t r e a t m e n t o f h V P C .


FOAMING OF WHEY PROTEIN CONCENTRA TES—43

Table 2 —Definition and levels of independen t variables

LevelsIndependent variable Symbol -2 -1 0 1 2Heating temperature (°C/30 min) x, 65 70 75 80 85pH x, 4.0 5.0 6.0 7.0 8.0Redox potential (V) X, -0.025 0.075 0.175 0.275 0.375Na oxalate (Ml X< 0.0500 0.0375 0.0250 0.0125 0.0000Na lauryl sulfate (% of solids) X, 0.00 0.05 0.10 0.15 0.20

using a U L attach m en t. Whey protein dénaturation was also investigated by urea starch gel electrophoresis, conducted as by M orr (1 9 7 1 ) and Cooney and M orr (1 9 7 2 ) . Protein zones were identified as: a -L a , a-lactalbu m in ; (3-Lg, |3-lactoglobulins A and B ; and B S A , bovine serum albumin.Foam ing experim ents

Sam ples were whipped w ithin 1 hr after com pletion o f the heating experim en t using a m od ification o f the standard egg album in whip test described by Matz (1 9 6 0 ) . 125 m l o f

sample w ere whipped for 9 0 sec at speed setting 2 in a H obart M odel C-5 m ixer, follow ed oy additional whipping at speed setting 3 until the endpoint was reached. During whipping, peaks o f foam were form ed initially in the cen ter o f the bow l and progressively appeared toward the outside o f the bow l. T h e tota l elapsed time required to produce peaks at the outside o f the bow l was recorded as whipping tim e. Whipping was then continued for an additional 2 min at speed setting 3 be fo re testing for m axim um overrun and foam stability .

Overrun was determ ined by weighing 5-oz paper cups o f foam and unwhipped sample and calculating by the expression:

w t liquid - wt foam% overrun = ----------------------------------x 100%

w t foam

The foam cups w ere im m ediately inverted over glass funnels and the elapsed tim e for the first drop o f liquid to separate and the percent liquid separation during the first 3 0 m in were determ ined as a measure o f foam stability . E xperim ental design

A ppropriate com bin ations o f five independen t variables ( X , - X 5 ) , each at five d ifferent levels, were studied (T ab le 2 ) . A central com posite experim en tal design, described by C ochran and C o x (1 9 5 7 ) , was used to estim ate regression co effic ie n ts (B ^ ) according to the m odel given in Figure 2. M athem atical analyses w ere cond ucted on a U M ST A T 5 0 0 statistical program o f the U niversity o f M innesota C om puter C en ter. S ta tistica l significance was determ ined by the “ t test,”

t = Bjj/S.D.

w here S .D . is standard deviation.

Table 3—Effect of independent variable combinations upon nine major dependent variables

Independent variables3 Dependent variablesb

Trialno. x, X2 X3 x4 Xs Y, Y, Y, y 4 Ys y 6 Y,

Initialviscosity

(CP)

Heatedviscosity

(CP)

1 -1 -1 -1 -1 1 4.75 1082 100 4.5 80.6 81.4 44.3 2.38 3.352 1 -1 -1 -1 -1 4.00 824 100 7.5 67.9 69.6 44.8 2.07 8.363 -1 1 -1 -1 -1 5.00 953 85 8.3 83.1 105 45.6 1.65 1.784 1 1 -1 -1 1 9.50 759 22 17.0 38.1 81.2 46.8 1.64 16.95 -1 -1 1 -1 -1 4.00 1163 92 6.7 79.7 80.8 45.0 1.81 3.006 1 -1 1 -1 1 5.00 839 100 9.5 74 7 76.3 45.3 1.80 5.687 -1 1 1 -1 1 3.00 1343 65 12.0 71 2 103 46.0 1.80 1.828 1 1 1 -1 -1 7.00 736 0 36.0 36 8 76.9 46.9 1.57 43.49 -1 -1 -1 1 -1 5.25 1027 100 4.0 81 7 87.2 44.9 2.32 3.52

10 1 -1 -1 1 1 5.00 836 100 5.0 66 8 74.0 44.1 1.70 6.5711 -1 1 -1 1 1 3.00 1272 59 12.5 73 0 98.5 45.7 1.72 1.6712 1 1 -1 1 -1 6.50 825 14 20.0 40.5 94.1 46.1 1.74 29.213 -1 -1 1 1 1 3.25 1363 44 15.0 74.9 95.9 45.5 1.64 1.7114 1 -1 1 1 -1 5.00 855 100 7.5 74.2 76.8 45.5 1.87 7.8815 -1 1 1 1 -1 2.75 1284 35 18.5 63.5 100 46.5 1.67 1.8416 1 1 1 1 1 5.00 851 48 12.0 42.8 104 45.3 1.60 3.8417 -2 0 0 0 0 3.75 1283 81 12.0 80.9 100 45.5 1.61 1.6218 2 0 0 0 0 11.0 651 100 8.5 42.4 50.5 47.2 1.72 >10019 0 -2 0 0 0 4.50 1217 100 4.5 73.4 71.2 45.5 2.74 3.8620 0 2 0 0 0 4.00 982 54 10.5 45.0 101 45.5 1.72 2.6721 0 0 -2 0 0 5.00 884 100 9.0 66.0 85.8 45.9 1.70 5.0022 0 0 2 0 0 3.75 1147 94 9.0 71.7 103 45.0 1.66 1.8023 0 0 0 -2 0 3.75 1081 76 9.0 77.5 104 45.2 1.65 1.8124 0 0 0 2 0 4.75 1036 100 10.0 76.3 89.4 45.0 1.67 3.4825 0 0 0 0 -2 4.00 1213 38 16.0 67.4 105 45.0 1.58 1.8326 0 0 0 0 2 3.50 1103 82 8.5 86.5 113 45.4 1.66 1.6527 0 0 0 0 0 4.00 - 85 11.0 77.4 102 44.5 1.65 1.7728 0 0 0 0 0 3.50 1179 72 9.0 74.6 104 45.1 1.65 1.7429 0 0 0 0 0 3.50 1183 69 9.0 79.8 107 45.1 1.73 1.7730 0 0 0 0 0 4.00 1120 100 10.0 78.3 104 45.5 1.66 1.7231 0 0 0 0 0 3.50 1180 77 9.5 74.8 101 45.0 1.67 1.7832 0 0 0 0 0 3.00 1195 79 11.0 80.9 103 45.4 1.68 1.89

3 Independent variab les defined in Tab le 2 b Dependent variab les defined in Tab le 4


RESULTS & DISCUSSIONG en era l o b se rv a t io n s

H ea t in g WPC d isp e rs io n s a t > 7 0 ° C fo r 30 m in caused p r o t e in aggrega tion as ev id en ced by an increase in tu rb id i ty . T h e t u r b id i ty a p p e a re d to be m o re p r o n o u n c e d at pH 4 a n d 5, in d ic a t in g th a t p r o t e in ag g reg a tio n was fav o red near t h e isoe lec tr ic pH.

In genera l, t h e m o re severe h e a t t r e a t m e n t s im p a i red th e fo a m in g c h a r a c t e r istics o f WPC d ispers ions , i.e., h ighly h e a t e d WPC d isp e rs io n s re q u ire d longer w h ip p in g t im e s an d p r o d u c e d lo w e r o v e rr u n fo a m s w i th h igh ly var iab le s tab i l i ty . T r e a tm e n t 8 (T ab le 3 ) WPC d ispe rs ion had u n iq u e and in te res t in g fo a m in g p r o p er ties , e.g., t h e d isp e rs io n was e x t r e m e ly v iscous an d th e fo a m was den se and re sem b led w h ip p e d cream .

R aw e x p e r im e n ta l d a ta fo r n ine d e p e n d e n t var iab les (Y ) o b ta in e d f ro m 32 c o m b in a t io n s o f i n d e p e n d e n t variables (X ) a re t a b u la te d in T ab le 3.

R egress ion analysis

Analysis o f va r iance (A N O V A ) s u m m aries are given in T ab le 4 fo r n ine i m p o r t a n t d e p e n d e n t variables. M ult ip le c o r re la t io n c o e f f ic ie n ts give t h e co r re la t io n b e tw e e n p re d ic te d re sp o n ses and o b se rv ed re sponses . T h u s , t h e h ig h er th e m u l t i p l e c o r re la t io n c o e f f ic ie n t , th e b e t t e r t h e m a th e m a t ic a l m o d e l (Fig. 2) a c c o u n ts fo r t h e v a r ia t io n o b se rv ed fo r t h e d e p e n d e n t variable . Sim ila r ly , th o se d e p e n d e n t variables w i th h ighes t m u l t ip le c o r re la t io n co ef f ic ie n ts ( Y j , Y 2 , Y s , Y 6 a n d Y 8 ) w ere m o s t s ign if ican tly a f fe c te d by a l te r ing each o f th e i n d e p e n d e n t var iab les (P ro b a b i l i ty < 0 .0 1 ) . Regression analys is o f su rface t e n s io n (Y 7 ) d id n o t ex p la in a s ign if ican t a m o u n t o f th e va r ia t io n , in d ic a t in g t h a t t h e in d e p e n d e n t var iab les used in th is e x p e r im e n t d id n o t a p p re c ia b ly a l te r it.

R e g r e s s io n c o e f f ic ie n ts and th e i r s ta tis t ica l s ign if icance (p ro b a b i l i ty level) fo r six o f th e d e p e n d e n t variables are given in Tab le 5. These d e p e n d e n t variables w ere c h o se n b e cau se t h e y w ere m o s t c losely re la te d to t h e ob jec t ive s o f t h e e x p e r im e n t a n d becau se t h e y w ere m o s t su i tab le fo r regress ion analysis. R egress ion c o e f f ic ie n ts are th e least sq u a res e s t im a te s o f t h e “ B y ” te rm s in t h e m o d e l (Fig. 2). It is i m p o r t a n t to n o te t h a t t h e c o e f f ic ie n ts a re in t e rm s o f co d ed levels o f i n d e p e n d e n t variables , i.e., - 2 , — 1 , 0 , 1 a n d 2 (see T ao le 2). C o d ed levels o f i n d e p e n d e n t var iab les a re d e f in e d by th e fo l low ing re la t io n :

, , , , a c tu a l level - c e n te r levelc o d ed level = ---------------------------------------

i n c r e m e n t b e tw ee nlevels

Table 4 - D e f in i t io n s and analysis of variance (ANOVA) summaries of nine d e p e n d e n t variables

Dependentvariable Description

Multiplecorrelationcoefficient

Meansquarefrom

regression

Meansquareerror Probability

Y, Whipping time (min) 0.964 1.516 0.200 7.57 0.01y 2 Maximum overrun (%) 0.968 57034 7129 8.00 0.01

y 3 % drainage at 30 mih 0.903 1.33 0.58 2.29 0.1Y, Time at first drop (min) 0.919 1.37 0.440 3.13 0.05Ys % undenatured protein 0.982 335.6 21.68 15.48 0.01y 6 % soluble protein 0.960 313.0 45.91 6.82 0.01y 7 Surface tension (dynes/cm) 0.851 0.562 0.369 1.52 n.s.Y5 Log, 0 heated viscosity 0.977 0.3257 0.0265 12.30 0.01Y, Log, 0 heated viscosity 0.905 165.4 63.3 2.61 0.1

Log , 0 initial viscosity

in t h e m o d e l is c h an g e d o n e in c re m e n t . P re d ic t io n s o f e x p e c te d va lues o f d e p e n d en t var iab les m ay be c a lcu la te d by su b s t i tu t in g a p p r o p r i a te va lues o f c o d ed in d e p e n d e n t var iab les an d regress ion c o e f f ic ie n ts in to t h e m o d e l .

T he o n ly tw o i n d e p e n d e n t variables th a t s ign if ican tly a f fe c te d o v e r ru n ( Y 2 ) o f the WPC d ispers ions were h e a t in g t e m p e ra tu r e ( X ( ) an d r e d o x p o te n t i a l ( X 3 ). R esponse su rface p lo ts w ere g en era ted by c o m p u t e r to aid in visualiz ing the e f fec ts o f these tw o in d e p e n d e n t variables (Fig. 3). These p lo ts r e p re se n t p re d ic t io n s o f X ! a t 115 levels a n d X 3 a t 115 levels while h o ld in g X 2 , X 4 a n d X 5 c o n s ta n t . These d a ta sh o w t h a t h igher h e a t in g t e m p e ra tu r e s and m o re re d u c in g c o n d i t ions adverse ly a f fec t fo a m v o lu m e , w h ich agrees w i th th e r e p o r t o f H ansen and Black (1 9 7 2 ) th a t h y d ro g e n p e ro x id e t r e a tm e n t e n h a n c e d the fo a m in g p r o p e r ties o f C M C -w hey p ro te in c o n c e n t r a te .

F o a m s tab il i ty (Y 4 ) was s ign if ican tly a f fe c te d by p H (X 2 ) an d by f o u r in te r a c t io n e f fe c ts (T ab le 5). T h e posi t ive regress ion c o ef f ic ien t o f 3 .6 9 fo r pH in d ic a te s t h a t a d ju s t in g WPC t o h ig h er pH va lues re su l ted in a m o r e s tab le fo a m . T he s ign if ican t e f fec t o f pH o n fo a m s tab il i ty , while n o t a f fec t in g fo a m v o lu m e (Y 2 ), d e m o n s t r a t e s t h e necess i ty o f using b o th p a ra m e te r s in th e se WPC fo a m in g s tu d ies. In t e r a c t io n e f fec ts b e tw e e n tw o in

d e p e n d e n t var iab les o c c u r w h e n b o t h variables s im u l ta n e o u s ly a f fec t t h e o b served va lue fo r a d e p e n d e n t va r iab le . Th is c an be easily v isua lized by c a l c u l a t ing p re d ic t io n s o f d e p e n d e n t v a r iab .es at c o m b in a t io n s o f high anci lo w in d e p e n d e n t va r iab le levels (T ab le 6). T h e p os i t ive i n te r a c t io n e f fe c t o f X i X 2 ( B 12 = 2 .1 5 ) m ea n s t h a t c h ang ing p H has a g re a te r e f fec t u p o n fo a m s ta b i l i ty t im e a t h igher t h a n a t lo w e r h e a t in g t e m p e ra tu r e s . Spec if ica l ly , t h e e f fe c t u p o n f o a m s ta b i l i ty t im e (Y 4 ) p r o d u c e d by increas ing pH f ro m 6 .0 to 7 .0 was 1.41 m in a t "70 °C ,3 .5 6 m in a t 7 5 ° C an d 5.71 m in a t 8 0 °C . These successive fo a m s ta b i l i ty va lues each d if fe r b y 2 .1 5 m in , t h e va lue f o r t h e regress ion c o e f f ic ie n t . S ign if ican t in te r a c t io n e f fec ts involving h e a t in g t e m p e r a t u r e ( X j X 2 , X j X 4 a n d X ^ s ) suggest t h a t th e e f fec t iveness o f h e a t t r e a t m e n t u p o n f o a m s ta b i l i ty is also d e p e n d e n t u p o n o t h e r e x p e r im e n ta l c o n d i t i o n s , e.g., pH , c a lc iu m c o n c e n t r a t i o n a n d so d iu m lau ry l su l fa te c o n c e n t r a t io n .

T e m p e r a tu r e o f h e a t in g (X j ) a n d p H (X 2 ) had m a jo r e f fe c ts u p o n p r o t e i n d é n a tu r a t io n ( Y 5 ) a n d p r o t e in so lu b i l i ty ( Y 6 ). R e sp o n se su rface c o n to u r s f o r th e se variables are given in F ig u re s 4 a n d 5. In i t ia lly , 8 7 .1 % o f t h e to ta l WPC p r o te in s w ere u n d e n a t u r e d (T ab le 1). W hey p r o te ins w e re m o r e su sce p t ib le t o hea t d é n a t u r a t i o n a t h igh p H t h a n lo w pH

Y = B0 + BlXl + B2 X2 + B3 X3 + B4 X4 + B5 X5 + B1 2 XlX2 + B1 3 X1 X3 + B j ^ +

B 1 5 X1 X5 + B 2 3 X2 X3 + B 2 4 X2 X4 + B 2 5 X2X5 + B 3 4 X 3X4 + B 3 5 X3 X5 + B 4 5 X4 X5 +

31 1 X1 + B 2 2 X2 + B 3 3 X3 + B 4 4 X4 + B5 5 XJ

R egress ion c o e f f ic ie n ts m a y be in te r - Fig . 2 —M a th e m a tic a l m o d e ! used to de scribe va ria b le response to v a r ia t io n in in d e p e n d e n t

p re te d as t h e c h an g e e x p e c te d in t h e variab les, w here Y = va lue o f d e p e n d e n t v a r ia b le ; X j = value o f in d e p e n d e n t v a r ia b le ; B // =d e p e n d e n t va r iab le w h e n th e t e r m va lue regress ion c o e ff ic ie n t.

FOAMING OF WHEY PROTEIN CONCENTRATES- 4 5

Table 5 —Regression analysis of six dep en d en t variables3

Regression coefficients (B^)

term Y2 Y, y 5 y 6 Y, Y,Constant 1178.0** 9.44** 77.80** 103.72** 0.2365** 1.7018**x, -176.1 ** 1.08 -10.10** -8.24** 0.3795** 7.3701**X2 -18.2 3.69** -8.66** 7.51** 0.0175 1.6951x 3 57.6** 1.60 -0.08 2.45 -0.0732* -0.0605X, 21.8 -0.21 -0.69 1.38 -0.0190 -0.6846Xs 19.1 -1.50 1.35 1.66 -0.0802* -1.4252X ,X2 -25.0 2.15* -0.62 -0.11 0.1443** 2.7075x , x 3 -48.9* -0.46 2.74 0.47 -0.0157 0.1615x ,x , -12.3 -2.75* 1.74 2.09 -0.0333 -1.0350x , x 5 -36.8 -2.13* 0.74 0.77 -0.0775 -2.0018X2X3 -2.9 0.19 -1.71 -0.78 -0.0019 0.2362X3X„ 16.8 -0.85 -0.29 0.29 -0.0408 -1.1977x , x 5 11.0 -2.35* -0.51 -0.16 -0.0507 -1.8726x , x 4 -4.4 -0.96 -0.01 1.44 -0.0798 -1.9384x 3x 5 2.4 -1.21 1.54 4.09* -0.0658 -1.2587x „ x s -1.0 0.63 0.06 0.29 -0.0497 0.2002X? -57.0** 0.56 —4.16** -7.28** 0.2271** 6.4362**X̂ -23.8 -0.13 -4.77** -4.57** 0.0775* -0.5369X23 ^14.8* 0.24 -2.36* -2.61 0.0708* -0.4038X42 -34.1 0.37 -0.35 -2.29 0.0508 -0.5094X? -9.2 1.05 -0.34 1.16 0.0109 -0.6380a Dependent variab les defined in Tab le 4

* P < 0.05 * * P < 0.01

Table 6—Predicted values of foam stability(Y4 ) as functions of X, and X2

X j-pHXj —Heating temperature (°C)

70 75 805.0 7.25 5.62 5.116.0 8.92 9.44 11.087.0 10.33 13.00 16.79

Table 7 - and foaming centratea

Protein dénaturation and solubility properties of whey protein con-

Heat 0//o 0//o Maximumtreatment Undenatured Soluble overrun

(°C/30 min) proteinb protein0 (%)d65 80 est. 92 est. 150070 85 est. 100 150075 77 100 135080 62 87 115085 40 58 800 est.

a S ta u ffe r Chem ical Co., Rochester, M in n . b F ro m Fig. 4 a t m id p o in t levels, pH 6 .0 c F ro m Fig. 5 a t m id p o in t levels, pH 6 .0 d F ro m Fig. 3 at m id p o in t levels, redox p o te n

tia l + 0 .1 7 5 V .

(Fig. 5), w h ic h h as b e e n sh o w n p rev ious ly fo r u n f r a c t io n a t e d w h e y sy s te m s (G u y et al., 19 6 7 ; N ie lsen et al., 1973) . R e sponse su rface c o n to u r s fo r % so lu b le p r o te in (Fig. 5 ) sh o w an a p p a r e n t s tab i l i ty m a x im u m a t a p p r o x im a te ly p H 7 a n d 7 0 °C . This m a y be e x p la in e d as a b a lan ce b e tw e e n th e d isaggrega tion e f fec t o f p H ’s a b o u t 7 a n d t h e t e n d e n c y t o w a r d s g re a te r p ro te in a ggrega tion at t h e h ig h e r t e m p e r a tu res o f h e a t in g ( > 7 0 ° C ) . While th e f i t te d m o d e l p re d ic ts g rea te r so lu b i l i ty at 7 5 ° C a n d p H 6 t h a n a t 6 5 ° C , it is q u i te

u n l ik e ly t h a t th is is t ru e . In fac t , th e o b se rv ed va lue a t 6 5 ° C a n d p H 6 was 100% (T ab le 3, T ria l 17). M ore l ikely , the t r u e r e sp o n se is s t a t io n a ry a t p H > 6 and h e a t in g t e m p e r a t u r e s < 75 C. A m o re c o m p le x m o d e l w o u ld be re q u ire d to m o re a cc u ra te ly d ep ic t th is re sponse .

O n e e x a m p le o f t h e re la t io n sh ip b e tw e e n p r o t e i n d é n a t u r a t i o n a n d so lu b i l i ty p r o d u c e d b y h ea t t r e a tm e n t s w i th m a x i m u m o v e r ru n fo r WPC d ispers ions is given in T ab le 7. H igher t e m p e ra tu r e t r e a tm e n t s ab o v e 7 0 ° C r e d u c e d th e

u n d e n a t u r e d a n d so lu b le p r o t e i n levels an d also i ih p a i red fo a m in g p ro p e r t i e s o f t h e WPC.

C hanges in WPC d isp e rs io n viscosities u p o n h e a t in g w ere c o n s id e re d as a poss ible c o n v e n ie n t m e t h o d f o r fo l lo w in g th e e x t e n t o f h e a t i n d u c e d p r o t e i n d é n a t u r a t io n . T h e lo g a r i th m o f v iscosi ty was s u b s t i tu t e d fo r v iscosi ty as d e p e n d e n t variable , s ince t h e f o r m e r p ro v id e d a b e t t e r fi t t o t h e m o d e l (Fig. 2). As sh o w n in T ab le 5, all var iab les e x c e p t o x a la te c o n c e n t r a t i o n (X 4 ) s ign if ican tly a f fe c te d

Fig. 3 -R e s p o n s e su rface c o n to u r fo r te m p e ra tu re IX J a n d re d o x

p o te n t ia l ( X 3) e f fe c t o n m a x im u m o v e rru n ( Y 2I. A l l o th e r in d e p e n d e n t

variab les (X ) w ere a t m id p o in t levels.

pH

Fig. 4 - Response su rface c o n to u rs fo r te m p e ra tu re ( X J a n d p H IX 3 ) e f fe c t o n % u n d e n a tu re d p ro te in ( Y j . A H o th e r in d e p e n d e n t variab les

were a t m id p o in t levels.


t h e v isco s i ty o f t h e h e a t e d WPC disp e rs io n . T h u s , th e se in te r fe r in g fa c to r s m u s t e i t h e r b e c o n t r o l l e d o r c o r re c te d b e fo re v iscosi ty c a n be used to e s t im a te p r o t e in d é n a t u r a t i o n in h e a te d WPC desp e rs io n s . T h e ra t io o f log h e a te d v iscosi ty t o log in it ia l v iscosi ty ( Y 9 ) was s ign if ican tly a f fe c te d b y o n ly h e a t in g t e m p e r a t u r e (X j ), in d ic a t in g t h a t th is in d e x m ig h t be u se fu l as a q u ic k m e t h o d fo r a p p r o x im a t in g h e a t d é n a t u r a t i o n o f W PC d ispers ions .

Table 9—Effect of heat treatments upon foaming properties of WPC

Foaming properties

TreatmentWhipping

time (min)Maximum

overrun (%)Foam

stability (min)

Unheated 9 800 4Heated 50°C/30 min 3 1260 12Heated 60°C/30 min 3 1290 15Heated 65°C/30 min 2-3/4 1270 14Heated 65°C/30 min. 7 770 not stable

held overnight

C o r re la t io n o f d e p e n d e n t variablesT h e c o m p u t e r o u t p u t f ro m th e

U M S T A T 5 0 0 sta t is t ica l p ro g ra m in c lu d es a c o r r e l a t i o n m a t r ix a m o n g each o f th e d e p e n d e n t var iab les a n d c h eck s t h a t i n d e p e n d e n t va r iab les a re n o t co r re la te d . C o r r e la t io n c o e f f i c ie n ts fo r n in e o f th e d e p e n d e n t var iab les used in th is e x p e r i m e n t a re given in T ab le 8. T hese co r re la t io n s a re o f p r im a r y c o n c e r n to th is s tu d y since o n e o f t h e m a jo r ob jec t ive s was to d e te r m in e th e re la t io n s h ip b e tw e e n p r o te in d é n a t u r a t i o n an d fo a m in g p ro p e r t i e s o f WPC. F o r e x a m p le , t h e m a x im u m o v e r ru n (Y 2 ) w as s ign if ican tly c o r re la te d w i th % u n d e n a t u r e d p r o t e i n (Y s ) a n d % so lub le p r o t e in ( Y 6 ), in d ic a t in g th a t

increas ing p r o t e in d é n a t u r a t i o n a n d agg reg a t io n decreases fo a m v o lu m e . Sm alle r c o r re la t io n s w ere f o u n d b e tw e e n fo a m s ta b i l i ty (Y 3 ) a n d d é n a t u r a t i o n ( Y 5 ), w h e rea s s ign if ican t c o r re la t io n s w ere n o t o b se rv ed b e tw e e n fo a m s ta b i l i ty a n d p r o te in so lu b i l i ty ( Y 6 ). Th is l a t t e r e f fec t is n o t u n e x p e c te d , s ince th e r e w as no c o r re la t io n b e tw e e n fo a m v o lu m e (Y 2 ) an d f o a m s ta b i l i ty ( Y 3 a n d Y 4 ). G o o d c o r re la t io n s w ere o b ta in e d b e tw e e n visco s i ty o f t h e h e a te d p r o d u c t an d p ro te in d é n a t u r a t i o n an d so lu b i l i ty , f u r t h e r in

Table 8—Correlation of dependent variables3

Y, y 2 y 3 Y, Ys y 6 Y, Y» Y,

Y, 1.000y 2 -0.779** 1.000y 3 -0.169 0.036 1.000Y, 0.236 -0.139 -0.861** 1.000Y s -0.675** 0.619** 0.633** 0.593** 1.000y 6 -0.648** 0.617** -0.261 0.081 0.338 1.000Y, 0.533** -0.297 -0.578** 0.635** -0.622 -0.182 1.000y 8 0.880** -0.831** -0.205 0.362* -0.737** -0.768** 0.518** 1.000Y, 0.787** -0.589** -0.158 0.322 -0.614** -0.614** 0.643** 0.851** 1.000a D ependent variab les defined in Tab le 4* S ign ificance P < 0.05* * S ign ificance P <0.01

Table 10-Comparison of foaming properties of different WPC products

WPC product

Sephadexa

Electro-dialyzed,

delactosedbUltra

filtration0

Polyphosphate

precipitated6

Polyphosphate

precipitated^Protein content (%) 40.3 35.0 66.0 54.2 76.2Undenatured protein (%)f 88.6 94.4 55.8 30.0 102Whipping time (min)S 9 -3 2.5-2.25 no foam no foam 8.0-no foaiMaximum overrun (%)6 800-1290 1060-1300 - - 610- -Foam stability (min)g 4-15 10-17 - - 5- -a S ta u ffe r Chem ical Co., Rochester, M in n , b F o re m o s t Foods C om pany, San Francisco, C a lif. c A b co r, Inc., Cambridge, Mass, d Borden Inc., E lg in , III. (sod ium ne u tra lized ). e R iche rt, 1972. ̂ Percent o f to ta l p ro te in

£ F irs t num b er, unheated W PC d ispersion va lue; second num ber, 6 0 °C /3 0 m in heated WPC dispersion va lue

d ica t in g t h a t v iscosi ty m a y be u se fu l in eva luat ing h e a t d a m a g e to w h e y p r o t e in s in WPC.

Revers ib le im p r o v e m e n t o f fo a m in g p ro p e r t i e s

I t was o b se rv ed t h a t h e a t in g WPC d ispers ions a t t e m p e r a t u r e s o f 6 5 - 70 C great ly im p r o v e d th e f o a m in g p ro p e r t i e s , whereas, h ig h er t e m p e ra tu r e t r e a tm e n t s im p a i red fo a m in g p ro p e r t i e s (T ab le 7). T o f u r t h e r inves t iga te th is p h e n o m e n o n , WPC d ispers ions w ere a d ju s te d to pH 7.0 and h e a t e d to 50, 6 0 a n d 6 5 ° C w i t h o u t a d d in g o x a la te , l au ry l su l fa te o r su lf ide . R esu lts o f these tr ia ls are given in Tab le9. All th ree h e a t in g t r e a tm e n t s vas t ly im p ro v e d fo a m in g p ro p e r t i e s o f th e WPC d ispers ion , w i th n o s ign if ican t d i f fe re n ce a m o n g the th re e t r e a tm e n ts . H o w ev e r , the i m p r o v e m e n t in fo a m in g p ro p e r t i e s was t e m p o r a r y a n d revers ib le , in t h a t a f te r h o ld in g th e 6 5 ° C h e a te d d isp e rs io n o v e rn ig h t , tire fo a m in g p ro p e r t i e s w ere th e n p o o re r t h a n b e fo re h ea t in g . T h ese s u b d e n a tu r a t io n h e a t t r e a tm e n t s m ay im p ro v e fo a m in g p ro p e r t i e s o f WPC d ispers ions by a l te r in g (a) p ro te in - l ip id - p h o sp h o l ip id c o m p le x e s , (b ) p ro te in h y d r a t io n , c r (c) p ro te in c o n f o r m a t i o n , th e re b y fav o rin g p ro te in a b s o r p t io n at th e a ir- l iquid in te r fac e w i th in t h e f o a m lamella, im p r o v e m e n t o f f o a m in g p r o p e r ties o f p a r t ia l ly d e la c to se d w h e y c o n c e n t r a te has been o b se rv ed p re v io u s ly (P e te r an d Bell, 19 3 0 ) . T h e p h e n o m e n o n was n o t o b se rv ed to be revers ib le , h o w ever.

C o m p ar iso n o f WPC p r o d u c t sFive d i f fe re n t WPC p r o d u c t s were

c o m p a re d fo r p ro te in c o m p o s i t i o n , d é n a tu ra t io n (so lu b i l i ty a t pH 4 .6 ) a n d f o a m ing p ro p e r t i e s (T ab le 10). P r o t e in c o m p o s i t io n o f t h e d i f fe re n t WPC p r o d u c t s varied f ro m 3 5 - 7 6 % . T h e u l t r a f i l t r a t io n WPC w as h ig h ly d e n a t u r e d , w h e r e as th e p o l y p h o s p h a te - p r e c ip i t a te d WPC (R ic h e r t , 19 7 2) t r e a te d to re d u c e res idua l p o ly p h o s p h a te , w as c o m p le te ly u n d e n a tu red . T he se c o n d p o l y p h o s p h a te - p r e c ip i t a te d WPC gave an a p p a r e n t ly high p ro te in d é n a tu r a t io n value, based on so lu b i l i ty a t pH 4 .6 , w h ic h m a y have been d u e to re s idua l p o l y p h o s p h a te ions

FOAMING OF WHEY PROTEIN CONCENTRATES—4 7

Fig. 5 —Response surface con tours fo r tem perature ( X 11 and p H (X 21 e ffe c t on % solub le p ro te ins (Y 6). AH o th e r independen t variables were a t m id p o in t levels.

in ad d ition to actual p ro te in denaturation (M o rr et al., 1973). Urea starch gel electrophoresis patterns o f various WPC, dispersed in water at the ir in it ia l pH value, ranging fro m 6 .0 -8 .3 , and a fte r ad justing to pH 4.6 and cen trifug ing to remove denatured prote ins, are in Figures 6 and 7. |3-lactoglobulins A and B in Sephadex WPC (F ig. 6 a - d ) were in com ple te ly resolved, bu t otherw ise the prote ins appeared undenatured, except fo r some m in o r streaking between a-La and /3-Lg zones. U ltra fi lt ra t io n WPC (Fig. 7a and b) had tw o add itiona l, heat- produced p ro te in zones at pH 8.3. Polyphosphate prec ip ita te (F ig . 7c and d) and electrod ia lysis (F ig . 7e and f ) WPC exh ib ite d considerable p ro te in denaturation and/or reduced s o lu b ility due to residual po lyphosphate ions. R iche rt’s (1972 )

Fig. 6 -U re a starch gel e lectrophoresis o f Sephadex WPC (S tau ffe r Fig. 7 -U re a starch gei e lectrophoresis o f 1% WPC dispersions in waterChemical Co., Rochester, M in n .): a and b, superna tan t ob ta ined fro m a a t th e ir in it ia l p H and a fte r ad justing to p H 4 .6 and ce n trifu g in g a t1% WPC dispersion in water, ad justed to p H 4 .6 and cen trifug ed a t 1,000 X G fo r 10 m in to remove denatu red (inso lub le) p ro te ins : a,1,000 X G fo r 10 m in ; c and d, same as a and b, except p H 4 .6 disper- u ltra f i lt ra t io n WPC (A bco r, Inc., Cambridge, Mass.), p H 4 .6 ; b, a a tsion was n o t cen trifug ed ; and e and f, fresh p H 4 .6 w hey standard. in it ia l pH , 8 .3 ; c, po lyphosphate p re c ip ita te (Borden, Inc ., E lg in, III.),

p H 4 .6 ; d, c a t in it ia l oH , 6 .7 5 ; e, e lectrod ia lys is (F o rem ost Foods Co., San Francisco, C a lif.), p H 4 .6 ; f, e a t in it ia l p H , 6 .6 5 ; g, po lyphosphate p re c ip ita te (R iche rt, 1972), p H 4 .6 ; h, g a t in it ia l pH , 6 .0 ; and i, fresh p H 4 .6 whey standard.

48-JOURNAL OF FOOD SCIENCE-Vo/ume 39 (1974)

polyphosphate precip ita te WPC (F ig. 7g and h) was least denatured o f all WPC exam ined. These la tte r data con firm that i t is possible to prepare an undenatured WPC by carefu l processing and removal o f residual po lyphosphate ions. Experience in our labo ra to ry indicates tha t urea starch gel electrophoresis, conducted w ith reducing cond itions in the gel, is superior to po lyacry lam ide gel electrophoresis fo r s tudy ing p ro te in denaturation in WPC (M o r re ta l. , 1973).

Each WPC produc t was adjusted to pH7.0 w ith o u t adding oxalate, la u ry l sulfate or sulfide and a po rtion was heated at 60°C fo r 30 m in . B oth unheated and heated dispersions were w h ipped as above and the data are in Table 10. E lectro- dia lyzed WPC exh ib ited the best foam ing properties and Sephadex WPC was second best. U ltra fi ltra t io n and polyphosphate WPC products exh ib ited poor or no foam ing properties. The heat trea tm ent im

proved the foam ing properties o f bo th Sephadex and e lectrod ia lyzed WPC to comparable w h ipp ing times, m axim um overrun and foam s tab ility .

REFERENCESA O A C . 1970 . “ O ff ic ia l M ethods o f A n a lys is ,”

1 1 th ed. A ssoc ia tion o f O ff ic ia l A n a ly tic a l Chem ists, W ash ing ton, D .C.

C ochran, W .G . and C ox, G .M . 1957 . “ E x p e r im e n ta l Designs.” Jo hn W ile y and Sons, Inc., N ew Y o rk , N .Y .

C ooney, C .M . and M o rr, C .V . 1972 . H ydrogen perox id e a lte ra t io n o f w hey p ro te in s in w h e y and concen tra ted w h e y systems. J. D a iry Sci. 55 : 567.

Guy. E .J ., V e t te l, H .E . and Pallansch, M .J. 1967 . D e n a tu ra tio n o f cottage cheese w hey p ro te in s b y heat. J. D a iry Sci. 50: 828.

Hansen, P .M .T . and B lack, D .H . 1972 . W h ip p ing p roperties o f sp ray-dried com plexes fro m w h e y p ro te in and c a rb o x ym e th y lc e l- lu lose. J. Fo od Sci. 37 : 452.

Jenness, R . 1953 . T it ra t io n o f ca lc ium and m agnesium in m ilk and m ilk fra c tio n s w ith e th y le n e d ia m in e te traaceta te . A na l. Chem . 25: 966 .

M atz, S .A . 1960 . “ B akery T e chno log y and

E ng inee ring .” Th e A v i Pub lish ing C o m p a n y , W estport. Conn.

M o rr, C .V . 19 71 . C om p arison o f p ro te in p re p a ra tion procedures and starch versus p o ly a c ry la m id e gel e lec trop hores is fo r exa m in in g casein d eg radation p ro d uc ts o f cheese. J. D a iry Sci. 54: 339.

M o rr , C .V ., Sw enson, P .E . and R ic h te r . R .L .1973. F u n c tio n a l charac te ristics o f w h e y p ro te in concentrates. J. F o o d Sci. 38 324.

N ielsen, M .A ., C o u lte r, S .T ., M o rr , C .V . and Rosenau, J .R . 19 73 . A fo u r fa c to r response surface e xp e rim e n ta l design fo r eva lu a ting th e ro le o f processing variab les u p o n p ro te in d e n a tu ra tio n in heated w h e y system s. J. D a iry Sci. 56 : 76.

Peter, P .N . and B e ll, R .W . 1 9 3 0 . N o rm a l and m o d ifie d foam in g p ro p erties o f w h e y -p ro te in and egg-album in so lu tio n s . In d . Engr. C hem . 22: 1124.

R ic h e rt, S .H . 1972 . W hey p ro te in iso la te — P re p a ra tion and fu n c t io n a l p ro p erties . Ph .D . thesis, U n iv e rs ity o f W isconsin , M adison.

S um ner, J.B . 1944. A m e thod fo r th e c o lo r im e t r ic d e te rm in a t io n o f phosphorus. Science 10 0 : 413.

Ms received 6 /2 8 /7 3 ; revised 8 /1 3 /7 3 ; accepted8 /1 6 /7 3 .____________________________________________

Th e a u th o rs th a n k the W PC supp lie rs, and especially M r. Joseph F e m in e lla , S ta u ffe r C hem ical Co., w h o also c o n tr ib u te d va luab le c o n su lta tio n and advice th ro u g h o u t th e s tu d y .

T. B E V E R ID G E ,' S.J. TO M A and S. N A K A I Dept, o f F o o d Science, U n ive rs ity o f B ritish C olum bia, Vancouver 8, Canada

DETERM INATION OF SH- AND SS-GROUPS IN SOME FOOD PRO TEIN S USING ELLM AN'S REAGENT

INTRODUCTIONS U L F H Y D R Y L ( S H ) a n d d i s u l f i d e ( S S ) g r o u p s h a v e b e e n w i d e l y i m p l i c a t e d a s i m p o r t a n t f u n c t i o n a l g r o u p s i n m a n y f o o d p r o t e i n s . T h e f o r m a t i o n o f / i - l a c t o g l o b u - l i n - K - c a s e i n c o m p l e x e s w h e n m i l k i s

h e a t e d m a y o c c u r t h r o u g h t h e S S i n t e r

c h a n g e r e a c t i o n ( S a w y e r , 1 9 6 9 ) . T h e t h i c k g e l o f e g g w h i t e i s c o n s i d e r e d t o c o n s i s t o f l a r g e g l y c o p r o t e i n m o l e c u l e s

c r o s s l i n k e d b y S S b o n d s a n d a r e d u c t i v e

m e c h a n i s m h a s b e e n p r o p o s e d t o e x p l a i n t h e t h i n n i n g r e a c t i o n w h i c h o c c u r s o n a g i n g o f e g g s ( S h e n s t o n e , 1 9 6 8 ) . F u r t h e r , i t i s w e l l d o c u m e n t e d t h a t a d d i t i o n o f S H

c o m p o u n d s t o f l o u r d r a s t i c a l l y w e a k e n s t h e r h e o l o g i c a l p r o p e r t i e s o f t h e f l o u r

( K u n i n o r i a n d S u l l i v a n , 1 9 6 8 ) . I n t e r a c t i o n s b e t w e e n S S a n d S H g r o u p s o f f l o u r h a v e b e e n s u g g e s t e d t o e x p l a i n , i n p a r t , t h i s c h a n g e .

A s i m p l e m e t h o d f o r t h e m e a s u r e m e n t

o f S H a n d S S g r o u p s i n f o o d s w o u l d c l e a r l y b e d e s i r a b l e . A s s a y p r o c e d u r e s u t i l i z i n g E l l m a n ’s r e a g e n t ( E l l m a n , 1 9 5 9 ) h a v e t h e a d v a n t a g e o f b e i n g s i m p l e , r a p i d a n d d i r e c t ( K a l a b , 1 9 7 0 ) a n d h a v e y i e l d e d v a l u e s c o m p a r a b l e w i t h o t h e r a n a l y t i c a l m e t h o d s f o r S H g r o u p s ( F e r n a n d e z - D i e z

e t a l . , 1 9 6 4 ) a n d f o r S S g r o u p s ( A t a s s i e t a l . , 1 9 7 0 ) i n p u r i f i e d p r o t e i n s .

I n t h i s r e p o r t a m e t h o d f o r t h e d e t e r m i n a t i o n o f b o t h S H a n d S S g r o u p s u t i l i z i n g E l l m a n ’s r e a g e n t i s a p p l i e d t o f l o u r , e g g w h i t e , m i l k a n d t h e i r p r o d u c t s .

EXPERIMENTALReagents and m aterials

Guanidine hydrochloride (GuH Cl) was ultra pure reagent obtained from Mann Research Laboratories. (¡-Lactoglobulin was purchased from N utritional B iochem icals In c. Ovalbumin was isolated from egg w hite by th e m ethod o f K ekw ick and Cannan ( 1 9 3 6 ) , a s l -casein by the m ethod o f Z ittle and Custer (1 9 6 3 ) , a S5-casein by the m ethod o f Annan and M anson ( 1 9 6 9 ) , and K-casein by the m ethod o f Nakai e t al.(1 9 7 2 ) . F lou r, instantized skim m ilk and spray- dried egg w hite were local com m ercial products and eggs and m ilk were obtain ed from the U niversity farm . R eagen t grade chem icals were used to prepare the follow ing: Tris-glycine bu ffer (1 0 .4 g Tris, 6 .9g glycine and 1.2g E D TA per liter, pH 8 .0 , denoted as T ris-G ly), 0 .5% sodium

1 Present address: Dept, of AgriculturalChemistry, Macdonald College of McGill University, Ste. Anne de Bellevue, Quebec, Canada

u

to

Fig. 1—E ffe c t o f urea concen tra tion on tne to ta l SH groups o f several p ro te in m aterials a fte r in cub a tion fo r 1 h r a t 2 5 °C in the presence o f 2-m ercaptoe thanoi. M ilk and egg w h ite re ported on d ry weigh t basis.

d odecylsulfate (SD S ) in Tris-G ly , 1% NaCl in Tris-G ly , E llm an ’s reagent (5 ,5 '-dithiobis-2- n itro ben zoic acid) in Tris-G ly (4 m g/m l), 12% trich lo roacetic acid (T C A ), 8M and 10M urea in Tris-G ly and 8M urea contain ing 5M GuHCl in Tris-G ly (U rea-GuH C l).

F lou r and gluten. A 75-m g sample was suspended in 1 ml o f Tris-G ly ; 4 .7g o f GuHCl was added, and the volum e made to 10 m l. F o r SH , to 1 ml o f this slightly turbid solution was added 4 ml o f Urea-GuH Cl and then 0 .05 ml o f E llm an’s reagent was added. F o r S S , to 1 m l o f the flou r or gluten solution was ad dec 0 .0 5 ml o f 2-m ercaptoethan ol and 4 ml o f Urea-GuHCl and the m ixture was incubated for 1 hr at 25° C. A fter an additional 1 hr incu bation with 10 ml o f 12% T C A , the tubes were centrifuged at 5 0 0 0 X G in an in tern atio n al C linical C en trifuge for 10 min. T he precip itate was tw ice resuspended in 5 ml o f 12% TC A and centrifuged to rem ove 2-m ercap toethan ol. T he precip itate was dissolved in 10 m l o f 8M urea in Tris-G ly and the co lor was developed w ith 0 .0 4 ml o f E llm an’s reagent.

Egg w hite. Thoroughly blended egg w hite (1 .5 g ) was diluted to 10 ml w ith 1% NaCl in Tris-G ly . F o r SH , 2 .9 m l o f 0 .5% SD S in Tris- G ly was added to 0 .1 ml o f diluted egg w hite and 0 .0 2 ml o f E llm an ’s reagent developed co lo r. F or S S , 0 .2 ml o f diluted sam ple, 1 m l o f

10M urea and 0 .0 2 ml o f 2-m ercap toethan ol w ere m ixed and allow ed to stand fo r 1 hr. A fter p recip itation o f p ro tein w ith 10 m l o f TCA and washing as described for flou r and gluten, the precip itate was dissolved in 3 m l o f 0 .5% SD S or 8M urea in Tris-G ly , then 1 m l was diluted to 10 ml with the same solvent. C olor was developed by adding 0 .0 5 m l o f E llm an’s reagent. F o r dried egg w hite, 2 0 0 mg was dissolved in 10 m l o f 1% NaCl in Tris-G ly . M ixing should be gentle to avoid surface d enaturation .

Skim m ilk. F o r SH , 0 .5 ml o f skim m ilk was added to 2 .5 ml o f 8M urea in Tris-G ly and 0 .0 2 ml o f E llm an’s reagent. F o r S S , 0 .2 ml o f skim m ilk, 1 ml o f 10M urea in Tris-G ly and 0.02 m l o f 2-m ercap toeth an ol were incubated at 2 5 °C fo r 1 hr. A fter precip itation and washing o f protein as for flour and g luten , the precip itate was dissolved in 3 m l o f 8M urea in Tris-G ly and 0 .0 3 ml o f E llm an’s reagent was added for co lor developm ent.

A bsorbance was measured at 4 1 2 nm on a B eckm an DB sp ectrop h otom eter. T o ta l solids o f food products were analyzed by drying at 105°C fo r 24 hr. T h e co n cen tratio n s o f the purified protein s w ere determ ined from the absorbances using their absorptivities, A j ^ : 9 .5 and 7 .3 5 at 2 8 0 nm for (3-lactoglobulin and ovalbum in (Su zuki e t al., 1 9 6 9 ), 11 .7 at 2 8 0 nm for K-casein (N akai e t al., 1 9 6 5 ) and 10.7 and 10.1 at 2 7 8 nm for a s l - and a S5-caseins (A nnan and M anson, 1 9 6 9 ) . T h e SH and SS levels o f these e lectrop h oretically pure proteins were determ ined on 1% solutions using the same procedure as that o f skim m ilk exce p t for ovalbum in which was determ ined by the same procedure as egg w hite.

Calculations

7 3 .5 3 A 412 • D mM SH /g = -------------------------

where A 4 j 2 = the absorbance at 4 1 2 nm ; C = the sample co n cen tratio n in mg solids/m l; D = the d ilution fa cto r, 5 .0 2 , 6 .0 4 and 3 0 .2 for SH and 10, 15 and 15 0 fo r to ta l SH (SH + reduced S S ) in flou r or g luten, m ilk and egg w hite, respectively; and 7 3 .5 3 is derived from 10 6 / (1 .3 6 x 10 4 ) ; 1 .36 x 10 4 is the m olar absorptivity (E llm an , 1 9 5 9 ) and 10 6 is for conversions from the m olar basis to the juM/ml basis and from mg solids to g solids.

RESULTS & DISCUSSIONReduction of SS groups

R e d u c t i o n o f S S g r o u p s i n p r o t e i n s p r i o r t o t h e i r e s t i m a t i o n a s S H h a s c o m m o n l y e m p l o y e d s u l f i t e o r b o r o h y d r i d e a s a r e d u c i n g a g e n t . A t t e m p t s t o u s e s o d i u m b o r o h y d r i d e f o r e s t i m a t i o n o f S S g r o u p s i n K - c a s e i n r e s u l t e d i n v a l u e s a p p r o x i m a t e l y o n e - h a l f t h a t e x p e c t e d f r o m l i t e r a t u r e ( M a c K i n l a y a n d W a k e , 1 9 7 1 ) .

Volume 39 (19741-JOURNAL OF FOOD SCIENCE-49


Table 1—S u lfh y d ry l and d isu lfid e values fo r pu rifie d p ro te ins

SH (m oles/m o le p ro te in ) SS (m oles/m o le p ro te in )

P ro te in3 Found L ite ra tu re Found L ite ra tu re

(3-Lactoglobulin 0.95 0.9 (Fernandez-D iez e t al., 1964)

2.0 -2 .0 (M cKenzie, 1971)

O valbum in 4.0 CO bo (Fernandez-D iez e t al., 1964)

1.0 1.0 (F o the rg ill and F o the rg ill, 1970)

«-Casein 0 0 (M acK in lay and Wake, 1971 ) 1.0 1.0 (M acK in lay and Wake, 19 711a ss-Casein 0 0 (Hoagland e t al., 1971 ) 1.9 1.8 (H oaglard e t al., 1971)crsl -Casein 0 0 (Beveridge and Nakai, 1970) 0 0 (Beveridge and Nakai, 1970)

a Assumed molecular weight: 0-Lactoglobulin, 18,000 (McKenzie, 1971); Ovalbumin, 45,000 (Fothergill and Fotherglll, 1970); K-Casetn, 20,000 (MacKInlay and Wake, 1971); as5-Casein, 65,750 (Toma and Nakal, 1973) and asl-Casein, 27,000 (McKenzie, 1967)

Table 2—S u lfh y d ry l and d isu lfid e values fo r fo o d p roducts

SH (pM /g d ry w eigh t) SS (pM /g d ry w eigh t)

P roduct Found L ite ra tu re Found L ite ra t are

Skim m ilk 2.13 2 .0 6 -2 .4 (Y osh ino et al., 1962) 10.8 13.3 (Y osh ino et al., 1962)Fresh 5.8 (Po-ahl and Vakaleris, 1968) 48 .0 (Pofahl and Vakaleris, 1968)

1.78 (Sasago e t al., 1963) 10.8 (Sasago et al., 1963)2 .0a 9 .7 a

Instantized 1.33 0.98 (Kalab, 1970) 11.3 1 4 -1 5 (M anning et al., 1969)pow der 1 .0 2 -1 .3 8 (Lyste r, 1964) 4.01 (Kalab, 1970)

1.22 (Sasago et al., 1963) 10.1 (Sasago et al., 1963)Freeze dried 2.20 10.4

Egg w h iteFresh 50.7 50.0 (Feeney et al., 1966) 79.7 84 .5a

51.6 (M acD onnell e t al., 1951)Powdered 48.2 79.4

F lou r1 2.18

-O1̂*Tq

(Tsen and Bushuk, 1968) 14.6 8 .5 —16.9b (Tsen and Bushuk, 1968)2 2.65 12.8Crude g luten 3.1 60.9 70 (Kasarda et al., 1971 )

a Calculated values, see text 13 Depending on variety

C o n s e q u e n t l y , t h e p o s s i b l e u s e o f 2 - m e r c a p t o e t h a n o l a s a r e d u c i n g a g e n t w a s

e x p l o r e d . T h e e f f e c t o f u r e a c o n c e n t r a t i o n o n t h e t o t a l S H v a l u e s f o r j 3 - l a c t o - g l o b u l i n , o v a l b u m i n , K - c a s e i n , m i l k , e g g

w h i t e a r e s h o w n i n F i g u r e 1 . I t i s a p p a r e n t t h a t 1 h r i n 8 M u r e a i s s u f f i c i e n t t o r e d u c e S S g r o u p s i n a l l p r o t e i n s o l u t i o n s t e s t e d . F u r t h e r i n c u b a t i o n u p t o 3 . 5 h r s l i g h t l y i n c r e a s e d t o t a l S H l e v e l s b u t e x t e n d e d r e d u c t i o n f o r 8 . 5 h r d e c r e a s e d t h e m p r e s u m a b l y d u e t o a i r o x i d a t i o n o f t h e S H g r o u p s . T h i s r e d u c i n g c o n d i t i o n w a s a l s o a p p l i c a b l e t o f l o u r a s i n c u b a t i o n b e y o n d 1 h r d i d n o t i n c r e a s e t o t a l S H v a l u e .

SH and SS of purified proteinsA s s h o w n i n T a b l e 1 , t h e v a l u e s f o r a l l

o f t h e p r o t e i n s a g r e e d w e l l w i t h p u b l i s h e d d a t a . N o f a l s e p o s i t i v e v a l u e f o r a s l - c a s e i n , f o r w h i c h n e i t h e r S H n o r S S h a s

b e e n r e p o r t e d b e f o r e , i n d i c a t e s t h a t t h e w a s h i n g p r o c e d u r e w i t h T C A w a s s u f f i c i e n t .

SH and SS of food productsS a t i s f a c t o r y a g r e e m e n t w a s o b t a i n e d

b e t w e e n S H a n d S S o f f o o d p r o d u c t s a n d r e p o r t e d v a l u e s ( T a b l e 2 ) . T h e S H a n d S S c o n t e n t s o f f r e s h s k i m m i l k a g r e e d w i t h m o s t l i t e r a t u r e v a l u e s a n d w i t h c a l c u l a t e d o n e s a s s u m i n g t h a t a l l S H a n d S S w a s d e r i v e d f r o m K - c a s e i n , | 3 - l a c t o g l o b u l i n a n d a - l a c t a l b u m i n . H o w e v e r , t h e v a l u e s r e

p o r t e d b y P o f a h l a n d V a k a l e r i s ( 1 9 6 8 ) a s d e t e r m i n e d b y a f l u o r o m e t r i c p r o c e d u r e w e r e c o n s i d e r a b l y h i g h e r t h a n o u r s . T h e v a l u e s o b t a i n e d f o r i n s t a n t i z e d s k i m m i l k p o w d e r a r e i n g o o d a g r e e m e n t w i t h p u b

l i s h e d d a t a , e x c e p t f o r a l o w v a l u e f o r S S r e p o r t e d b y K a l a b ( 1 9 7 0 ) w h o u s e d s o d i u m b o r o h y d r i d e f o r r e d u c t i o n o f S S . N o

s i g n i f i c a n t c h a n g e w a s o b s e r v e d d u r i n g

f r e e z e d r y i n g o f f r e s h s k i m m i l k . W i t h t h e

c o e f f i c i e n t o f v a r i a t i o n o f 2 % f o r t h i s m e t h o d , i t i s d i f f i c u l t t o d e t e c t d i f f e r e n c e s i n t o t a l S H f o r f r e s h s k i m m i l k ,

i n s t a n t i z e d s k i m m i l k p o w d e r a n d f r e e z e - d r i e d s k i m m i l k , a l t h o u g h S H w a s s i g n i f i

c a n t l y l o w e r i n c o m m e r c i a l i n s t a n t i z e d p o w d e r t h a n i n f r e s h s k i m m i l k . S u l f h y d r y l c o n t e n t s o f f r e s h a n d p o w d e r e d e g g w h i t e w e r e i n g o o d a g r e e m e n t w i t h l i t e r a t u r e v a l u e s f o r f r e s h e g g w h i t e . T h e d i s u l f i d e c o n t e n t s o b t a i n e d a g r e e d r e a s o n a b l y w e l l w i t h a v a l u e c a l c u l a t e d f r o m a m i n o a c i d c o m p o s i t i o n ( F e v o l d , 1 9 5 1 ) a n d t h e p r o t e i n c o n c e n t r a t i o n i n t h e t o t a l s o l i d s o f e g g w h i t e ( F e e n e y , 1 9 6 4 ) . N o d i r e c t d a t a w e r e a v a i l a b l e i n t h e l i t e r a t u r e . A s w i t h m i l k p r o d u c t s i t i s u n l i k e l y t h a t d r i e d a n d f r e s h e g g w h i t e d i f f e r s i g n i f i c a n t l y i n t h e i r S H o r S S c o n t e n t s . T h e S H c o n t e n t s f o r f l o u r w e r e s l i g h t l y h i g h e r t h a n t h e r e p o r t e d v a l u e s . S o m e u n c e r -

SH- AND SS-GROUPS IN FOOD PROTEINS-51

t a i n t y i n t h e v a l u e s w a s u n a v o i d a b l e b e

c a u s e l o w a b s o r b a n c e s h a d t o b e u s e d f o r c a l c u l a t i o n , a s h i g h c o n c e n t r a t i o n s o f f l o u r d e v e l o p e d t u r b i d i t y . T h e S S c o n t e n t s o f f l o u r a g r e e d w i t h t h e p u b l i s h e d d a t a . T h e c r u d e g l u t e n u s e d i n t h i s s t u d y

h a d a p p r o x i m a t e l y 9 0 % p u r i t y s o t h a t t h e v a l u e o b t a i n e d f o r t o t a l S H i s w i t h i n t h e a g r e e a b l e r a n g e ( K a s a r d a e t a l . , 1 9 7 1 ) .

A p r o b l e m e n c o u n t e r e d d u r i n g t h e c o u r s e o f t h e s e m e a s u r e m e n t s w a s t h e f a i l u r e o f t h e r e d u c e d p r o t e i n s t o d e v e l o p c o l o r a f t e r d i s s o l v i n g i n u r e a o r S D S a n d a d d i t i o n o f E l l m a n ’s r e a g e n t . T h i s w a s p a r t i c u l a r l y n o t i c e a b l e i n t h e c a s e o f f l o u r w h e r e a c r y s t a l l i n e p r e c i p i t a t e , p r e

s u m a b l y o f G u H C l , a p p e a r e d a f t e r a d d i t i o n o f T C A . T h i s p r e c i p i t a t e w a s s o m e w h a t s o l u b l e i n T C A a n d a l m o s t d i s a p p e a r e d a f t e r t h e s e c o n d w a s h i n g . T h e f a i l u r e t o d e v e l o p c o l o r c o u l d b e o v e r c o m e b y f u r t h e r d i l u t i o n o f t h e r e a c

t i o n m i x t u r e w i t h T r i s - G l y b u f f e r . T h e e x p l a n a t i o n o f t h i s d i f f i c u l t y a p p e a r s i n F i g u r e 2 . T h e p H o f t h e r e a c t i o n m i x t u r e h a s a m a r k e d e f f e c t o n t h e r a t e o f c o l o r d e v e l o p m e n t . A p p a r e n t l y , e n o u g h T C A s o m e t i m e s c l i n g s t o t h e t e s t t u b e w a l l o r i s a b s o r b e d i n t h e p r e c i p i t a t e , p a r t i c u l a r l y w h e n c r y s t a l s o f G u H C l a r e p r e s e n t , t o l o w e r t h e p H o f t h e r e a c t i o n m i x t u r e . T h i s s l o w s o r p r e v e n t s t h e c o l o r d e v e l o p m e n t . T h e p r o b l e m w a s l a r g e l y s o l v e d b y

i n c r e a s i n g t h e b u f f e r c o n c e n t r a t i o n . W h e n t h e p H w a s m a i n t a i n e d a t a b o u t 8 , c o l o r d e v e l o p m e n t w a s c o m p l e t e i n 5 m i n f o r

a l l p r o t e i n s t e s t e d , a n d w a s s t a b l e f o r a t l e a s t 2 h r .

T h e r e c o v e r i e s o f S H a n d S S a d d e d a s ( 3 - l a c t o g l o b u l i n t o f o o d p r o d u c t s r a n g e d f r o m 9 1 - 9 8 . 3 % a n d f r o m 8 9 — 1 0 2 % f o r S H a n d S S , r e s p e c t i v e l y ( T a b l e 3 ) .

REFERENCESAnnan, W.O. and Manson, W. 1969. A frac

tionation of the as-casein complex of bovine milk. J. Dairy Res. 36: 259.

Atassi, M.Z., Habeeb, A.F.S.A. and Rystedt, L. 1970. Lack of immunochemical cross-reaction between lysozyme and a-lactalbumin and comparison of their conformations. Biochim. Biophys. Acta 200: 184.

Beveridge, H.J.T. and Nakai, S. 1970. Effects of chemical modification with 2-phenyl-l,4- dibromoacetoin on asl-casein. J. Dairy Sci. 53: 1532.

Ellman, G.D. 1959. Tissue sulfhydryl groups.Arch. Biochem. Biophys. 82: 70.

Feeney. R.E. 1964. Egg proteins. “Symposium on Food Proteins and Their Reactions,” Ed. Schultz, H.W. and Anglmeir, A.F. Avi Publishing Co., Inc., Westport, Conn.

Fig. 2 —E ffe c t o f p H on c o lo r deve lopm ent fo r egg w h ite in the presence o f 0.5% SDS. C ond itions: 0.1 m l o f d ilu te d egg w h ite (12 .6 m g p ro te in /m l) , 2 .9 m l SDS b u ffe r, 0 .0 2 m l El¡man's reagent. SH = 5 2 .6 pM /g p ro te in .

Table 3—Recovery o f su lfh y d ry l and d isu lfide groups o f /3-lactoglobulin fro m foodproducts

Recovery (%)

P roduct SH SS

Raw skim m ilk 98.3 102Fresh egg w h ite 91.0 101Crude gluten 91 .0 89

Feeney, R.E., Osuga, D.T., Lind, S.B. and Miller, H.T. 1966. The egg white proteins of the Adelie penquin. Comp. Biochem. Physiol. 18: 121.

Fernandez-Diez, M.J., Osuga, D.T. and Feeney, R.E. 1964. The sulfhydryls of avian ovalbumin, bovine /3-lactoglobulin and bovine serum albumin. Arch. Biochem. Biophys. 107: 449.

Fevold, H.L. 1951. Egg Proteins. Adv. Protein Chem. 6 : 187.

Fothergill, L.A. and Fothergill, J.E. 1970. Thiol and disulfide contents of hen ovalbumin. C-Terminal sequence and location of disulfide bond. Biochem. J. 116: 555.

Hoagland, P.D., Thompson, M.P. and Kalan, E.B. 1971. Amino acid composition of (*-3 -, aS4- and (Xg5 -caseins. J. Dairy Sci. 54: 1103.

Kalab, M. 1070. Factors affecting the Ellman determination of sulfhydryl groups in skimmilk powder and gels. J. Dairy Sci. 53: 711.

Kasarda, D.D., Nimmo, C.C. and Kohler, G.O.1971. Protein and the amino acid composi

tion of wheat fractions. In “Wheat Chemistry and Technology,” Ed. Pomeranz, Y. American Association of Cereal Chemists, Inc., St. Paul, Minn.

Kekwick, R.A. and Cannan, R.K. 1936. The hydrogen ion dissociation curve of the crystalline albumin of the hen’s egg. Biochem. J. 30: 227.

Kuninori, T. and Sullivan, B. 1968. Disulfide- sulfhydryl interchange studies of wheat flour. 2. Reaction of glutathione. Cereal Chem. 45: 486.

Lyster, R.L.J. 1964. The free and masked sulfhydryl groups of heated milk and milk powder and a new method for their determination. J. Dairy Res. 31: 41.

MacDonnell, L.R., Silva, R.B. and Fenney, R.E. 1951. The sulfhydryl groups of ovalbumin. Arch. Biochem. Biophys. 32: 288.

MacKinlay, A.G. and Wake, R.G. 1971. k- Casein and its attack by rennin (chymosin). In “Milk Proteins,” Ed. McKenzie, H.A., Vol 2, p. 175. Academic Press, New York and London.

Manning, P.B. Heinselman, A.L., Jenness, R. and Coulter, S.T. 1969. Method for determining sulfhydryl and disulfide groups in milk proteins. (Abstr.) J. Dairy Sci. 52: 8 8 6 .

McKenzie, H.A. 1967. Milk proteins. Adv. Protein Chem. 22: 55.

McKenzie, H.A. 1971. /3-Lactoglobulin. In “Milk Proteins,” Ed. McKenzie, H.A., Vol 2, p. 257. Academic Press, New York and London.

Nakai, S., Wilson, H.K. and Herreid, E.O. 1965. Effect of changes in sulfur compounds on stability and gelation of caseins and of sterile concentrated milk. J. Dairy Sci. 48: 431.

Nakai, S., Toma, S.J. and Nakahori, C. 1972. Fractionation of caseins directly from skim milk by gel chromatography. 2. Elution with phosphate buffers. J. Dairy Sci. 55: 30.

Pofahl, T.R. and Vakaleris, D.G. 1968. Effects of heat on sulfhydryl and disulfide groups of milk proteins as measured by the speetro- fluorometric method. J. Dairy Sci. 51: 1345.

Sasago, K., Wilson, H.K. and Herreid, E.O. 1963. Determination of sulfhydryl and disulfide groups in milk by p-chloromercuribenzoate-dithizone method. J. Dairy Sci. 46: 1348.

Sawyer, W.H. 1969. Complex between /3-lactoglobulin and k-casein. A review. J. Dairy Sci. 52: 1347.

Shenstone, F.S. 1968. The gross composition, chemistry and physico-chemical basis of organization of the yolk and white. In “Egg Quality—A Study of the Hen’s Egg,” Ed. Carter, T.C. Oliver and Boyd, Edinburgh.

Suzuki, M., Coombs, T.L. and Vallee, E.L. 1969. Determination of protein thio groups by atomic absorption spectrometry. Anal. Biochem. 32: 106.

Toma, S.J. and Nakai, S. 1973. Calcium sensitivity and molecular weight of aS5 -casein. J. Dairy Sci. In press.

Tsen, C.C. and Bushuk, W. 1968. Reactive and total sulfhydryl and disulfide contents of flours of different mixing properties. Cereal Chem. 45: 58.

Yoshino, U., Wilson, H.K. and Herreid, E.O. 1962. Amperometric titration of sulfhydryl and disulfide groups in milk. J. Dairy Sci. 45: 1459.

Zittle, C.A. and Custer, J.H. 1963. Purification and some of the properties of as-casein and K-casein. J. Dairy Sci. 46: 1183.

Ms received 6/26/73; revised 8/18/73; accepted8/24/73.____________________________

Grants from the National Research Councilof Canada are gratefully acknowledged.

Ft. E. M U D G E T T , A . C. S M IT H ,' D. I. C. W ANG and S. A . G O L D B L IT H

Dept, o f N u tr it io n & F o od Science, Massachusetts In s t itu te o f Technology, Cambridge, M A 02 739

PREDICTION OF DIELECTRIC PRO PERTIES IN NONFAT MILK AT FREQ UENCIES AND TEM PERATURES OF INTEREST IN MICROWAVE PROCESSING

INTRODUCTION

C H E M I C A L S I M U L A T I O N s t u d i e s b y t h e a u t h o r s ( M u d g e t : e t a l . , 1 9 7 1 ) s h o w e d t h a t d i e l e c t r i c l o s s e s i n n o n f a t m i l k s o l u t i o n s c o u l d n o t b e d i r e c t l y p r e d i c t e d b y c h e m i c a l c o m p o s i t i o n i n t e r m s o f p r o x i m a t e a n a l y s i s . R e s u l t s s u g g e s t e d , h o w e v e r , t h a t l o s s b e h a v i o r m i g h t b e p r e d i c t e d b y c o n s i d e r i n g m i l k a s a c o m p l e x a q u e o u s i o n i c s o l u t i o n w h o s e e f f e c t i v e d i s s o l v e d s a l t s l e v e l w a s m u c h l o w e r t h a n t o t a l a s h c o n t e n t , d u e t o i o n b i n d i n g a n d c o m p l e x i n g e f f e c t s . S u c h e f f e c t s w e r e d e m o n s t r a t e d b y d i e l e c t r i c m e a s u r e m e n t s o f m i l k a n d i t s c h e m i c a l a n a l o g s a n d b y t o t a l a n d f r e e c a t i o n m e a s u r e m e n t s , w h i c h s h o w e d a s u b s t a n t i a l f r a c t i o n o f m i l k s a l t s t o b e i n a n a s s o c i a t e d s t a t e .

T h e s e e f f e c t s a p p e a r t o r e s u l t f r o m s o l u t e - s o l u t e a n d s o l u t e - s o l v e n t i n t e r a c t i o n s w h i c h r e d u c e f r e e c h a r g e d e n s i t y i n s o l u t i o n . B i n d i n g e f f e c t s a r e b e l i e v e d t o r e s u l t f r o m i n c o m p l e t e d i s s o c i a t i o n o f s p a r i n g - l y - s o l u b l e m i l k s a l t s , e . g . , c a l c i u m p h o s p h a t e , a n d , p e r h a p s t o a l e s s e r e x t e n t , f r o m i o n b i n d i n g b y p r o t e i n s a t c h a r g e d s u r f a c e s i t e s . P a r e n t h e t i c a l l y , W a u g h a n d N o b l e ( 1 9 6 5 ) h a d o b s e r v e d t h e f o r m a t i o n o f m i l k m i c e l l e s , e . g . , c a l c i u m - p h o s p h a t e - c a s e i n c o m p l e x e s , a n d t h e c o m m o n i o n e f f e c t o n s p a r i n g l y s o l u b l e s a l t s i s t o f u r t h e r r e p r e s s t h e i r s o l u b i l i t y , e . g . , t h e f o r m a t i o n o f c a l c i u m p h o s p h a t e b y m a s s a c t i o n

f r o m t h e s e c o n d p h o s p h o r i c a c i d e q u i l i b r i u m . B i n d i n g i n t e r a c t i o n s t h u s r e d u c e f r e e c h a r g e d e n s i t y w i t h r e s p e c t t o a s h c o n t e n t b a s e d o n a n i n i t i a l a s s u m p t i o n o f c o m p l e t e m i l k s a l t s i o n i z a t i o n . N o n b i n d i n g e f f e c t s , o n t h e o t h e r h a n d , a r e b e l i e v e d t o r e s u l t f r o m t h e f o r m a t i o n o f h y d r a t i o n c o m p l e x e s b y h y d r o g e n b o n d i n g o f d i s s o l v e d i o n s . S u c h c o m p l e x e s a r e s e e n t o m i g r a t e b y c o n d u c t i o n m o r e s l o w l y t h a n i n d i v i d u a l i o n s , d u e t o r e d u c e d e l e c t r o p h o r e t i c m o b i l i t y . T h e s e s t u d i e s s u g g e s t t h a t n o n f a t m i l k m a y b e h a v e d i e l e c t r i c a l l y a s a n a q u e o u s i o n i c s o l u t i o n . S u c h a p o s s i b i l i t y i s o f m o r e g e n e r a l i n t e r e s t i n p r e d i c t i n g t h e h e a t i n g c h a r a c t e r i s t i c s o f l o w c o l l o i d a l c o n t e n t l i q u i d s y s t e m s i n m i c r o w a v e p r o c e s s i n g .

I n c o m p r e h e n s i v e s t u d i e s , C o l l i e e t a l( 1 9 4 8 ) m e a s u r e d t h e d i e l e c t r i c p r o p e r t i e s o f w a t e r w i t h t e m p e r a t u r e a n d f o u n d t h a t

1 Dept, of Animal Industries, University of Connecticut, Storrs, Conn.

t h e s e w e r e a c c u r a t e l y p r e d i c t e d b y t h e

D e b y e m o d e l s f o r p o l a r l i q u i d s :

= (K* - K° ), K 1 + (Xs/X) 2 ( 1 )

q u e n c i e s a n d t e m p e r a t u r e s o f m e a s u r e

m e n t .

EXPERIMENTAL

„ = (Ks - K 0 )(AS/X)1 + (X s /X )2 ( 2 )

w h e r e K 'w a n d a r e t h e r e l a t i v e d i e l e c

t r i c c o n s t a n t a n d l o s s o f p u r e w a t e r , K s a n d K 0 a r e t h e s t a t i c a n d o p t i c a l d i e l e c

t r i c Xs i s t h e c r i t i c a l w a v e l e n g t h , a n d X is t h e w a v e l e n g t h o f m e a s u r e m e n t . H a s t e d

e t a l . ( 1 9 4 8 ) a l s o s h o w e d t h a t d i e l e c t r i c

b e h a v i o r i n a q u e o u s s o l u t i o n s o f d i s s o l v e d i n o r g a n i c s a l t s c o u l d b e p r e d i c t e d b y m o d

i f y i n g t h e D e b y e m o d e l s t o r e f l e c t t w o e f f e c t s o f s a l t s d i s s o c i a t i o n o n w a t e r

b e h a v i o r . F i r s t , t h e b i n d i n g o f w a t e r m o l e c u l e s i n h y d r a t i o n s h e a t h s b y d i s s o l v e d

i o n s d e p l e t e s f r e e w a t e r a n d d e p r e s s e s t h e d i e l e c t r i c c o n s t a n t . S e c o n d , s a l t s i o n i z a t i o n i n c r e a s e s f r e e c h a r g e d e n s i t y i n s o l u t i o n a n d c a u s e s w a t e r t o a c t a s a c o n d u c t o r . T h e H a s t e d - D e b y e m o d e l s a r e g i v e n a s :

K 's = K'W- 2 8 C ( 3 )

„ „ A CK s = + --------------

1 0 0 0 w e o ( 4 )

w h e r e K 's a n d K s a r e t h e r e l a t i v e d i e l e c t r i c c o n s t a n t a n d l o s s o f a n a q u e o u s i o n i c s o l u t i o n , K w a n d a r e t h e c o r r e s p o n d i n g p r o p e r t i e s o f p u r e w a t e r , 5 i s a n a v e r

a g e h y d r a t i o n n u m b e r , i . e . , t h e a v e r a g e n u m b e r o f f r e e w a t e r m o l e c u l e s b o u n d b y

c o u n t e r i o n s o f t h e d i s s o l v e d s a l t s , A i s t h e e q u i v a l e n t c o n d u c t i v i t y o f t h e s o l u t i o n , C i s t h e d i s s o l v e d s a l t s c o n c e n t r a t i o n , co i s t h e a n g u l a r f r e q u e n c y a n d e G i s t h e d i e l e c t r i c c o n s t a n t o f v a c u u m . V a l u e s o f e q u i v a l e n t c o n d u c t i v i t y a r e f o u n d i n t h e I n t e r n a t i o n a l C r i t i c a l T a b l e s f o r a w i d e v a r i e t y o f i n o r g a n i c s a l t s .

T o c o n f i r m e s s e n t i a l m i l k b e h a v i o r a s a n a q u e o u s i o n i c s o l u t i o n , o r i g i n a l s t u d i e s w e r e e x t e n d e d o v e r a r a n g e o f f r e q u e n c i e s a n d t e m p e r a t u r e s o f i n t e r e s t i n m i c r o - w a v e p r o c e s s i n g . T h e s e i n c l u d e s t a n d i n g

w a v e m e a s u r e m e n t s o f m i l k a n d i t s c h e m i c a l a n a l o g s , m i l k c o n d u c t i v i t y m e a s u r e m e n t s a n d c a t i o n b i n d i n g s t u d i e s . D i e l e c t r i c p r o p e r t i e s o b t a i n e d f r o m t h e m e a s u r e m e n t s w e r e t h e n c o m p a r e d w i t h H a s t e d - D e b y e m o d e l p r e d i c t i o n s a t f r e

Sam plesSam ples w ere prepared at co n cen tra tio n s

(w /v) o f 6% casein , 10% lacto se , 2% sodium chloride, 18% sy n th etic m ilk and 18% n o n fat dried milk with three or m ore m easurem ents at these co n cen tratio n s and on e-h alf co n cen tra tion levels obtained w ith distilled w ater dilutions. Preparation and co m p osition o f n o n fat dried m ilk solids and ch em ical analogs used have been previously reported.

D ielectric m easurem ents (slo tted line techn iqu e)

Standing wave m easurem ents were m ade at frequencies o f 3 0 0 , 1 ,0 0 0 and 3 ,0 0 0 MHz at tem peratures o f 2 5 , 3 5 , 4 5 and 5 5 °C in w ater- jack ete d sam ple holders w ith tem perature co n trol by a therm ostated circu lating w ater bath . The m ethod (R o b erts and V o n H ippel, 1 9 4 6 ) and equipm ent used have been previously described by Pace e t al. ( 1 9 6 8 ) .

Digital com pu tation

D ielectric properties from standing wave m easurem ents and H asted-D ebye m odel predictions were calculated by m eans o f F o rtran programs w ritten for the IBM 3 6 0 and 1 1 3 0 digital com puters.

C ond uctance m easruem ents

C onductivity m easurem ents w ere m ade at tem peratures o f 2 5 , 3 5 , 4 5 and 5 5 °C in a w ater- jack ete d cell w ith electrod es 1 cm 2 m ounted 1 cm apart with tem perature co n tro l by a therm ostated circu lating w ater bath . T he electrod es were platinized by e lectrod ep osition from ch loroplatin ie acid solutions w ith an added trace o f lead aceta te . T h e cell was calibrated w ith standard co n cen tratio n s o f aqueous sodium chloride. C ell con stan ts at each tem perature were determ ined from values o f equ ivalent co n ductivity published in the In tern ation al C ritical Tables by resistance m easurem ents. R esistances were m easured by a G eneral R ad io 6 5 0 A im pedance bridge driven by an extern al 1 KHz oscillator to ob tain m axim um sensitivity . Bridge balance was observed on an R C A W5 1A oscilloscop e.

T ota l and free cation con cen tration s

Free cation co n cen tratio n s fo r sodium , potassium ar.d calcium were determ ined by ion- selective electrod e m easurem ents at tem peratures o f 2 5 , 3 5 , 45 and 5 5 °C in a w ater-jack eted sample holder w ith tem peratu re co n tro l by a therm ostated circu lating w ater bath . M easurem ents were made against a Corning silver/silver chloride referen ce electrod e at co n tro lled levels o f pH and ion ic strength calibrated by standard dilutions o f chloride salts at each tem peratu re. E lectrod es were m anufactured by the Corning

5 2 -JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

DIELECTRIC PROPERTIES IN NONFAT MILK-53

8 0 1—

7 0 -

6 0 -

5 0 -

4 0 -

JO O O M H z------- / r - — ,* - 3 0 0 0 MHz

a^ IO O O M H z

3 0 0 0 MHz Û

0 >0 )

b

3 0 -

2 0 -

1 0 -o Model p red ic tions

à • Standing wave measurements

2 5 3 5 4 5Tem perature (°C )

5 5

Fig. 1-P re d ic te d vs. measured d ie le c tric loss in n o n fa t m ilk fro m Fig. 2 -P re d ic te d vs. measured d ie le c tric con s tan t in n o n fa t m ilk a t3 0 0 -3 .0 0 0 M H z. 2 5 - 5 5° C. 1.000 and 3 ,0 00 M H z. 2 5 - 5 5 ° C.

Glass Co. o f M edfield, Mass. M illivolt m easurem ents were made on an expanded scale PH M 26C m eter m anufactured by the R adiom eter C orp ., Copenhagen, D enm ark. T o ta l cation con cen tration s were measured by a tom ic ab sorption or flam e p h otom eter in chem ical simulation studies w hich have been previously reported (M udgett et al., 1 9 7 1 ).


A N I N I T I A L d i s s o l v e d s a l t s c o n c e n t r a

t i o n o f 0 . 2 5 M s o d i u m c h l o r i d e e q u i v a

l e n t s w a s e s t i m a t e d b y a s s u m i n g t h a t m i l k s a l t s w e r e c o m p l e t e l y i o n i z e d a n d c o u l d t h e r e f o r e b e a p p r o x i m a t e d b y t o t a l a s h c o n t e n t . T h i s a s s u m p t i o n w a s m o d i f i e d b y d i e l e c t r i c m e a s u r e m e n t s o f m i l k a n d

i t s c h e m i c a l a n a l o g s , a n d b y c a t i o n b i n d i n g s t u d i e s w h i c h s h o w e d e f f e c t i v e d i s s o l v e d s a l t s c o n c e n t r a t i o n s t o b e m u c h l o w e r . T h e s e r e s u l t s h a v e b e e n v e r i f i e d i n

t w o w a y s b y c u r r e n t s t u d i e s . F i r s t , m i l k c o n d u c t i v i t y m e a s u r e m e n t s f r o m 2 5 — 5 5 ° C s h o w e f f e c t i v e d i s s o l v e d s a l t s l e v e l s o f 0 . 1 0 0 ± 0 . 0 0 2 M s o d i u m c h l o r i d e

e q u i v a l e n t s a t a l l t e m p e r a t u r e s o f m e a s u r e m e n t . T h e s e w e r e d e t e r m i n e d b y c o m p a r i s o n o f m i l k c o n d u c t i v i t i e s w i t h s t a n d a r d c o n c e n t r a t i o n s o f s o d i u m c h l o r i d e . S e c o n d , m i l k c a t i o n b i n d i n g l e v e l s f r o m

2 5 — 5 5 ° C w e r e a l s o e s s e n t i a l l y c o n s t a n t , a s s h o w n i n T a b l e 1 . T h e s e r e s u l t s w e r e s o m e w h a t u n e x p e c t e d s i n c e i t i s k n o w n t h a t t h e s o l u b i l i t y o f i n o r g a n i c s a l t s

g e n e r a l l y i n c r e a s e s o r d e c r e a s e s w i t h t e m p e r a t u r e . B u t r e s u l t s a r e c o n s i s t e n t w i t h o b s e r v e d c o n d i t i o n s f o r t h e f o r m a

t i o n o f m i l k m i c e l l e s b y N o b l e a n d W a u g h( 1 9 6 5 ) a n d w i t h t h e p o s s i b i l i t y o f t i g h t b i n d i n g o f i o n s b y d i s s o l v e d p r o t e i n s .

I n a d d i t i o n , H a s t e d - D e b y e m o d e l p r e d i c t i o n s o f d i e l e c t r i c p r o p e r t i e s f o r e f f e c t i v e s a l t s c o n c e n t r a t i o n s o f 0 . 1 M s o d i u m c h l o r i d e e q u i v a l e n t s a g r e e c l o s e l y w i t h

v a l u e s o b t a i n e d f r o m s t a n d i n g w a v e a n d

c o n d u c t i v i t y m e a s u r e m e n t s ( 3 0 0 —3 , 0 0 0 M H z , 2 5 —5 5 ° C ) . F i g u r e 1 c o m p a r e s p r e d i c t e d d i e l e c t r i c l o s s e s w i t h t h o s e o b

t a i n e d f r o m c o n d u c t i v i t y m e a s u r e m e n t s . M e a s u r e d v a l u e s a r e w i t h i n 1 0 % o f p r e d i c

t i o n s a t a l l f r e q u e n c i e s a n d t e m p e r a t u r e s

Table 1—C ation b ind ing levels in no n fa t m ilk fro m 25—55°C

C ation concentra tions(m g/100 m l)

C ation T o ta l3

Freeb (°C)

25 35 45 55

Potassium 280 234 253 233 195Sodium 85 69 69 69 76Calcium 123 36 47 43 44

a Atomic absorption b Ion-selective electrode

a n d a r e g e n e r a l l y w i t h i n 5 % o f p r e d i c

t i o n s a t 1 , 0 0 0 a n d 3 , 0 0 0 M H z . S t a n d i n g

w a v e m e a s u r e m e n t s ( n o t s h o w n ) w e r e a l s o w i t h i n 1 0 % o f p r e d i c t i o n s a t 1 , 0 0 0 a n d 3 , 0 0 0 M H z b u t w e r e 1 0 —2 5 % l o w e r t h a n p r e d i c t i o n s a t 3 0 0 M H z . T h i s i s a t t r i b u t e d t o e l e c t r o d e p o l a r i z a t i o n

e f f e c t s a t f r e q u e n c i e s b e l o w 1 , 0 0 0 M H z d u e t o l o n g e r e x c i t a t i o n p e r i o d s . W h e n c o n d u c t i v i t y m e a s u r e m e n t s a r e c o n s i d

e r e d i n t e r m s o f s t a n d a r d d e v i a t i o n s , c l o s e

a g r e e m e n t i s i n d i c a t e d b e t w e e n m e a s u r e d a n d p r e d i c t e d l o s s e s .

F i g u r e 2 c o m p a r e s p r e d i c t e d d i e l e c t r i c c o n s t a n t s w i t h t h o s e o b t a i n e d f r o m s t a n d i n g w a v e m e a s u r e m e n t s a t 1 , 0 0 0 a n d 3 , 0 0 0 M H z , 2 5 — 5 5 ° C . W h i l e t h e s e m e a s u r e m e n t s a r e w i t h i n 1 5 % o f m o d e l p r e d i c t i o n s , t h e y a r e s o m e w h a t l o w e r t h a n e x p e c t e d . T h i s s u g g e s t s a d d i t i o n a l b i n d i n g o r e x c l u s i o n o f f r e e w a t e r m o l e c u l e s b y n o n i o n i c c o n s t i t u e n t s . W h e n m o d e l p r e d i c t i o n s w e r e c o r r e c t e d f o r t h e s e e f f e c t s b y c a s e i n a n d

l a c t o s e f r o m a n a l o g d e p r e s s i o n m e a s u r e m e n t s a t 3 , 0 0 0 M H z , 2 5 ° C , m e a s u r e d v a l u e s w e r e w i t h i n 5 % o f p r e d i c t i o n s f o r

1 , 0 0 0 a n d 3 , 0 0 0 M H z a t a l l t e m p e r a t u r e s .

M o d e l c o r r e c t i o n s a r e o m i t t e d f r o m F i g u r e 2 f o r t h e s a k e o f c l a r i t y a n d a r e s h o w n i n T a b l e 2 . S t a n d i n g w a v e m e a s u r e m e n t s a t 3 0 0 M H z c o u l d n o t b e m a d e w i t h t h e a v a i l a b l e s a m p l e h o l d e r . H o w e v e r , s t a n d i n g w a v e m e a s u r e m e n t s o f

w a t e r a t 3 0 0 M H z c o m b i n e d w i t h c o n

5 4 -JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

d u c t i v i t y m e a s u r e m e n t s o f m i l k f r o m 2 5 — 5 5 ° C a g r e e c l o s e l y w i t h t h e m o d e l

p r e d i c t i o n s .T h e s e r e s u l t s c o n f i r m t h a t t h e n o n f a t

d r i e d m i l k s o l u t i o n s u n d e r s t u d y b e h a v e e s s e n t i a l l y a s a q u e o u s i o n i c s o l u t i o n s w i t h a n e f f e c t i v e d i s s o l v e d s a l t s c o n c e n t r a t i o n o f a p p r o x i m a t e l y 0 . 1 M s o d i u m c h l o r i d e

e q u i v a l e n t s w h i c h i s c o n s t a n t f o r t h e r a n g e o f t e m p e r a t u r e s s t u d i e d . M o r e

i n t e r e s t i n g l y , t h e y s u g g e s t t h a t d i e l e c t r i c

b e h a v i o r f o r l i q u i d f o o d s y s t e m s o f l o w

c o l l o i d a l c o n t e n t m a y b e p r e d i c t e d f r o m

c o n d u c t i v i t y m e a s u r e m e n t s a t t e m p e r a t u r e s o f i n t e r e s t f o r a p a r t i c u l a r m i c r o - w a v e p r o c e s s , w i t h c a l c u l a t i o n o f d i e l e c

t r i c p r o p e r t i e s a s c o n t i n u o u s f u n c t i o n s o f f r e q u e n c y a n d t e m p e r a t u r e b y t h e

H a s t e d - D e b y e m o d e l s .

E f f e c t i v e d i s s o l v e d s a l t s c o n c e n t r a t i o n s

i n s o d i u m c h l o r i d e e q u i v a l e n t s m a y b e

o b t a i n e d f r o m l i q u i d c o n d u c t i v i t y m e a s u r e m e n t s a s a f u n c t i o n o f t e m p e r a t u r e .

W a t e r - p r o p e r t i e s a t c o r r e s p o n d i n g t e m p e r a t u r e s m a y t h e n b e o b t a i n e d f r o m t h e

d a t a o f C o l l i e e t a l . ( 1 9 4 8 ) . L i q u i d c o n d u c t i v i t y m e a s u r e m e n t s c a n b e m a d e o v e r w i d e t e m p e r a t u r e r a n g e s q u i c k l y , i n e x

p e n s i v e l y , a n d a c c u r a t e l y ; c o n v e r s e l y ,

Table 2—D ie lec tr ic constan t depression by no n io n ic constituen ts a t 3 ,0 00 M H z, 25°C

C once n tra tionC o n s titu e n t (% w/v) A K '

Casein 6.0 - 2 .9Lactose 10.0 - 2 .0Tota l 16.0 - 4 .9

s t a n d i n g w a v e m e a s u r e m e n t s a r e t i m e

c o n s u m i n g a n d e x p e n s i v e . C o n d u c t i v i t y

m e a s u r e m e n t s a l s o p e r m i t d i r e c t o b s e r v a t i o n o f t e m p e r a t u r e e f f e c t s o n e f f e c t i v e d i s s o l v e d s a l t s l e v e l s . T h e r e s t r i c t i o n o n c o l l o i d a l c o n t e n t is e m p h a s i z e d , a l t h o u g h

n o t r i g o r o u s l y d e f i n e d a t t h i s t i m e , s i n c e d i e l e c t r i c b e h a v i o r i n l i q u i d s i s k n o w n t o

b e m o d i f i e d b y r e l a t i v e l y s m a l l v o l u m e f r a c t i o n s o f s u s p e n d e d c o l l o i d s . A s u b

s e q u e n t p a p e r w i l l c o n s i d e r s u c h b e h a v i o r

a n d i t s p o s s i b l e i m p l i c a t i o n s i n m i c r o w a v e f o o d p r o c e s s i n g .

REFERENCESCollie, C.H., Hasted, J.B. and Ritson, D.M.

1948. The dielectric properties of water and heavy water. Proc. Roy. Phys. Soc. (London) 60: 145.

Hasted, J.B., Ritson, D.M. and Collie, C.H. 1948. Dielectric properties of aqueous ionic solutions. Parts 1 and 2. J. Chem. Phys. 16: 1 .

Mudgett, R.E., Smith, A.C., Wang, D.I.C. and Goldblith, S.A. 1971. Prediction on the relative dielectric loss factor in aqueous solutions of nonfat dried milk through chemical simulation. J. Food Sci. 36: 915.

Noble, R.W. Jr. and Waugh, D. 1965. 1. Casein micelles. Formation and structure. J. Am. Chem. Soc. 87: 2236.

Pace, W.E., Westphal, W.B. and Goldblith, S.A. 1968. Dielectric properties of commerical cooking oils. J. Food Sci. 33: 30.

Roberts, S. and Von Hippel, A. 1946. A new method for measuring dielectric constant and loss in the range of centimeter waves. J. Appl. Phys. 17: 610.

Waugh, D. and Noble, R.W. Jr. 1965. 2. Casein micelles. Formation and structure. J. Am. Chem. Soc. 87: 2246.

Ms received 6/18/73; revised 9/7/63; accepted9/11/73.

This work was supported by U.S. Public Health Service, National Institutes of Health Grant No. FD-00166-02 (formerly UI-00784) entitled ’‘Measurement and Prediction of Dielectric Properties of Foods.” R.E.M. gratefully acknowledges support by an NDEA Title IV Graduate Fellowship during a portion of this work, and the 1972—73 Nestle Graduate Fellowship award by the Institute of Food Technologists. The authors also gratefully acknowledge the generous assistance of Mr. William B. Westphal of the M.I.T. Insulation Research Laboratory.

P A U L B. M c N U L T YDept, o f A g r ic u ltu ra l Engineering, U n ive rs ity College, D ub lin , Ire land

and H O W A R D R. M O S K O W ITZ Pioneering Research La bo ra to ry , U.S. A rm y N a tick Labs, N a tick , M A 01760

INTENSITY-TIM E CURVES FOR FLAVORED OIL-IN-WATER EM ULSIONS

INTRODUCTIONM c N U L T Y A N D K A R E L ( 1 9 7 3 a , b , c ) s t u d i e d t h e e f f e c t s o f v a r y i n g s o m e p h y s i c a l - c h e m i c a l p r o p e r t i e s o n t h e e x t e n t a n d r a t e o f f l a v o r r e l e a s e i n a n i n v i t r o , o i l - i n - w a t e r ( o / w ) e m u l s i o n . T h e y p r e d i c t e d t h a t f l a v o r r e l e a s e i n t h e m o u t h f r o m l i q u i d o / w e m u l s i o n s s h o u l d n o r m a l l y b e r a p i d , t ( l / 2 ) < 1 5 s e c i . e . , e q u i l i b r a t i o n h a l f - l i f e s h o u l d n o r m a l l y b e l e s s t h a n 1 5 s e c . T h e y a l s o p r e d i c t e d t h a t t h e p o t e n t i a l e x t e n t o f f l a v o r r e l e a s e i n t h e m o u t h i n c r e a s e s w i t h i n c r e a s e i n ( a ) t h e o i l - w a t e r p a r t i t i o n c o e f f i c i e n t , K p , i . e . , a s t h e f l a v o r s o l u b i l i t y i n t h e o i l i n

c r e a s e s ; ( b ) t h e i n i t i a l o i l v o l u m e f r a c t i o n , 0 j ; ( c ) t h e e m u l s i o n d i l u t i o n f a c t o r , D F e m , d u e t o m i x i n g w i t h s a l i v a .

N e i l s o n ( 1 9 5 7 ) r e p o r t e d t h a t f l a v o r s , s u c h a s t h a t i m p a r t e d

b y b i t t e r s u b s t a n c e s , c h a n g e i n i n t e n s i t y o v e r t i m e . T h e m e t h o d o f i n t e n s i t y - t i m e e v a l u a t i o n c a n b e u s e d t o r e l a t e t h e p e r c e i v e d t a s t e i n t e n s i t y o f a s u b s t a n c e a s t i m e v a r i e s . T h e p a n e l i s t m a y b e i n s t r u c t e d t o s w a l l o w t h e s u b s t a n c e , e x p e c t o r a t e i t , o r k e e p i t i n t h e m o u t h . T y p i c a l l y , f o r s u b s t a n c e s d i s s o l v e d i n a q u e o u s m e d i a , t a s t e i n t e n s i t y i n c r e a s e s r a p i d l y a n d t h e n d e

c l i n e s s l o w l y a n d s y s t e m a t i c a l l y a s t h e t o n g u e i s s t i m u l a t e d c o n t i n u o u s l y ( M e i s e l m a n , 1 9 7 2 ) . T h e o b j e c t i v e s o f t h i s s t u d y w e r e ( a ) t o d e t e r m i n e a p p r o p r i a t e f u n c t i o n s r e l a t i n g t a s t e i n

t e n s i t y t o t i m e ; a n d ( b ) t o e v a l u a t e t h e e f f e c t o f f l a v o r r e l e a s e o n i n t e n s i t y - t i m e f u n c t i o n s .

Table 1—Parameters o f in te n s ity -tim e curves fo r flavored o /w emulsions

Linear fu n c tio n E xponentia l fu n c tio nppm X 10 3 1 == K T + C Log I = K T + C

</>i anetho le K C R F K C R F

0.125 0. .23 7.88 .98 217.6 9.5 -0 .71 .97 143.22.0 .16 6.49 .93 81.3 6.9 -0 .0 4 .98 328.24 .0 .38 6.86 .99 462.6 16.0 -7 .8 5 .98 219.48.0 .24 9.40 .96 109.9 10.8 -0 .7 9 .99 1087.0

16.0 .45 13.16 .99 397.1 19.5 -4 .4 0 .97 176.20.25 0 .50 6.28 .99 1188.0 19.5 -1 0 .7 8 .97 149.8

2.0 .32 6.92 .93 66 .4 13.6 -5 .3 5 .93 60 .04.0 .17 13.67 .89 36.9 8.2 5.66 .97 185.28.0 .36 4.47 .99 371.2 15.2 -9 .1 7 .96 105.5

16.0 .37 7.09 .99 648.8 14.5 -5 .1 6 .94 77.50.5 0 .34 6.02 .97 161.5 14.2 -6 .8 5 .93 70.4

2.0 .36 6.50 .99 603.7 15.6 -7 .61 .97 176.14.0 .12 8.72 .96 114.0 8.4 3.77 .96 136.18.0 .32 9.51 .97 197.9 14.3 —4.03 .99 695.9

16.0 .27 8.84 .96 252.5 11.8 -2 .3 6 .98 235.8Lo garith m ic fu n c tio n Power fu n c tio n

i = K logT + C Logl = K logT + C

0.125 0. 11.81 -1 .8 9 .97 143.2 0.41 0.26 .99 328.72.0 8.62 -0 .9 0 .98 320.2 0.45 0.21 .94 77.134.0 20.25 -9 .8 7 .98 219.4 0.57 0.11 .98 205.58.0 13.47 -2 .1 4 .99 1087.0 0.45 0.36 .99 372.6

16.0 24.23 -6 .8 2 .97 176.2 0.46 0.61 .99 816.1

0.25 0. 24 .42 -1 3 .2 2 .97 149.8 0.51 0.22 .99 1124.0

2.0 16.82 -7 .0 2 .93 60 .0 0.48 0.16 .98 219.9

4.0 10.17 4.65 .97 185.2 0.36 0.49 .92 58.5

8.0 19.10 -1 0 .0 8 .96 105.5 0.53 0.21 .97 187.1

16.0 18.29 -6 .9 9 .94 77.5 0.54 0.31 .99 1004.0

0.5 0. 18.08 -8 .6 5 .93 70.4 0.56 -0 .1 0 .99 383.9

2.0 19.55 -9 .5 6 .97 176.1 0.45 -0 .2 8 .96 122.9

4.0 6.70 3.10 .96 136.1 0.28 0.45 .96 111.9

8 .0 18.21 -5 .8 5 .99 695.9 0.43 0.45 .98 199.3

16.0 14.79 -4 .8 4 .98 235.8 0.45 0.39 .99 203.4

Volume 39 (1974)-JOURNAL OF FOOD SCIENCE- 5 5


EXPERIMENTALSam ple preparation

Liquid vegetable o/w em ulsions were prepared as described by M cN ulty ( 1 9 7 2 ) using Wesson oil and the Span 60-Tw een 60 em ulsifier system . T he em ulsifier H LB was 1 1 .8 , and the co n cen tratio n was 4 .3 8 % w/v o f oil plus em ulsifier. E m u lsification was by the “ agent-in-oil” m ethod (B ech er, 1 9 6 5 ) using a Waring Blendor at an autotran sform er setting o f 100 . F ive con cen tration s o f aneth ole (0 , 2 0 0 0 , 4 0 0 0 , 8 0 0 0 and 1 6 0 0 0 Mg/ml em ulsion), and three oil volum e fractions C0i = 0 .1 2 5 , 0 .2 5 and 0 .5 ) were used. An aqueous standard was prepared containing 3 0 0 Mg/ml anethole and 5 0 0 0 Mg/ml Tw een 6 0 . T he standard was assigned an arbitrary taste in tensity value o f 10.

In ten sity-tim e d eterm inationsAn untrained panel o f four fem ales and ten m ales sampled the em ul

sions at room tem perature (2 4 ± 1°C ). T he observers began by dipping their tongue, w ithout stirring, in to approxim ately 50 m l o f standard contained in a 50 ml Tri-Pour plastic beaker. A fter rinsing with distilled w ater the observers dipped their tongue, w ith out stirring, in to approxim ately 5 0 ml o f test em ulsion in a similar beaker, a t the presentation o f an audible 1-sec signal. A signal was presented every 5 sec (1-sec signal,4-sec silence). When th e second signal began, the observer made the first rating o f relative taste in tensity (with the standard = 1 0 ). T h e estim ates were made by the m ethod o f m agnitude e s tim a tio n -ra tio s o f estim ates reflected ratios o f relative taste intensities, so that an estim ate o f 20 designated 2x the in tensity o f the standard, and an estim ate o f 2 designated 1/5 the in ten sity o f the standard (M oskow itz, 1 9 7 0 ) .

T h e observers kept their tongues imm ersed in the em ulsion for a total o f 60 sec, and recorded their estim ate every 5 sec, for a to ta l o f 12 in tensity-tim e estim ates per em ulsion. A fter rinsing w ith distilled w ater at the end o f each test, the observer rested for at least 2 m in, and then proceeded to the n ext random ly chosen sam ple. T he observers were perm itted to recheck the standard before or after any set o f evaluations.

Prediction o f flavor release

The potential ex ten ts o f flavor release in the m ou th , Cw e/C w d, were predicted from Eq (1 ) derived from the m odel o f M cNulty and Karel (1 9 7 3 a ):

Cwe _ [4>i (K p ~ 1) + 11 (D Fem ~ Qj) , j .Cw d [0 i ( K p - 1) + D F em ] (1 - 0 j)

where Cwd = aqueous flavor con cen tration in the m outh im m ediately a fter em ulsion dilution with saliva; Cwe = potential equilibrium aqueous co n cen tratio n in the m outh which may be attained i f equilibrium is restored ; D F em and are defined elsew here and K p is given in equation 2:

“ L oe/C we (2)

where C o e , Cwe = flavor concen trations in the oil and aqueous phases at equilibrium , respectively. P artition co effic ien ts w ere determ ined by the m ethod o f M cN ulty ( 1 9 7 2 ) . Aqueous sam ples o f aneth ole were assayed by absorbance at 2 0 2 nm on a H itachi-Perkin Elm er Sp ectro p h otom eter, Model 139 .

RESULTS & DISCUSSIONT A B L E 1 p r e s e n t s t h e p a r a m e t e r s o f t h e i n t e n s i t y - t i m e c u r v e s , f i t t e d b y f o u r f a m i l i e s o f e q u a t i o n s ( l i n e a r , l o g a r i t h m i c , e x p o n e n t i a l a n d p o w e r f u n c t i o n s ) a s s h o w n b y E q ( 3 ) t o ( 6 ) :

L i n e a r : I = K T + C ( 3 )L o g a r i t h m i c : I = K l o g T + C ( 4 )E x p o n e n t i a l : l o g I = K T + C ( 5 )P o w e r : l o g I = K l o g T + C ( 6 )

w h e r e I i s i n t e n s i t y ; K i s t h e s l o p e ; T i s t i m e ; C i s a c o n s t a n t .

T h e j u d g m e n t s o f t h e 1 4 o b s e r v e r s w e r e a v e r a g e d a r i t h m e t i c a l l y t o y i e l d t h e m e a n e s t i m a t e s f o r t h e l i n e a r a n d l o g a

r i t h m i c f u n c t i o n s , a n d a v e r a g e d l o g a r i t h m i c a l l y ( g e o m e t r i c m e a n ) f o r t h e e x p o n e n t i a l a n d p o w e r f u n c t i o n s . I n e a c h i n s t a n c e t h e i n d e p e n d e n t v a r i a b l e w a s t o t a l t i m e e l a p s e d , a n d t h e d e p e n d e n t v a r i a b l e w a s m e a n m a g n i t u d e e s t i m a t i o n f o r a g i v e n

e m u l s i o n t a s t e a t o n e o f t h e 1 2 t i m e s . T h e s t a t i s t i c s f o r g o o d - n e s s - o f - f i t a r e t h e P e a r s o n c o r r e l a r i o n c o e f f i c i e n t ( r ) a n d t h e F

s t a t i s t i c ( d f = 1 , 1 2 ) f o r t h e s i g n i f i c a n c e o f r e g r e s s i o n . F i g u r e s 1 —4 p r e s e n t t h e f u n c t i o n s i n g r a p h i c a l f o r m , a n d i n d i c a t e t h e

p r e s e n c e o f d e p a r t u r e s f r o m s t r i c t l i n e a r i t y .T h e s l o p e o f e a c h f u n c t i o n i n T a b l e 1 i n d i c a t e s h o w r a p i d l y

t h e t a s t e i m p r e s s i o n i n c r e a s e s w i t h t i m e o f s t i m u l a t i o n . B o t h t h e l i n e a r a n d p o w e r f u n c t i o n s a r e g o v e r n e d b y s l o p e s l e s s t h a n 1 . 0 . F o r t h e l i n e a r f u n c t i o n t h i s m e a n s t h a t a 1 u n i t i n c r e a s e i n t h e t i m e o f s t i m u l a t i o n p r o d u c e s l e s s t h a n a 1 u n i t i n c r e a s e i n a p p a r e n t t a s t e i n t e n s i t y . T h e l i n e a r f u n c t i o n d e p e n d s u p o n t h e

u n i t s o f t i m e c h o s e n , a n d t h e p o w e r f u n c t i o n i s m o r e p r e f e r a b l e b e c a u s e t h e e x p o n e n t d o e s n o t d e p e n d u p o n t h e u n i t o f

t i m e o r t h e m o d u l u s n u m b e r .

Fig. 1 — R elation between the mean estim ate o f taste in te n s ity and elapsed tim e o f s tim u la tio n (by m agnitude estim ation). Each o f five concentra tions o f anetho le (0—16,000 p p m ) and three phase volumes (0 .125, 0 .25, 0 .5 ) was eva luated over a p e r io d o f 60 sec b y 14 observers (L inear fu n c tio n ). Points are connected b y a p p ro x im a tin g curves and straigh t lines.

Fig. 2 —R elation between the mean estim ate o f taste in te n s ity an d the loga rithm o f elapsed tim e (L oga rithm ic fu n c tio n ).

FLAVORED OIL-IN-WATER EMULSION-57

Fig. 3 -R e la t io n between the geom etric mean estim ate o f taste in te n s ity and the elapsed tim e o f s tim u la tio n (E xpo nen tia l fu n c tion ).

F i g u r e s 1—4 r e v e a l t h a t e a c h c o n t r o l e m u l s i o n ( i. e ., z e r o p p m a n e t h o l e ) s t i m u l a t e d a n a p p r e c i a b l e a n d u n e x p e c t e d r e s p o n s e . I t w a s f o u n d t h a t T w e e n 6 0 , a l t h o u g h a c o m m o n

f o o d s u r f a c t a n t , w a s r e s p o n s i b l e d u e t o i t s s t r o n g l y b i t t e r a n d

u n p l e a s a n t t a s t e . A t t h e l o w e r a n e t h o l e c o n c e n t r a t i o n s ( 2 0 0 0

a n d 4 0 0 0 p p m ) t h e t a s t e r e s p o n s e s w e r e g e n e r a l l y s l i g h t l y

l o w e r t h a n c o n t r o l p r e s u m a b l y b e c a u s e t h e t a s t e s e n s a t i o n s a r e m u t u a l l y s u p p r e s s e d o r m a s k e d . A t h i g h e r c o n c e n t r a t i o n s ( 8 0 0 0 a n d 1 6 0 0 0 p p m ) t h e a n e t h o l e t a s t e g e n e r a l l y b e c a m e

m o r e p r e d o m i n a n t b u t o n l y s l i g h t l y s o .

T h e n e g l i g i b l e d e p e n d e n c e o f t a s t e i n t e n s i t y o n a n e t h o l e

c o n c e n t r a t i o n s u g g e s t e d a n a n e t h o l e - T w e e n 6 0 i n t e r a c t i o n . T h i s w a s s u p p o r t e d b y i n v i t r o d a t a w h i c h r e v e a l e d t h a t t h e

p a r t i t i o n c o e f f i c i e n t s o f a n e t h o l e i n t h e v e g e t a b l e o i l - w a t e r s y s t e m s w e r e 1 5 . 7 a n d 5 0 9 i n t h e p r e s e n c e a n d a b s e n c e o f a q u e o u s T w e e n 6 0 r e s p e c t i v e l y . T h u s T w e e n 6 0 g r e a t l y i n

c r e a s e s t h e a q u e o u s s o l u b i l i t y o f a n e t h o l e p r o b a b l y d u e t o s o l u b i l i z a t i o n .

U s i n g K p = 1 5 . 7 a n d D F e m = 2 ( i . e . , a o n e - t o - o n e d i l u t i o n o f e m u l s i o n w i t h s a l i v a i n t h e m o u t h ) , t h e p o t e n t i a l e x t e n t s o f f l a v o r r e l e a s e w e r e p r e d i c t e d t o b e 1 . 5 9 , 1 . 9 2 a n d 2 . 6 8 f o r o i l

v o l u m e f r a c t i o n s o f 0 . 1 2 5 , 0 . 2 5 a n d 0 . 5 , r e s p e c t i v e l y . U n f o r -

Fig. 4—Relation between the geometric mean estimate o f taste intensityand the logarithm o f elapsed time (Power function).

t u n a t e l y i t w a s n o t p o s s i b l e t o r e l a t e t h e s e p r e d i c t i o n s t o i n t e n s i t y - t i m e o r i n t e n s i t y - c o n c e n t r a t i o n d a t a d u e t o t h e t a s t e i n t e r a c t i o n .

I n s u m m a r y , t h e i n t e r a c t i o n o f a n e t h o l e a n d T w e e n 6 0 s u g g e s t s t h a t s o l u t e - s u r f a c t a n t i n t e r a c t i o n s i n m u l t i c o m p o n e n t f o o d s y s t e m s m a y h a v e a n i m p o r t a n t a n d c o m p l e x e f f e c t o n f l a v o r p e r c e p t i o n T h i s o b s e r v a t i o n a u g m e n t s t h e f i n d i n g s o f M c N u l t y a n d K a r e l ( 1 9 7 3 b , c ) w h o r e p o r t e d t h a t s o l u t e -

s u r f a c t a n t i n t e r a c t i o n s a l t e r e d b o t h t h e e x t e n t s a n d r a t e s o f f l a v o r r e l e a s e i n t h e i r i n v i t r o s t u d i e s w h e r e S p a n 6 0 a n d T w e e n 6 0 w e r e t h e s u r f a c t a n t s a n d n - a l c o h o l s ( C 3 - C 8 ) a n d

c h o l e s t e r o l w e r e t h e m o d e l f l a v o r c o m p o u n d s . W e s u g g e s t t h a t t h e s e e x p e r i m e n t s s h o u l d b e r e p e a t e d u s i n g a n e m u l s i f i c a t i o n s y s t e m t h a t i s t a s t e l e s s i . e . , t h e c o n t r o l e m u l s i o n s w o u l d e x h i b i t n e g l i g i b l e t a s t e i n t e n s i t y . H e n c e w e c o u l d a s s e s s t h e e f

f e c t s o f t i m e a n d f l a v o r c o n c e n t r a t i o n o n p r e c e i v e d i n t e n s i t y i n s y s t e m s w h e r e t a s t e i n t e r a c t i o n s s h o u l d n o t o c c u r . I t w o u l d a l s o b e u s e f u l t o e v a l u a t e i n t e n s i t y - t i m e c u r v e s u s i n g t h e t e r

n a r y s y s t e m , f l a v o r - s u r f a c t a n t - w a t e r . T h i s w o u l d c o n s t i t u t e a n a d d i t i o n a l c h e c k o n t h e i n f l u e n c e o f f l a v o r r e l e a s e f r o m s u s

p e n d e d e m u l s i o n o i l d r o p l e t s o n f l a v o r p e r c e p t i o n . A n a l t e r n a t e a p p r o a c h w c u l d b e t o r e t a i n T w e e n 6 0 a s s u r f a c t a n t a n d a d d a n a p p r o p r i a t e s w e e t e n i n g a g e n t t o m a s k t h e b i t t e r s u r f a c t a n t t a s t e . H o w e v e r , t h i s w o u l d i n c r e a s e t h e c o m p l e x i t y o f

t h e s y s t e m a n d m i g h t a l s o r e s u l t i n m a s k i n g t h e f l a v o r a s w e l l a s t h e s u r f a c t a n t t a s t e .

REFERENCESBecher, P. 1965. “Emulsions: Theory and Practice.” Reinhold Publish

ing Corp., New York.McNulty, P.B. 1972 Factors affecting flavor release and uptake in O/W

emulsions. Ph.D. thesis, Massachusetts Institute of Technology, Cambridge.

McNulty, P.B. and Karel, M. 1973a. Factors affecting flavor release and uptake in O/W emulsions. 1. Release and uptake models. J. Food Technol. 8: 309.

McNulty, P.B. and Karel, M. 1973b. Factors affecting flavor release and uptake in O/W emulsions. 2. Stirred cell studies. J. Food Technol. 8: 319.

McNulty, P.B. and Karel, M. 1973c. Factors affecting flavor release and uptake in O/W emulsions. 3. Scale-up model and emulsion studies. J. Food Technol. In press.

Meiselman, H.L. 1972. Human taste perception. CRC Critical Reviews in Food Technol. 3: 89.

Moskowitz, H.R. 1970. Ratio scales of sugar sweetness. Perception & Psychophysics. 7: 315.

Neilson, A.J. 1957. Time-intensity studies. In “Flavor Research and Food Acceptance.” Reinhold Publishing Corp., New York.

Ms received 6/19/73; revised 8/31/73; accepted 9/6/73.The authors acknowledge the assistance of the panel who were

mostly students of the Dept, of Nutrition & Food Science, Massachusetts Institute of Technology, Cambridge and Professor M. Karel who made the laboratory facilities of the Department available for the study.

J. F. S U L L IV A N , Ft. P. K O N S T A N C E , M . J. C A L H O U N , F. B. T A L L E Y , J. C O R D IN G JR . a n d 0 . P A N A S IU K

U S D A E aste rn R e g io n a l Research C en te r, A R S , P h ila d e lp h ia , PA 1 9 1 1 8

FLAVOR AND STORAGE STABILITY OF EXPLOSION-PUFFED POTATOES: Nonenzymatic Browning

INTRODUCTION

N O N E N Z Y M A T I C b r o w n i n g p h e n o m e n a o c c u r f r e q u e n t l y d u r i n g f o o d p r o c e s s i n g a n d s t o r a g e . I n t h e m a j o r i t y o f c a s e s , t h i s n o n e n z y m a t i c b r o w n i n g i s a d i s t i n c t s i g n

o f a d e t e r i o r a t i o n o f t h e f l a v o r a n d t h e n u t r i t i o n a l v a l u e o f t h e f o o d i n q u e s t i o n

( B r a v e r m a n , 1 9 6 3 ) .

O n e o f t h e m a j o r c a u s e s o f t h e d e g e n e r a t i o n o f f l a v o r i n d e h y d r a t e d p o t a t o

p r o d u c t s i s t h e M a i l l a r d r e a c t i o n . T h i s a m i n o - c a r b o n y l r e a c t i o n o f r e d u c i n g

s u g a r s a n d a m i n o a c i d s r e s u l t s i n t h e f o r m a t i o n o f 2 - m e t h y l p r o p a n a l ( 2 M P ) a n d

2 - a n d 3 - m e t h y l b u t a n a l ( 2 + 3 M B ) , t h e S t r e c k e r d e g r a d a t i o n a l d e h y d e s , a s w e l l a s m a n y o t h e r v o l a t i l e c o m p o u n d s .

T h i s f o r m a t i o n o f t h e s e S t r e c k e r a l d e

h y d e s i n e x p l o s i o n - p u f f e d p o t a t o d i c e i s a c c e l e r a t e d i n t h e p u f f i n g g u n w h e r e t h e b r o w n i n g p r e c u r s o r s a r e e x p o s e d t o f a v o r a b l e r e a c t i o n c o n d i t i o n s ( i . e . , h i g h t e m p e r a t u r e , l o w m o i s t u r e c o n t e n t ) . T h i s a c c e l e r a t i o n o f b r o w n i n g d u r i n g p u f f i n g w a s e v i d e n c e d b y t h e c h a r a c t e r i s t i c b r o w n i n g o d o r ( b u r n t a n d t o a s t e d ) a n d w a s f u r t h e r s u b s t a n t i a t e d b y g a s - l i q u i d c h r o m a t o g r a p h y ( S a p e r s e t a l . , 1 9 7 0 ,

1 9 7 1 ) .T h e e x p l o s i o n p u f f i n g p r o c e s s r e l i e s

u p o n s t e a m c o n d e n s a t i o n , u n d e r h i g h p r e s s u r e - h i g h t e m p e r a t u r e c o n d i t i o n s , t o

c r e a t e a n e x p a n d e d p o t a t o p i e c e . T h e e n e r g y s u p p l i e d b y t h i s c o n d e n s a t i o n s u p e r h e a t s t h e w a t e r i n t h e i n d i v i d u a l

p i e c e s . W h e n t h e p r e s s u r e i s r e l e a s e d t h e w a t e r w i t h i n t h e d i c e i s f l a s h e d a n d a

p o r o u s s t r u c t u r e i s c r e a t e d ( T u r k o t e t a l . ,

1 9 6 7 ) . T h i s p o r o u s s t r u c t u r e r e m a i n s i n t a c t d u r i n g t h e f i n a l d e h y d r a t i o n s t e p a l l o w i n g r a p i d d r y i n g a n d p e r m i t t i n g a m u c h m o r e r a p i d p e n e t r a t i o n o f w a t e r u p o n r e h y d r a t i o n ( E i s e n h a r d t e t a l . ,1 9 6 2 ) .

R e s e a r c h d o n e b y C o r d i n g a n d S u l l i v a n ( 1 9 7 3 ) , i n d i c a t e s t h a t d i l u t i o n o f t h e s u p e r h e a t e d s t e a m w i t h a n o n c o n d e n s a b l e

g a s ( 2 : 1 s t e a m t o g a s ) i n t h e p u f f i n g s y s t e m i s e f f e c t i v e i n i n h i b i t i n g t h e f o r m a t i o n o f t h e b r o w n i n g a l d e h y d e s d u r i n g t h e p u f f i n g o p e r a t i o n .

T h e p r e s e n t p a p e r c o m p a r e s t h e r e l a t i v e a m o u n t s o f 2 + 3 M B i n p o t a t o e s u n d e r s t o r a g e t h a t h a v e b e e n ( 1 ) c o n v e n t i o n a l l y p r o c e s s e d , ( 2 ) p u f f e d w i t h s u p e r h e a t e d s t e a m a l o n e a n d ( 3 ) p u f f e d w i t h a s u p e r h e a t e d s t e a m / n i t r o g e n m i x t u r e . T h e l a c k o f q u a n t i t a t i v e o r g a n o l e p t i c d a t a i n

Table 1—Sugar contents o f raw potatoes

g/100g,MFB

Kennebec Russet Burbank

High Low High Low

Glucose 2.688 0.380 3.000 0.800Fructose 2.872 0.820 3.842 1.610Sucrose 3.355 1.010 2.548 1.250

t h i s s t u d y n e c e s s i t a t e d a s e c o n d s t o r a g e

t e s t .T h i s s u b s e q u e n t s t o r a g e s t u d y e x a m

i n e s t h e d e v e l o p m e n t o f t h e S t r e c k e r

a l d e h y d e s i n h i g h a n d l o w s u g a r p o t a t o e s b y m e a n s o f c h r o m a t o g r a p h i c a n a l y s e s

a n d o r g a n o l e p t i c e v a l u a t i o n s . H e x a n a l , w h i c h d e v e l o p s d u e t o t h e a u t o x i d a t i o n o f p o t a t o l i p i d s , w a s a l s o f o u n d . I t s d e v e l

o p m e n t w a s f o l l o w e d t h r o u g h o u t t h e s t o r a g e p e r i o d s b e c a u s e o f t h e r a n c i d f l a v o r i t i m p a r t s t o t h e p o t a t o d i c e . T h e c o n t r o l o f t h i s o f f - f l a v o r w i l l b e t h e s u b j e c t o f a l a t e r s t u d y .

EXPERIMENTALTY PIC A L COM M ERCIAL processing varieties o f p o ta to es were used fo r b o th storage tests: (a) T est # 1 , Idaho R usset B urbank; (b) T est # 2 , Maine K ennebec and M aine R usset B urbank .

T he p o ta to es were subm erged in a 20% (by w eight) caustic so lu tion to loosen o u te r skins. These skins were th en rem oved by a ro ta ry w asher w ith high pressure w ater je ts . T rim m ing to rem ove ro t, sunburn and o th e r blem ishes, was fo llow ed by a 1/2% sodium bisu lfite , 1/2% c itric acid dip to p reven t enzy m atic brow ning. T his d ip was app lied each tim e a new surface o f p o ta to was exposed . T he p o ta to e s w ere then cu t in to nom inal 3 /8 in . cubes and traversed over a 3 /16 in. shaker screen to provide a fractio n o f m ore un ifo rm size. T he dice were w ashed tho ro u g h ly to rem ove surface starch . P recooking a t 71°C for 15 m in and cooling (C ording e t al., 1955, 1959) in cold w ater (< 21°C ) fo r 10 m in preceded a 15 m in a tm ospheric steam blanch . T he dice w ere th en d ipped in a 1/4% N a H S 0 3-l/4 % N a2S 0 3 so lu tion fo r 1 m in to co n tro l nonen zy m atic b row ning during the d rying cycle. The p o ta to dice w ere dried to 2 5 -3 0 % m oistu re in a ba tch th rough-flow air drier a t 93°C. E qu ilib ra tion a t 23°C for an 1 8 - 2 4 hr period to insure a un ifo rm m oistu re d is tr ib u tio n w ith in and am ong dice was the last step before exp losion puffing.Storage te s t #1

Idaho R usset B urbank p o ta to es were p ro cessed in th is tes t. A fter in itia l processing (as above) the partia lly dried p o ta to es w ere separa ted in to three frac tions. T he firs t frac tio n was conventionally air dried to 4.5% m oistu re . The

second frac tio n w as exp losion -puffed w ith superhea ted steam alone. T he th ird frac tio n was exp losion-puffed in a superhea ted s te a m /n itro gen m ix tu re (2 :1 steam to N 2).

A fter puffing , the p o ta to e s w ere p laced in a h o t air, th rough-flow tray d rier and d ried a t 66°C to 5 -4 % m oistu re . All m aterial w as th en packed u nder n itrogen (less than 2% 0 2) or air a tm ospheres in 211 X 414 cans, 120g/can and sto red at - 1 8 and 23°C.Storage te s t # 2

E valuations o f b o th high and low sugar level Maine p o ta to es w ere m ade in th is test. D ue tc the low sugar level o f the p o ta to e s h a lf was processed upo n receip t. T he rem aining h a lf was s to red a t 3°C for 12 w k to increase the sugar c o n te n t. Sugar levels appear in T able 1.

T he p o ta to dice w ere exp losion -puffed in a superhea ted steam /n itro g en m ix tu re (2 :1 steam to N 2) and dried.

Sam ples w ere packed und er air and n itrogen atm ospheres in 211 x 414 cans and sto red a t - 1 8 ° C , 23°C and 38°C . Sam ples w ere analyzed by tw o m eth o d s co n cu rren tly : (a) gas liquid ch rom atography and (b) o rg an o lep tic evaluations.C hrom atograph ic analyses

Head space vapor analyses by gas-liquid ch rom atography w ere used to m easure the volatiles above the re c o n s titu te d p ro d u c t (Sapers e t al., 1970). R esults w ere reco rded as ra tio s o f the areas o f 2 + 3 MB and h exanal to the area o f the in ternal stan d ard , e th y lb u ty ra te . 2 MP resu lts w ere n o t rep o rted due to inaccuracies resulting from the co incidence o f the 2 MP and acetone re ten tio n tim es (Sapers, 1970). O rganolep tic evaluations

Sensory evaluations were accom plished by the use o f a m odified K ram er test (K ram er anc Twigg, 1962) in w hich each sam ple was co m pared w ith a s tandard and judged accordingly . The m o d ifica tion consists o f an ad d itio n a l ca te gory in the “ b e tte r th an sta n d ard ” range. T he scoring system was as follow s:

(a) Less th an 4 , decreasingly w orse than standard (i.e., score o f 1, w hich is th e low er lim it, ind ica tes the sam ple is “ very m uch w orse” th an the s tandard ).

(b) E qual to 4, sam e as standard .(c) G reater th an 4 , increasingly b e tte r th an

standard (i.e., a score o f 6, w hich is the upp er lim it, ind icates the sam ple is “ m uch b e tte r” th an the s tandard).

All sam ples w ere reh y d ra te d un til tender and riced to m inim ize tex tu ra l d ifferences. They w ere served in sm all, o d o r free, a lu m inum dishes and p resen ted to a tra in ed pan e l averaging 15 m em bers. C olored lights w ere used during tasting to m ask any co lor d ifference in the sam ples. T he d a ta w ere tre a te d sta tis tica lly by an analysis o f variance and a m u ltip le range tes t (D uncan , 1955).P ro d u c t accep tab ility

P o ta to es w ere p rep ared w ith a b land cream

5 8 -J O U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

BROWNING OF PUFFED POTATOES IN STORAGE-59

Table 2—Storage test #1: Idaho Russet Burbank air and N2 pack

A ir N 2

Processing condition

Time(Months)

°CTemp Comp. Conv.

SteamPuff

Steam/l\l2 Puff Conv.

SteamPuff

Steam/NPuff

Mean peak area ratio3-18°C 2 + 3 MB 0.092 0.519 0.226 0.080 0.483 0.236

1 Hexanal 0.075 0.097 0.113 0.095 0 .1 1 2 0.08723° C 2 + 3 MB 0.118 0.524 0.235 0.117 0.507 0.220

Hexanal 0.083 0.130 0.085 0.074 0.086 0.089

-18°C 2 + 3 MB 0.100 0.519 0.191 0.090 0.513 0.1648 Hexanal 0.119 0.393 0.202 0.052 0 .210 0.058

23° C 2 + 3 MB 0.124 0.622 0.260 0.108 0.583 0.238Hexanal 0.718 1.690 1.370 - 0.067 0.105

a M e a n p e a k a r e a r a t i o — a r e a a l d e h y d e p e a k / a r e a i n t e r n a l s t a n d a r d p e a k

sauce and ra ted by a m inim um o f 45 un tra in ed tasters. T he sam ples w ere p resen ted as a single stim ulus and scored on a stan d ard 9 -po in t h edon ic scale (Peryam and Pilgrim , 1957).

RESULTS & DISCUSSIONS t o r a g e t e s t # 1

I n t h i s t e s t , c h r o m a t o g r a p h i c m e a s u r e m e n t s o f t h e d e v e l o p m e n t o f 2 + 3 M B a n d h e x a n a l w e r e m a d e f o r p o t a t o d i c e p r o c e s s e d u n d e r t h r e e d i f f e r e n t t e c h n i q u e s : ( a ) C o n v e n t i o n a l p r o c e s s i n g ; ( b )

E x p l o s i o n p u f f i n g ( s t e a m o n l y ) ; a n d ( c ) E x p l o s i o n p u f f i n g ( s t e a m / N 2 m i x t u r e ) .

E x a m i n a t i o n o f t h e i n i t i a l f o r m a t i o n o f t h e S t r e c k e r a l d e h y d e s ( s e e T a b l e 2 — 1

m o n t h ) r e v e a l e d t h a t t h e c o n v e n t i o n a l l y

p r o c e s s e d m a t e r i a l h a d t h e l e a s t a m o u n t

o f 2 + 3 M B . I n o r d e r o f i n c r e a s i n g a l d e

h y d e f o r m a t i o n t h e p r o c e s s e s a r e c o n v e n t i o n a l , e x p l o s i o n p u f f ( s t e a m / N 2 ) a n d f i n a l l y e x p l o s i o n p u f f ( s t e a m o n l y ) .

I t s h o u l d b e n o t e d t h a t u n d e r t h e

s t o r a g e c o n d i t i o n s s e t , t h e r e i s n o s i g n i f i c a n t i n c r e a s e i n t h e b r o w n i n g a l d e h y d e s

w i t h r e s p e c t t o t i m e .

I n a i r p a c k e d s a m p l e s ( T a b l e 2 ) , h o w e v e r , t h e a l d e h y d e , h e x a n a l , r e s u l t i n g f r o m t h e a u t o x i d a t i o n o f p o t a t o l i p i d s , i n c r e a s e d r a p i d l y w h e n s t o r e d a t 2 3 C

w i t h a l l p r o c e s s i n g t e c h n i q u e s .T h e r e w a s a s u b s t a n t i a l d e c r e a s e i n t h e

i n i t i a l f o r m a t i o n o f 2 + 3 M B w h e n a s t e a m / N 2 m i x t u r e w a s e m p l o y e d d u r i n g

p u f f i n g r a t h e r t h a n s t e a m a l o n e . T h i s d e c r e a s e i s m e a n i n g f u l o n l y i f t h e o f f -

f l a v o r i m p a r t e d b y t h e s e c o m p o n e n t s i s b e l o w t h e t a s t e t h r e s h o l d l e v e l a n d r e

m a i n s s o f o r a r e a s o n a b l e p e r i o d o f t i m e i n s t o r a g e .

T a s t e e v a l u a t i o n s o f t h e s a m p l e s w e r e m a d e m o n t h l y . T h e b r o w n i n g o f f - f l a v o r

w a s d e t e c t e d i n t h e s t e a m - p u f f e d p r o d u c t b y 7 5 % o f a g r o u p o f 1 0 — 1 5 j u d g e s w h i l e t h e m a t e r i a l p u f f e d i n a s t e a m / N 2 m i x t u r e h a d n o d e t e c t a b l e b r o w n i n g f l a v o r d u r i n g t h e 8 - m o n t h s t o r a g e . A r a n c i d f l a

v o r , w h i c h c a n b e a t t r i b u t e d t o t h e f o r

m a t i o n o f h e x a n a l , w a s n o t e d i n t h e 2 3 ° C a i r - p a c k s a m p l e s w i t h i n t h e f i r s t 3 m o n t h s o f s t o r a g e .

T h e s e t a s t e e v a l u a t i o n s c a n n o t b e p r e s e n t e d a s q u a n t i t a t i v e d a t a b e c a u s e p a n e l m e m b e r s h i p a n d t r a i n i n g h a d n o t b e e n c o m p l e t e d . T r a i n i n g s e s s i o n s w e r e h e l d i n p r e p a r a t i o n f o r t h e s e c o n d s t o r a g e t e s t .

S t o r a g e t e s t # 2

I n o r d e r t o s u b s t a n t i a t e t h e “ b e l o w t h r e s h o l d l e v e l s ” o f t h e b r o w n i n g i n s t e a m / N 2 p u f f e d p o t a t o d i c e , a s e c o n d s t o r a g e t e s t w a s i n i t i a t e d . I n a d d i t i o n t o c h r o m a t o g r a p h i c a n a l y s e s , o r g a n o l e p t i c e v a l u a t i o n s w e r e m a d e m o n t h l y b y 1 5

t r a i n e d t a s t e p a n e l i s t s . M a i n e K e n n e b e c s a n d M a i n e R u s s e t B u r b a n k s w e r e s t u d i e d a t b o t h h i g h a n d l o w s u g a r l e v e l s . A l l s a m

p l e s w e r e c o m p a r e d t o a c o n t r o l w h i c h w a s s t o r e d a t — 1 8 ° C u n d e r a n i t r o g e n a t m o s p h e r e . U n d e r t h e s e c o n d i t i o n s , p o t a t o d i c e r e m a i n e d s t a b l e w i t h r e g a r d

t o b r o w n i n g a n d a u t o x i d a t i o n t h r o u g h o u t ' t h e s t o r a g e p e r i o d ( s e e T a b l e s 3 —6 ) .

A n a l y t i c a l r e s u l t s

H i g h s u g a r . S a m p l e s o f b o t h v a r i e t i e s s t o r e d a t 2 3 ° C u n d e r a i r o r n i t r o g e n a t m o s p h e r e s r e m a i n e d s t a b l e w i t h r e g a r d t o 2 + 3 M B t h r o u g h o u t t h e i r r e s p e c t i v e s t o r a g e p e r i o d s ( s e e T a b l e s 3 a n d 4 ) . A n i n c r e a s e i n h e x a n a l w a s n o t e d i n t h e a i r - p a c k e d s a m p l e s a l m o s t i m m e d i a t e l y . T h e i n c r e a s e o f t h i s c o m p o n e n t w a s d r a m a t i c a f t e r o n l y 2 m o n t h s s t o r a g e ( T a b l e 3 ) .

S a m p l e s s t o r e d a t 3 8 C w e r e c a n n e d i n a n i t r o g e n a t m o s p h e r e . S i g n i f i c a n t i n c r e a s e s i n 2 + 3 M B w e r e s e e n a f t e r s t o r a g e f o r 1 m o n t h ( s e e T a b l e s 3 a n d 4 ) .

L o w s u g a r . U n d e r a l l s t o r a g e c o n d i t i o n s , l o w s u g a r s a m p l e s e x h i b i t l o w e r i n i t i a l l e v e l s o f 2 + 3 M B t h a n t h e i r h i g h s u g a r c o u n t e r p a r t s . T h e S t r e c k e r d e g r a d a t i o n a l d e h y d e r e m a i n s s t a b l e t h r o u g h o u t t h e s t o r a g e p e r i o d r e g a r d l e s s o f s t o r a g e

Table 3—Maine Kennebec high sugar

Storagecondition

Time in storage (months)

Comp. 0 2 3 5 12

2 + 3 MB 0 .2 10

Mean peak area ra tio30.216 0.194 0.270

-1 8 ° C, N 2 Hexanal 0.027 0.028 0.026 0.082 —Taste*3 4.00 3.93 4.00 4.21 3.872 + 3 MB 0 .210 0.244 0.292 0.296 -

23° C, N2 Hexanal 0.027 0.030 0.033 0.100 —Taste*3 4.00 4.15 - 4.08 3.872 + 3 MB 0 .210 0.270 0.261 0.291 —

23° C, A ir Hexanal 0.027 0.222 0.395 0.565Taste*3 4.00 3.69 3.44c 2.92c 2.47c2 + 3 MB 0 .2 10 0.646 0.670 0.794 —

38° C, N 2 Hexanal 0.027 0.032 0.024 0.043 -Taste*3 4.00 SAO0 3.36c 3.14C -

a M e a n p e a k a r e a r a t i o - a r e a a l d e h y d e p e a k / a r e a i n t e r n a l s t a n d a r d p e a k

B T a s t e s c o r e a s p e r K r a m e r a n d T w i g g , 1 9 6 2

° I n d i c a t e s a c o n f i d e n c e l e v e l o f 9 5 % o r g r e a t e r r e l a t i v e t o c o n t r o l a t

same storage t ime

Table 4—Maine Russet Burbank high sugar

Storagecondition Comp.


0 1 3 12Mean peak area ratios3

-18 ° C, N2 2 + 3 MB 0.513 0 .542 0.516 -Taste*3 4.09 4.36 4.06 4.07

23° C, N2 2 + 3 MB 0.513 0 .5 58 0.599 -Taste B 4.09 4.21 3.88 3.87

23°C, A ir 2 + 3 MB 0.513 0 .4 86 0.578 -Taste*3 4.09 4.14 3.25c 2.67c

38° C, N2 2 + 3 MB 0.513 1.005 1.248 -Taste*3 4.09 3.50c 3.43c -


h T a s t e s c o r e s a s p e r K r a m e r a n d T w i g g , 1 9 6 2

c I n d i c a t e s c o n f i d e n c e l e v e l o f 9 5 % o r g r e a t e r r e l a t i v e t o c o n t r o l a t

same storage t ime

6 0 -J O U R N A L OF FOOD S C /E N C E -V o /u m e 3 9 (1974)

Table 5—Maine Kennebec low sugar

Storagecondition


Comp. 1 4 6 12Mean peak area ratios3

2 + 3 MB 0.107 0 .12 2 0.114 --18 °C , N2 Hexanal 0.064 0.092 0.105 -

Taste*3 4.00 4.05 4.12 3.922 + 3 MB 0.130 0.132 0.158 -

23° C, l\l2 Hexanal 0.083 0 .10 2 0 .1 1 2 -Taste*3 4.13 3.94 - 3.622 + 3 MB 0.129 0.134 0.131 -

23°C, A ir Hexanal 0.461 1.276 1.170 -Taste*3 3.19c 2.72c 3.00c 2.62c2 + 3 MB 0.178 0.273 0.316 -

38°C, N2 Hexanal 0.048 0.047 0.051 -Taste*3 3.80 4.06 4.00 -


k T a s t e s c o r e s a s p e r K r a r r e r a n d T w i g g , 1 9 6 2

c I n d i c a t e s c o n f i d e n c e le v e l o f 9 5 % o r g r e a t e r r e l a t i v e t o c o n t r o l a t

s a m e s t o r a g e t i m e

Table 6—Maine Russet Burbank low sugar

Storagecondition


Comp. 1 3 6

Mean peak area ratios3

-18°C , N 2 2 + 3 MB 0.230 0.256 0.279Taste*3 4.00 4.13 3.82

23° C, N 2 2 + 3 MB 0.244 0.285 0.340Taste*3 4.00 4.07 3.80

23° C, A ir 2 + 3 MB 0.261 0.291 0.294Taste*3 3.79 3.07c 3.07c

38° C, N 2 2 + 3 MB 0.403 0.592 0.801Taste*3 3.67 4.13 4.06


b T a s t e s c o r e s a s p e r K r a m e r a n d T w i g g , 1 9 6 2

c I n d i c a t e s a c o n f i d e n c e l e v e l o f 9 5 % o r g r e a t e r r e l a t i v e t o c o n t r o l a t

s a m e s t o r a g e t i m e

t e m p e r a t u r e o r a t m o s p h e r e ( s e e T a b l e s 5

a n d 6 ) .

O r g a n o l e p t i c e v a l u a t i o n s

A l l a i r - p a c k e d s a m p l e s , r e g a r d l e s s o f v a r i e t y o r s u g a r c o n t e n t , s h o w e d r a p i d d e v e l o p m e n t o f a r a n c i d f l a v o r ( h e x a n a l ) . T h i s o f f - f l a v o r w a s d e t e c t a b l e a f t e r 1 - 3 m o n t h s s t o r a g e a t a 9 5 % o r h i g h e r c o n f i d e n c e l e v e l . A f t e r t h e t h i r d m o n t h t h e

f l a v o r s c o r e s r e m a i n e d r e l a t i v e l y c o n s t a n t ( T a b l e s 3 — 6 ) .

S a m p l e s s t o r e d a t 2 3 C u n d e r a n i t r o g e n a t m o s p h e r e , d i d n o t , i n a n y c a s e , e x h i b i t a f l a v o r c h a n g e .

A t 3 8 C h i g h s u g a r M a i n e K e n n e b e c s ( T a b l e 3 ) s h o w e d a r a p i d d e c r e a s e i n f l a v o r s c o r e s w i t h i n t h e f i r s t 2 m o n t h s o f s t o r a g e . T h e d e v e l o p i n g f l a v o r w a s d e s c r i b e d a s “ t o a s t e d ” o r “ b u r n t , ” i n d i c a t i n g b r o w n i n g . T h e l o w s u g a r K e n n e b e c s ( T a b l e 5 ) s h o w e d n o s i g n i f i c a n t c h a n g e i n f l a v o r t h r o u g h o u t t h e e x p e r i m e n t .

P r o d u c t a c c e p t a b i l i t y

P o t a t o d i c e , s t o r e d a t 2 3 ° C w e r e r e h y d r a t e d , d r a i n e d a n d a d d e d t o a b l a n d c r e a m s a u c e a n d s e r v e d w a r m t o 4 5 u n t r a i n e d t a s t e r s , a t t h e b e g i n n i n g a n d t h e e n d o f t h e s t o r a g e t e s t t o o b t a i n j u d g m e n t s o f a c c e p t a b i l i t y a s a p r e p a r e d f o o d . R e s u l t s o f t h i s t e s t i n d i c a t e t h a t t h e r a t i n g s g i v e n w e r e n o t a f f e c t e d b y a i r o r N 2 p a c k , h i g h o r l o w s u g a r l e v e l , o r a g e o f t h e p r o d u c t . S c o r e s a v e r a g e d b e t w e e n 6 . 8 a n d 7 . 4 o n a 9 - p o i n t h e d o n i c s c a l e . T h e s e s c o r e s f a l l i n t h e “ l i k e s l i g h t l y ” t o “ l i k e m o d e r a t e l y ” r a n g e . I t i s r e a l i z e d t h a t a p a n e l s o s m a l l c a n n o t p r o v i d e a p o s i t i v e j u d g e m e n t o f q u a l i t y ; h o w e v e r , a t r e n d c a n b e i n d i c a t e d .

CONCLUSIONST H E A N A L Y T I C A L a n d o r g a n o l p e t i c d a t a a r e p a r a l l e l u n d e r a l l c o n d i t i o n s a n d t h e s e d a t a i n d i c a t e t h a t l o w s u g a r p o t a t o e s a r e m o s t s u i t a b l e f o r e x p l o s i o n - p u f f i n g . T h e r e l a t i o n s h i p b e t w e e n s u g a r c o n

t e n t a n d i n i t i a l 2 + 3 M B l e v e l s is e v i d e n t . C o m p a r i s o n o f t h e s e f a c t o r s s h o w s t h a t i n a l l c a s e s a h i g h e r s u g a r c o n t e n t y i e l d s

g r e a t e r q u a n t i t i e s o f t h e S t r e c k e r a l d e h y d e .

T h e u s e o f a s t e a m / N 2 m i x t u r e i n e x p l o s i o n p u f f i n g i s a n e f f e c t i v e m e t h o d o f r e t a r d i n g i n i t i a l d e v e l o p m e n t o f 2 + 3

M B . W i t h t h e e x c e p t i o n o f 3 8 ° C , h i g h s u g a r s a m p l e s , t h e 2 + 3 M B l e v e l s r e m a i n r e l a t i v e l y s t a b l e t h r o u g h o u t s t o r a g e .

A l t h o u g h t h e N 2 p a c k i n g a t m o s p h e r e e f f e c t i v e l y r e t a r d s t h e a u t o x i d a t i o n o f p o t a t o l i p i d s , f u r t h e r r e s e a r c h i n t o a m o r e p r a c t i c a l m e a n s o f c o n t r o l l i n g t h i s a u t o x i d a t i o n i s p r e s e n t l y u n d e r s t u d y .

REFERENCESBraverman, J.B.S. 1963. Nonenzymatic brown

ing. In “ In troduction to the Biochemistry of Foods,” p. 302. Elsevier Publishing Co., New York.

Cording, J. Jr., Willard, M.J. Jr., Eskew, R.K., Edwards, P.W. and Sullivan, J.F . 1955. Potato flakes. A new form of dehydrated mashed potatoes. 2. Some factors influencing texture. U.S. Dept, of Agriculture, Agricultural Research Service, ARS 73-9.

Cording, J. Jr., Sullivan, J.F . and Eskew, R.K. 1959. Potato flakes. A new form of dehydrated mashed potatoes. 4. Effects of cooling after precooking. U.S. Dept, of Agriculture, Agricultural Research Service, ARS 73-25.

Cording, J. Jr. and Sullivan, J.F . 1973. Retarding the browning reaction in the explosionpuffing of po tato pieces. Food Eng. Accepted for publication.

Duncan, D.B. 1955. Multiple range and m ultiple F tests. Biometrics 11: 1.

Eisenhardt, N.H., Cording, J. Jr., Eskew, R.K. and Sullivan, J.F . 1962. Quick-cooking dehydrated vegetable pieces. 1. Properties of potato and carrot products. Food Technol. 16(5): 143.

Kramer, A. and Twigg, B.A. 1962. Taste testing. In “ Fundam entals of Quality Control for the Food Industry ,” p. 123. Avi Publishing Co., W estport, Conn.

Peryam, D.R. and Pilgrim, F.J. 19 57. Hedonic scale m ethod of measuring food preference. Food Technol. 11(4): Insert, p. 32.

Sapers, G.M. 1970. Flavor quality in explosion puffed dehydrated po tato . 2. Flavor contribution of 2-m ethylpropanal, 2-methyl- butanal and 3-m ethylbutanal. J. Food Sci. 35: 731.

Sapers, G.M., Sullivan, J.F . and Talley, F.B. 1970. Flavor quality in explosion puffed dehydrated potato . 1. A gas chrom atographic m ethod for the determ ination of aldehydes associated with flavor quality. J. Food Sci. 35: 728.

Sapers, G.M., Osman, S.F., Dooley, C.J. and Panasiuk, O. 1971. Flavor quality of explosion puffed dehydrated po tato . 3. C ontribution of pyrazines and o ther com pounds to the toasted off-flavor. J. Food Sci. 36: 93.

Turkot, V.A., Sullivan, J .F ., Cording, J. Jr., Eskew, R.K. and Heiland, W.K. 1967. Explosion puffed dehydrated potatoes. 3. Estimated cost of commercial production using shortened cycle. U.S. D epartm ent of Agriculture, Agricultural Research Service, ARS 73-55.

Ms received 6/23/73; revised 8 /2 1 /73; accepted8/24/73.


The authors acknowledge N.C. Aceto, Chief, Engineering & Development Lab.; E.S. Della- Monica, Research Leader, Product Analysis;G.M. Sapers, Research Leader, F ru it & Vegetable Technology; and C. Ross and B.S. Clark, Technicians, of this Laboratory for their invaluable assistance in this storage study.

Use of a company or product nam e does no t imply approval or recom m endation of the produc t to the exclusion of others which may also be suitable.

M . C. G A C U L A J R ., L E E A N N P A R K E R , J. J. K U B A L A a n d J U N E R E A U M E

A rm o u r F o o d Research L a b o ra to ry , 8 0 1 W. 2 2 n d S t., O ak B ro o k , IL 6 0 5 2 1

DATA ANALYSIS: A VARIABLE SEQUENTIAL TEST FOR SELECTION OF SENSORY PANELS

INTRODUCTIONI N O R D E R T O O B T A I N a r e l i a b l e r e s u l t f r o m s e n s o r y a s s e s s

m e n t o f f o o d s , t h e m e m b e r s o f t h e p a n e l o f j u d g e s s h o u l d b e s e l e c t e d f o r t a s t e a c u i t y a n d i n t e r n a l c o n s i s t e n c y . T h e t r i a n g l e t e s t h a s b e e n t h e u s u a l m e t h o d u s e d i n s c r e e n i n g p r o s p e c t i v e

p a n e l i s t s f o r t a s t e d i s c r i m i n a t i o n . T h e n u m b e r o f t e s t s n e c e s s a r y f o r a c c e p t a n c e o r r e j e c t i o n u s u a l l y w e r e a r b i t r a r i l y s e t o r w e r e d e t e r m i n e d f r o m p r e v i o u s e x p e r i e n c e . T h e p u r p o s e o f

s t a t i s t i c a l p a n e l s e l e c t i o n i s t o q u a n t i f y t h e s e l e c t i o n p r o c e d u r e a n d t o m a x i m i z e t h e u s e o f p a n e l r e c o r d s b y u t i l i z i n g p a s t t e s t p e r f o r m a n c e .

T h e u s e o f s t a t i s t i c s t o s e l e c t j u d g e s w a s f i r s t i n v e s t i g a t e d b y L o m b a r d i ( 1 9 5 1 ) u s i n g t h e s e q u e n t i a l a n a l y s i s m e t h o d . H i s

w o r k w a s l i m i t e d t o t h e t r i a n g l e t e s t a n d t h e d u o - t r i o t e s t

w h e r e t h e r e s p o n s e m e a s u r e m e n t w a s q u a l i t a t i v e a n d d i c h o t o

m o u s . W h e n o n e i s d e a l i n g w i t h a q u a n t i t a t i v e r e s p o n s e s u c h a s

m e a t t e n d e r n e s s , s e n s i t i v i t y a n d s a m p l e h e t e r o g e n e i t y i n t e r a c t t o i n v a l i d a t e t h e u s e o f a t r i a n g l e t e s t . T h i s p a p e r d e v e l o p s a p a n e l s e l e c t i o n p r o c e d u r e f o r a q u a n t i t a t i v e r e s p o n s e v a r i a b l e

u t i l i z i n g t h e t h e o r y o f W a l d ’s s e q u e n t i a l a n a l y s i s .

EXPERIMENTAL

s a y , f o r t e n d e r n e s s . I n a r a t i n g s c a l e o f 1 t o 8 , f o r a g i v e n s a m p l e , i t i s l i k e l y t h a t w e c a n o b t a i n 8 d i f f e r e n t s c o r e s . B y u s i n g a p a i r e d d e s i g n , o n e c a n m i n i m i z e t h e e f f e c t o f s a m p l e h e t e r o g e n e i t y a s t h e a n a l y s i s i s b a s e d o n d i f f e r e n c e s b e t w e e n h o m o g e n e o u s o r a l i k e s a m p l e s . T h e a s s e s s m e n t o f a g o o d j u d g e i s b a s e d o n t h e m a g n i t u d e o f h i s s c o r e d i f f e r e n c e b e t w e e n t h e

t w o i d e n t i c a l s a m p l e s t a s t e d .

L e t

d ; - A , — B j , i = 1 , 2 , . . . m ( 2 )

w h e r e A ; a n d B j a r e s c o r e s f r o m p a i r e d s a m p l e s . T h e e x p e c t e d v a l u e ( E ) o f A ; a n d B ; is

E(Aj) = n

E(Bj) = ¡j.(3)

a n d , b y v i r t u e o f t h e d e s i g n s a m p l i n g , i t f o l l o w s t h e r e f o r e t h a t

ECAj) = E(Bi) = yu (4)

AS SHOWN BY Wald (1947) and o thers (W etherill, 1966) the lower (L 0) and the upp er ( L , ) lim it o f a sequen tial p robab ility ratio test is

a 5 (5 Mo + Mi m <x2 1 - /3 M0 + Mi----------- l o g ------- + m (— - — ) < 2 Xi < ----------- l o g --------+ m ( — -— )M, - M 0 1 - “ 2 i M, — M0 “ 2

(1)T he n o ta tio n s are defined as follow s:

(1) a 2 is th e residual variance o f the observations. S u bsequen tly , it will be d en o ted by a d to ind ica te th e variance o f the d ifferences betw een iden tical sam ples in a paired d ifference test.

(2) a is the T yp e I error know n in the sta tis tical lite ra tu re as the significance level. In the p resen t w ork it is defined as the p robab ility o f accepting a judge as qualified w hen in fact he is n o t; /3 is the T ype II error defined as th e p robab ility o f rejecting a judge w hen in fact he is qualified.

(3 ) Mi - M0 >s the d ifference betw een iden tical sam ples th a t can be to lera ted a t the a and /3 levels o f risk. In the succeeding p resen ta tio n Mt will be d en o ted by Mdi and MO b j' MdO 1,1 paired d ifference tests o fidentical sam ples, MdO = 0- We will designate /ad l - MdO as s • m

(4) £ Xj^are the observed m easu rem en ts o f in terest. This sum is d en o ted by £ dj or sim ply S d j, since the observation in a paired d ifference design is in te rm s o f d ifferences, e.g., left vs. right.

It can be show n th a t the cum ulative value £ d 4 w hen p lo tted against m can be used as a basis fo r a sequential decision since the boundary lines o f £ d j are given by the inequalities in (1 ). T he app lica tion o f this result to variable panel se lection is the subject o f th e p resen t study .

RESULTS & DISCUSSIONT H E S E N S O R Y P A N E L s e l e c t i o n f o r a v a r i a b l e t h a t f o l l o w s t h e n o r m a l d i s t r i b u t i o n w i t h q u a n t i t a t i v e r e s p o n s e s i s m o r e c o m p l e x t h a n t h a t o f t h e b i n o m i a l c a s e w h e r e t h e r e s p o n s e i s d i c h o t o m o u s ( a c c e p t a b l e o r n o t a c c e p t a b l e , d e f e c t i v e o r n o n

d e f e c t i v e ) . E x p e r i e n c e h a s s h o w n t h a t i t i s i m p o s s i b l e t o p r e p a r e a h o m o g e n e o u s m e a t s a m p l e f o r u s e i n p a n e l s e l e c t i o n ,

w h e r e /a i s t h e t r u e u n k n o w n p o p u l a t i o n m e a n . L i k e w i s e t h e

e x p e c t e d v a l u e o f t h e d i f f e r e n c e , E ( d , ) s h o u l d e q u a l z e r o .

T h u s , i n m r e p e a t e d t e s t s , a t r u l y g o o d p a n e l i s t w o u l d h a v e

m2 d j = 0 (5)

1 = 1

a n d a s a c o n s e q u e n c e o f t h i s , t h e i n e q u a l i t i e s

m2 d; > 0 (6 )

2 dj < 0 (7)i = 1

r e p r e s e n t r e j e c t i o n r e g i o n s o f o p p o s i t e d i r e c t i o n . I n o r d e r t o a c c o m m o d a t e b o t h i n e q u a l i t i e s , a d o u b l e - s i d e d s e q u e n t i a l t e s t

i s n e c e s s a r y . T h i s t e s t i s o b t a i n e d f r o m t h e r e l a t i o n L 0 = — L 0 a n d L ! = - L ^ F r o m t h i s r e l a t i o n , E q ( 1 ) c a n b e w r i t t e n i n a f o r m o f r e g r e s s i o n l i n e , a n d i s s u m m a r i z e d b e l o w :

m ?d P M dO + M dl.2 d , > 0 : ^ )

i = 1

?d 1-/5 Mdo+MdiL j = — l o g — - + m ( — - — )

5 a / 2 2

(8 )

ui .2 d, < 0: L0 =

i - t

L i ' -

P

" s 8 1 - a l lMdO "*■ (*d 1

m ( -------- --------- )

Od 1-----log —

5 a/2P M d o + F d u

m ( -----------------)

(9)

Volume 3 9 (1974)—JO U R N A L OF FOOD SCIENCE- 6 1

5 2 -J O U R N A L OF FOOD S C IF N C E -V o lu m e 3 9 (1974)

L e t us d e n o t e t h e s lo p e (Udo + / i j [ ) / 2 b y s. W h e n E d j > 0 , s is p o s i t iv e a n d t h e b o u n d a r y l in e s a re p r o j e c t i n g u p w a r d . W h e n E d ; < 0 , t h e s l o p e s is n e g a t iv e a n d h e n c e t h e l in e s a re p r o j e c t i n g d o w n w a r d . S i n c e = 0 f o r i d e n t i c a l s a m p l e s , t h e

s lo p e s is e q u i v a le n t t o 6 / 2 b y t h e r e l a t i o n 6 = jUd l - Mdo-T h e s t e p s in a s e q u e n t i a l p a n e l s e l e c t i o n a r e as f o l l o w s :( 1 ) A p r o s p e c t i v e j u d g e , a f t e r u n d e r g o i n g i n s t r u c t i o n s o n

s e n s o r y t e r m i n o l o g i e s a n d d e f i n i t i o n s , is g iv en m p a ire d d i f f e r e n c e t e s t s u s in g i d e n t i c a l s a m p le s .

( 2 ) D e c id e o n t h e v a lu e s , o f a , (3, 6 a n d o j . B a s e d o n t h e s e p r e a s s ig n e d v a lu e s , c o m p u t e t h e r e g r e s s io n e q u a t i o n s u s in g ( 8 ) a n d ( 9 ) . C o n s t r u c t t h e b o u n d a r y l in e s o n a s q u a r e -g r id g r a p h ing p a p e r w i t h Z d j as t h e o r d i n a t e a n d m as t h e a b s c is s a .

( 3 ) I f t h e c u m u l a t i v e v a lu e o f t h e d i f f e r e n c e s , E d p l ies b e t w e e n L 0 a n d L j ( r e g i o n o f i n d e c i s i o n ) o r b e t w e e n L o ' a n d L [ , f u r t h e r t a s t e t e s t in g is r e q u i r e d .

( 4 ) I f Z d j is a b o v e L i o r b e l o w L j ' ( r e g i o n o f r e j e c t i o n ) , t h e c a n d i d a t e j u d g e is r e j e c t e d .

( 5 ) I f Z d j is b e t w e e n L 0 a n d L 0 ' ( r e g i o n o f a c c e p t a n c e ) , t h e c a n d i d a t e ju d g e is a c c e p t e d as a m e m b e r o f t h e p a n e l .

S t e p s ( 3 ) , ( 4 ) a n d ( 5 ) im p l y t h a t t h e l e n g t h o f s e n s o r y t e s t in g f o r e a c h p r o s p e c t i v e ju d g e is s e q u e n t i a l l y d e t e r m i n e d b y t h e m a g n i t u d e o f Z d j r a t h e r t h a n d e t e r m i n e d in a d v a n c e .

L e t us i l l u s t r a t e s t e p 2 u s in g t h e v a lu e s b e l o w :

ffd = 2 . 2 5 a = 0 . 1 0

5 = 1 . 0 |3 = 0 . 2 0

s = 5 / 2 = 0 . 5

B y s u b s t i t u t i n g t h e s e v a lu e s i n t o e q u a t i o n s ( 8 ) a n d ( 9 ) , t h e re g r e s s io n e q u a t i o n s f o r t h e b o u n d a r y l in e s ( F i g . 1 ) a re o b t a in e d . B e in g a l in e a r f u n c t i o n , o n l y t w o v a lu e s o f m a re n e e d e d t o c o n s t r u c t t h e l in e s . T h e e f f e c t o f i n c r e a s i n g (3 f r o m 0 . 2 0 t o 0 . 4 0 o n t h e t h r e e r e g io n s is i n d ic a t e d b y t h e b r o k e n re g r e s s io n l in e s . T h e l e n g t h o f t e s t in g b e c o m e s s h o r t e r as t h e

w id t h o f t h e r e g io n o f i n d e c i s i o n b e c o m e s n a r r o w e r . A s a c o n s e q u e n c e t h e p r o b a b i l i t y o f c o m m i t t i n g a T y p e II e r r o r is i n c r e a s e d , w h i c h m e a n s t h a t w e w ill b e l a b e l l in g c a n d i d a t e s as “ a c c e p t e d ” w h e n , in f a c t , t h e y are n o t t r u l y q u a l i f i e d . W h e n t h e r e a re a l im i t e d n u m b e r o f c a n d i d a t e s t o s e l e c t f r o m , it is an a l t e r n a t i v e t o r e l a x t h e r i s k o f a a n d j3 e r r o r s .

T h e r e is o t h e r i n f o r m a t i o n t h a t c a n b e d e r iv e d f r o m th is t e s t . O n e is t h e p r e s e n c e o f a s y s t e m a t i c b ia s . W h e n 2 d j < 0 , an d p r o g r e s s iv e ly in c r e a s in g t o w a r d s t h e r e g i o n o f r e j e c t i o n it is s u s p e c t e d a t t h e a a n d j3 lev e ls o f r i s k t h a t t h e p a n e l i s t is r a t in g t h e s e c o n d s a m p l e h ig h e r in t h e m a j o r i t y o f c a s e s . A b ias d u e t o c o d i n g , a f t e r - t a s t e , a n t i c i p a t i o n , a m o n g o t h e r s c a n b e h y p o t h e s i z e d . A s im i la r a r g u m e n t c a n b e d e v e lo p e d f o r E d ; > 0 .

In o r d e r f o r t h e t e s t t o b e v a l id , t h e e x p e c t a t i o n E ( d ; ) = 0 s h o u ld h o ld . T h i s c o n d i t i o n u s u a l ly h o l d s in a p a ir o f h o m o g e n e o u s s a m p le s . A d e v ia t i o n f r o m t h i s e x p e c t a t i o n r e f l e c t s t h e i n a b i l i t y o f t h e c a n d i d a t e t o d i s c r i m i n a t e b e t w e e n s a m p l e s . It is w o r t h w h i l e t o m e n t i o n t w o f e a t u r e s o f t h e t e s t p r o c e d u r e . S e q u e n t i a l t e s t s in v o lv in g q u a n t i t a t i v e r e s p o n s e s s c r e e n c a n d i d a t e s f o r c o r r e c t i d e n t i f i c a t i o n o f s a m p le a n d m a g n i t u d e o f d i f f e r e n c e ( p e r h a p s , a n i n d i c a t i o n o f d i f f e r e n t i a l t h r e s h o l d a m o n g in d iv id u a ls ) . N o t e t h a t t r i a n g le a n d d u o - t r i o t e s t s s c r e e n c a n d i d a t e s o n l y f o r c o r r e c t i d e n t i f i c a t i o n o f t h e o d d s a m p le .

E xam pleA d i f f e r e n c e t a s t e t e s t in g e x p e r i m e n t f o r t e n d e r n e s s o f b e e f

w a s c o n d u c t e d f o r t h e p u r p o s e o f s c r e e n i n g p a n e l m e m b e r s . P a ir e d s a m p le s , o n e f r o m e a c h s id e , w e r e se r v e d t o e a c h c a n d i d a te . A r a t in g s c a le ra n g in g f r o m 1 = e x t r e m e l y t o u g h t o 8 = e x t r e m e l y t e n d e r w a s u s e d . T h e p r e a s s ig n e d v a lu e s o f t h e t e s : p r o c e d u r e a r e :

a \ = 2 . 2 5 a = 0 . 1 0

5 = 1 .0 ( 3 = 0 . 2 0

s = 5 / 2 = 0 . 5

TEST NUMBER M

Fig. 1 -R e g io n s o f re jec tion , acceptance and in decision fo r variable sequentia l test. Preassigned values fo r the connected lines are: a j = 2.25, & = 1.0, a = 0.10, ¡3 = 0 .2 0 ; B roken lines: =2.25, 6 = 1.0, cl = 0.10, 0 = 0.40.

TEST NUMBER M

Fig. 2 —P lo t o f (m , Sd j fo r pane l candidates 8(A l and 12(o). The preassigned values are: ajj = 2.25, 6 = 1.0, a = 0.10, ¡3 = 0.20.

VARIA BLE SEQUENTIAL SELECTION OF P A N E L S -6 3

The data and some pertinent calculations are shown in Table 1 .

Substituting the preassigned values into Eq (8 ) and (9), we get the regression equations shown below:

2.25 0.20L0 = ----- log------ + 0.5m

1.0 0.95

= -1.5226 + 0.5m

2.25 0.80Lj = ------log-------+0.5m

1.0 0.05

= 2.7093 + 0.5m

L0' = 1.5226 - 0.5m

L, ' = -2.7093 - 0.5m

The graph of these equations is shown in Figure 2. The cumulative value of dj for both candidates is plotted in this graph. Using candidate 8 as an example, the cumulative value 2dj is obtained as follows: For m = 2

Sd2 = di + d2

= ( - 1 ) + ( 1 )= 0

and the plotting coordinate is thus (0, 2). For m = 4,

Sd4 = d) + d2 + d 3 + d4

= ( - l ) + ( l) + ( l) + (0 )= 1

and the coordinate is (1,4). These coordinates may be verified in Figure 2.

After seven tests, candidate 12 is accepted as a panel member since the plot of Sdj falls in the region of acceptance. The plot for candidate 8 falls in the region of indecision, therefore more taste testing is required. Reliable panels often follow the pattern of the plot of candidate 12. In practice, occasional

Table 1—Calculation o f p lo tting coordinate (m, Sdj) in a variable sequential panel selection

Date of testTestno.

Sample

A B dj = A j - Bj £ d i

5 -8 - 7 2 1

Candidate number 8

6 7 - 1 - 12 7 6 1 03 5 4 1 1

5 -1 6 -7 2 4 5 5 0 15 7 6 1 2

5 -1 0 -7 2 6 6 5 1 37 6 5 1 48 7 7 0 4

7 -5 - 7 2 9 6 6 0 410 7 6 1 5

5 -8 - 7 2 1

Candidate number 12

4 3 1 12 4 4 0 13 3 2 1 2

5 -1 6 -7 2 4 4 5 - 1 15 5 5 0 1

5 -1 0 -7 2 6 4 3 1 27 4 4 0 28 5 5 0 2

7 - 5 - 7 2 9 3 3 0 210 4 5 - 1 1

testing is encouraged to update an individual’s record. In so doing, the maximum records of an individual judge are fully utilized.

REFERENCESLombardi, G.J. 1951. The sequential selection of judges for organolep

tic testing. M.S. thesis, VPI and State University, Blacksburg, Va. Wald, A. 1947. “Sequential Analysis.” John Wiley, New York.Wetherill, G.B. 1966. “ Sequential Methods in Statistics.” Methuen and

Co., L td., London.Ms received 6/28/73; revised 8/27/73; accepted 8/28/73.

C. G. T A S S A N a n d G. F. R U S S E L L

D e p t, o f F o o d S c ience & T e c h n o lo g y , U n iv e rs ity o f C a lifo rn ia , D avis , C A 9 5 6 1 6

SENSORY AND GAS CHROMATOGRAPHIC PROFILES OF COFFEE BEVERAGE HEADSPACE VOLATILES ENTRAINED ON POROUS POLYMERS

INTRODUCTION

DESPITE the large number of studies on coffee volatiles, only a few investigators have used the actual coffee brew as the source of volatiles, probably due to analytical difficulties with dilute aqueous solutions. Other research on coffee brews has used an organic solvent as the volatile extracting and concentration agent. One difficulty encountered with this latter method is the use of solvents with boiling points near those of the aroma constituents, thus making it impossible to strip all solvent from the extract without substantially altering the composition of the extract (Schultz and Randall, 1970). Until recently, headspace sampling had not been found to be a widely useful technique due to the low concentration of volatiles and the large amount of water which may be present in collected samples. In 1966, Hollis introduced porous polymer beads which could be used as column packing for gas-liquid chromatography (GLC) (Hollis, 1966; Hollis and Hayes, 1966). Use of these porous polymers in sampling traps and as packing can avoid extraction difficulties as well as related problems of handling and transferring small quantities of volatile compounds. Used as an entrainment technique, the porous polymers have the ability to separate organic fractions from water, concentrate organic compounds, and give a GLC analysis all without difficult transfer of the sample.

MATERIALS & METHODSP r e p a r a t i o n o f c o f f e e b r e w s

C o f f e e b e a n s w e r e r o a s t e d , g r o u n d a n d v a c u u m p a c k e d in 1 lb ( # 4 0 1 ) c a n s b y t h e C o f f e e B r e w i n g C e n t e r , N e w Y o r k . B l e n d a n d g r i n d a n a l y s e s a r e g iv e n in T a b l e 1. S a m p l e s w e r e s h i p p e d b y a i r t r a n s p o r t t o s t o r a g e f a c i l i t i e s a t D a v is w h e r e t h e y w e r e k e p t a t 5 ° C . T o p r e p a r e t h e b r e w , a s p e c i f i c w e i g h t o f c o f f e e g r o u n d s a t r o o m t e m p e r a t u r e w a s p l a c e d in a h e a v y p l a s t i c c o n e ( D a v i d D o u g l a s a n d C o . , M a n i t o w a c , W ise . ) l i n e d w i t h a p o r o s i t y c o n t r o l f i l t e r ( “ F l a v - r - F l o w , ” B o y d C o f f e e C o . , P o r t l a n d , O r e g . ) . A t h e r m o m e t e r w a s p l a c e d in t h e f i l t e r c o n e a s f a r d o w n i n t o t h e g r o u n d s as p o s s i b l e . A 3 7 5 - m l p o r t i o n o f w a t e r a t 1 0 0 ° C w a s p o u r e d i n t o t h e c o n e o v e r t h e g r o u n d s . T h e m i x t u r e w a s s t i r r e d w i t h a g la ss r o d o n e fu l l t u r n , a n d t h e t e m p e r a t u r e w a s r e c o r d e d a s t h e w a t e r f i l t e r e d t h r o u g h t h e g r o u n d s . T h e i d e n t i c a l p r o c e d u r e w a s r e p e a t e d a t 3 0 s e c , 1 .5 m i n a n d 3 m i n i n t e r v a l s , f o r a t o t a l w a t e r v o l u m e o f 1 5 0 0 m l , a n d a l l o w e d t o f i l t e r f o r a t o t a l t i m e p e r i o d o f 7 m i n . A 5 0 0 - m l p o r t i o n w a s r e m o v e d f o r m e a s u r e m e n t o f e x t r a c t e d s o l id s a n d t h e r e m a i n i n g v o l u m e w a s i m m e d i a t e l y p l a c e d in t h e h e a d s p a c e t r a p p i n g s y s t e m .

W a t e r u s e d in t h i s s t u d y w e r e : d i s t i l l e d , h a r d ( 1 0 m e q / l i t e r ( c a t i o n i c ) e a c h o f N a C l . C a C l , a n d M g C l j ] a n d s o f t [ 3 0 m e q / l i t e r ( c a t i o n i c ) o f N a C l ( . W a t e r s t o c k s w e r e m a d e u s i n g d i s t i l l e d w a t e r a n d r e a g e n t g r a d e c h e m i c a l s .

C o f f e e b r e w s w e r e m a d e t o a c o n s t a n t e x t r a c t e d s o l i d s o f 0 . 9 6 ± 0 . 0 2 % . W e i g h t s o f g r o u n d s n e e d e d f o r e a c h w a t e r t y p e - d i s t i l l e d , h a r d a n d s o f t - w e r e 7 5 , 5 3 a n d 5 0 g p e r 1 5 0 0 m l o f l i q u i d , r e s p e c t i v e l y .

E x t r a c t e d s o l id s in e a c h s a m p l e w e r e d e t e r m i n e d in t r i p l i c a t e w i t h a h y d r o m e t e r s p e c i f i c a l l y d e s i g n e d f o r l i q u i d a t 6 0 ° C ( R a s c h e r &

B e t z o l d I n c . , C h i c a g o , 111.), u s i n g t h e p r o c e d u r e d e s c r i b e d in d e t a i l b y L o c k h a r t ( 1 9 6 7 ) .

H e a d s p a c e t r a p p i n g s y s t e m

E q u i p m e n t . A V a r i a n A e r o g r a p h M o d e l 2 0 0 t h e r m a l c o n d u c t i v i t y i n s t r u m e n t w a s i n t e r f a c e d t o a V a r i a n A e r o g r a p h M o d e l 1 2 0 0 f l a m e i o n i z a t i o n i n s t r u m e n t v i a a s t a i n l e s s s t e e l d e l i v e r y l in e a n d s a m p l e l o o p . T h e 2 0 0 t h e r m a l c o n d u c t i v i t y u n i t w a s f i t t e d w i t h a 3 5 - c m X 7 - m m ( o . d . ) g lass c o l u m n p a c k e d w i t h 6 0 - 8 0 m e s h P o r a p a k Q ( W a t e r s A s s o c i a t e s , F r a m i n g h a m , M as s . ) . T h e c a r r i e r g a s w a s n i t r o g e n , w i t h a f l o w r a t e o f 6 m l / m i n . I n j e c t o r t e m p e r a t u r e w a s 2 0 0 ° C , a n d d e t e c t o r t e m p e r a t u r e w a s 2 5 0 ° C , a n d t h e u n i t w a s o p e r a t e d w i t h t h e f i l a m e n t s t u r n e d o f f . T h e 1 2 0 0 u n i t w a s f i t t e d w i t h a C a r l e v a lv e w h i c h a l l o w e d t h e c a r r i e r gas t o f l o w d i r e c t l y t o t h e c o l u m n , o r t o b e s h u n t e d t h r o u g h t h e s a m p l i n g l o o p a n d t h e n o n t o t h e c o l u m n ( R i c h a r d e t a h , 1 9 7 1 ) . T h i s i n s t r u m e n t w a s f i t t e d w i t h a 1 0 - m x 3 - m m ( o . d . ) g la ss c o l u m n p a c k e d w i t h 3 % ( w / w ) C a r b o w a x 2 0 M o n 6 0 - 8 0 m e s h C h r o m o s o r b G . T h e d e t e c t o r t e m p e r a t u r e w a s h e l d a t 2 7 5° C . T h e c a r r i e r ga s w a s n i t r o g e n , w i t h a f l o w r a t e o f 2 9 . 3 m l / m i n ; h y d r o g e n a n d a i r f l o w r a t e s w e r e 3 5 . 7 a n d 3 0 0 m l / m i n , r e s p e c t i v e l y . T h e C a r l e v a lv e t e m p e r a t u r e w a s 2 2 5 ° C , t h e b y p a s s l o o p 2 3 0 ° C , a n d t h e d e l i v e r y l i n e 2 2 5 ° C . T h e s a m p l i n g l o o p w a s a 1 0 - in . X 0 . 2 5 - i n . ( o . d . ) s t a i n l e s s s t e e l t u b e p a c k e d w i t h c h r o m a t o g r a p h i c g r a d e g la ss w o o l a n d w e l d e d t o t h e v a lv e . A H o n e y w e l l E l c c - t r o n i k 1 9 4 r e c o r d e r w a s u s e d w i t h a c h a r t s p e e d o f 1 0 m i n / i n .

M e t h o d . A l i t e r o f c o f f e e b r e w w a s p l a c e d in a 3 - l i t e r , r o u n d - b o t t o m g la s s f l a s k i m m e r s e d in a 6 0 ° C w a t e r b a t h . N i t r o g e n , p u r i f i e d t h r o u g h f i r e b r i c k a n d a c t i v a t e d c h a r c o a l , w a s p u r g e d t h r o u g h t h e b r e w a t a f l o w r a t e o f 1 0 0 m l / m i n f o r 1 h r b y p a s s a g e t h r o u g h a s p a r g e r p o s i t i o n e d

Table 1—Blend and grind analyses fo r coffee

Blend Country o f origin Grade Percentage

Columbia Armenia Excelso 33.3El Salvador Central Standard 33.3Brazil Santos 2/3 33.3

Sieve No. Tyler mesh Percentage retentionGrinda U.S. Std equivalent Regular Drip

12 10 2.9 1.516 14 19.9 10.020 20 52.8 42.130 28 11.6 25.0Pan Pan 13.3 20.8

a P e r f o r m e d a c c o r d i n g t o U S D C S i m p l i f i e d P r a c t i c e R e c o m m e n d a t i o n 2 3 1 4 8 . R o a s t e d t o 5 . 5 %

r e f l e c t a n c e a c c o r d i n g t o C o f f e e B r e w i n g C e n t e r p u b l i c a t i o n # 5 3 , " R o a s t e d C o f f e e C o l o r M e a s u r e m e n t C l a s s i f i c a t i o n " .


COFFEE B E VE RAG E HEADSPACE V O L A T IL E S -6 5

b e l o w t h e l i q u i d s u r f a c e . T h e h e a d s p a c e v o l a t i le s w e r e p a s s e d t h r o u g h ( a n d e n t r a i n e d o n ) a 3 5 - c m X 7 - m m ( o . d . ) g la ss P o r a p a k c o l u m n . A t t h e e n d o f t h e 1 -h r c o l l e c t i o n p e r i o d , t h e n i t r o g e n f l o w w a s t u r n e d o f f . T h e f l a s k c o n t a i n i n g t h e c o f f e e b r e w w a s t h e n d i s c o n n e c t e d f r o m t h e P o r a p a k c o l u m n , r e m o v e d f r o m t h e w a t e r b a t h , a n d r e p l a c e d w i t h a n e m p t y 5 0 0 - m l E r l e n - m e y e r f l a s k . T h e s y s t e m w a s a g a i n c o n n e c t e d a n d n i t r o g e n w a s p u r g e d t h r o u g h t h e P o r a p a k c o l u m n f o r 15 m i n a t 1 0 0 m l / m i n t o e l u t e w a t e r . T h e P o r a p a k c o l u m n w a s t h e n d i s c o n n e c t e d a n d p l a c e d in r e v e r s e p o s i t i o n in t h e t h e r m a l c o n d u c t i v i t y G L C ( t h e e n d o f t h e c o l u m n o n w h i c h t h e h e a d s p a c e v o l a t i l e s w e r e c o l l e c t e d w a s c o n n e c t e d t o t h e d e t e c t o r ) . O v e n t e m p e r a t u r e w a s r a p i d l y p r o g r a m m e d t o 2 0 0 ° C .

U s i n g a f l o w r a t e o f 6 m l / m i n , v o l a t i l e s w e r e e l u t e d o f f t h e P o r a p a k c o l u m n a n d c o n d e n s e d in- t h e s a m p l i n g l o o p , i m m e r s e d in a d r y ice - e t h a n o l b a t h , f o r 1 5 m i n . A f t e r c o l l e c t i o n , t h e C a r l e v a lv e w a s t u r n e d s o t h a t n i t r o g e n c a r r i e r g as f r o m t h e f l a m e i o n i z a t i o n u n i t p a s s e d t h r o u g h t h e s a m p l i n g l o o p a t a f l o w r a t e o f2 9 . 3 m l / m i n . T h e d r y i c e - e t h a n o l b a t h w a s r e m o v e d , a n d t h e l o o p w a s h e a t e d w i t h a h o t a ir g u n f o r 2 m i n t o d i s c h a r g e t h e v o l a t i l e s o n t o t h e C a r b o w a x c o l u m n . T h e C a r l e v a lv e w a s t h e n t u r n e d t o i t s o r i g i n a l p o s i t i o n , a n d t h e c h r o m a t o g r a p h o v e n p r o g r a m m e d a t a r a t e o f 2 ° C / m i n f r o m 3 0 - 2 3 0 ° C . T h e a t t e n u a t i o n o n t h e i n s t r u m e n t w a s XI, w i t h a n i m p e d a n c e r a n g e o f 1 0 " ' 0 a m p .

A f t e r t h e v o l a t i l e s w e r e c o l l e c t e d o f f t h e P o r a p a k , t h e c o l u m n w a s h e a t e d o u t f o r l ' / i h r a t 2 1 0 ° C , w i t h a n i t r o g e n f l o w o f 1 0 0 m l / m i n . T h e c o l u m n w a s t h e n c o o l e d t o a m b i e n t t e m p e r a t u r e f o r u s e in c o l l e c t i n g t h e n e x t s a m p l e o f v o l a t i l e s . W h i le n i t r o g e n w a s s t i l l f l o w i n g t h r o u g h t h e s a m p l e l o o p , i t w a s h e a t e d w i t h t h e h o t a i r g u n f o r 5 m i n t o d i s c h a r g e a n y r e s i d u e o r c o l u m n b l e e d .

D a t a a n a l y s i s

P e a k a r e a s w e r e i n t e g r a t e d w i t h a n I n f o t r o n - ics C R S 1 0 4 d ig i t a l i n t e g r a t o r . T h e o u t p u t w a s r e c o r d e d o n a T - 3 3 s t a n d a r d t e l e t y p e , u t i l i z i n g p a p e r t a p e . T h i s t a p e w a s t h e n u s e d t o g e n e r a t e a m a g n e t i c t a p e o f s u i t a b l e f o r m a t f o r f u r t h e r a n a l y s i s b y c o m p u t e r . P r o g r a m s w r i t t e n s p e c i f i c a l ly f o r t h i s t y p e o f c h r o m a t o g r a p h i c d a t a w e r e u s e d t o c a l c u l a t e r e l a t i v e r e t e n t i o n , a r e a p e r c e n t a g e s , s t a n d a r d d e v i a t i o n s a m o n g a n a l y s e s , n o r m a l i z e d c h r o m a t o g r a m s f o r e a c h t y p e o f w a t e r , a n d t o p l o t h i s t o g r a m s o f e a c h p e a k ( R i c h a r d e t a l . , 1 9 7 1 ) . A B i o m e d c o m p u t e r p r o g r a m f o r a n a l y s i s o f v a r i a n c e w a s u t i l i z e d t o d e t e r m i n e i f t h e r e w e r e s i g n i f i c a n t d i f f e r e n c e s in t h e a r o m a o f c o f f e e m a d e w i t h t h e t h r e e t y p e s o f w a t e r .

S e n s o r y a n a l y s i s o f t h e a r o m a o f c o f f e e b r e w s

D i s c r i m i n a t i o n . T o d e t e r m i n e s e n s o r y d i s c r i m i n a t i o n a m o n g t h e c o f f e e b r e w s m a d e w i t h d i s t i l l e d , h a r d a n d s o f t w a t e r s , p a n e l i s t s ( t h r e e f e m a l e s , t w o m a l e s ) w e r e p r e s e n t e d w i t h t r i a n g le t e s t s . S a m p l e s w e r e p r e p a r e d b y t w o m e t h o d s : ( 1 ) t o a c o n s t a n t e x t r a c t e d s o l id s ( 0 . 9 6 ± 0 . 0 2 % ) a n d ( 2 ) t o a c o n s t a n t f o r m u l a t i o n ( 5 0 g g r o u n d s / l i t e r w a t e r ) . S ix t r i a n g l e t e s t s w e r e p r e s e n t e d in r a n d o m i z e d o r d e r in 6 0 ° C w a t e r b a t h s o n t h r e e c o n s e c u t i v e d a y s , f o r a t o t a l o f 9 4 .

D e s c r i p t i o n o f b r e w s . T o d e v e l o p a n e x t e n sive v o c a b u l a r y t o d e s c r i b e c o f f e e , e x p e r i m e n t a l s a m p l e s w e r e p r e p a r e d t o p r e s e n t a s m a n y d i s s im i l a r , y e t c o f f e e - l i k e o d o r s a s p o s s i b l e . T h e s e w e r e d r i p g r o u n d s ; a h e a d s p a c e c o l l e c t i o n f r o m

c o f f e e m a d e f r o m d i s t i l l e d w a t e r ; a r o m a - s t r i p p e d - d i s t i l l e d - w a t e r c o f f e e ; b u r n t g r o u n d s ; d r i p c o f f e e m a d e f r o m d i s t i l l e d , s o f t , a n d h a r d w a t e r s ; s t r i p p e d g r o u n d s f r o m s u c h c o f f e e s ; a n d f r o m i n s t a n t c o f f e e ; h a l f - s t r e n g t h c o f f e e ; d o u b l e - s t r e n g t h c o f f e e ; c o f f e e p l u s c r e a m ; c o f f e e p l u s s u g a r ; c o f f e e p l u s c r e a m a n d s u g a r ; b o i l e d r e g u l a r g r i n d c o f f e e ; p e r c o l a t e d r e g u l a r g r i n d c o f f e e g r o u n d s ; a n d c o l d - e x t r a c t e d c o f f e e . A h e a d s p a c e c o l l e c t i o n s a m p l e w a s p r e p a r e d as p r e v i o u s l y d e s c r i b e d , w i t h e f f l u e n t f r o m t h e P o r a p a k c o l u m n c o l l e c t e d a n d s e a l e d in a c a p i l l a r y p i p e t t e p a c k e d w i t h c h r o m a t o g r a p h i c g r a d e g lass w o o l . T h e s a m p l e t u b e w a s b r o k e n a n d g r o u n d in a c u p j u s t p r i o r t o s a m p l e p r e s e n t a t i o n . T h e a r o m a - s t r i p p e d c o f f e e w a s a s a m p l e o f t h e b r e w l e f t in t h e h e a d s p a c e t r a p p i n g s y s t e m a f t e r a I - h r c o l l e c t i o n o f v o l a t i l e s . D r i p g r o u n c s w e r e p l a c e d in 2 0 0 ° C o v e n f o r 2 0 m i n t o p r o d u c e a b u r n t - g r o u n d s a m p l e . T h e s t r i p p e d g r o u n d s w e r e t h o s e l e f t o n t h e f i l t e r p a p e r a f t e r t h e 1 5 0 0 m l o f t h e v a r i o u s w a t e r s h a d p a s s e d t h r o u g h . F o r a b o i l e d c u p o f c o f f e e , t h e b r e w w a s p r e p a r e d in a n o n e l e c t r i c p e r c o l a t o r , o n e t a b l e s p o o n r e g u l a r g r o u n d s / t w o c u p s b e v e r a g e .

O n c e p e r c o l a t i o n c o m m e n c e d it w a s m a i n t a i n e d f o r 6 m i n . T h e g r o u n d s w e r e t h e n r e m o v e d a n d u s e d a s t h e p e r c o l a t e d r e g u l a r g r o u n d s s a m p l e . T h e b r e w w a s b o i l e d f o r 2 h r a n d t h e n r e m o v e d t o a 6 0 ° C w a t e r b a t h . 5 0 g o f d r i p g r o u n d s w e r e p l a c e d in I l i t e r o f d i s t i l l e d w a t e r a n d e x t r a c t e d o v e r n i g h t . T h e b r e w w a s t h e n f i l t e r e d a n d t h e l i q u i d u s e d a s o n e o f t h e s a m p l e s .

S a m p l e s w e r e p r e s e n t e d t o t h e p a n e l i n 8 0 m l c o b a l t b l u e g la s s e s c o v e r e d w i t h i n n e r a l u m i n u m l i n e r s p u n c h e d w i t h s m a l l h o l e s w h i c h , in t u r n , w e r e c o v e r e d b y c o d e d s o l i d a l u m i n u m lids . T h e g la s s e s c o n t a i n e d 3 5 m l o f t h e l i q u i d s a m p l e s , o r a p p r o x i m a t e l y lO g o f t h e g r o u n d s a m p l e s . S a m p l e p r e s e n t a t i o n t e m p e r a t u r e w a s m a i n t a i n e d a t 6 0 ° C t h r o u g h o u t e a c h s e s s i o n b y k e e p i n g t h e g l a s s e s i n s t a i n l e s s s t e e l w a t e r b a t h s m a i n t a i n e d a t 6 0 ° C . T h e h e a d s p a c e s a m p l e w a s p r e s e n t e d a t r o o m t e m p e r a t u r e .

P a n e l s e s s i o n s w e r e c o n d u c t e d e a c h d a y f r o m 2 : 0 0 - 3 : 0 0 P M in a t e s t r o o m m a i n t a i n e d a t 2 2 ° C , a n d u n d e r b l u e i l l u m i n a t i o n . F r e s h a ir w a s c o n t i n u a l l y c i r c u l a t e d t h r o u g h o u t t h e r o o m . P a n e l i s t s w e r e s e a t e d a t a r o u n d t a b l e , a n d r e f e r e n c e m a t e r i a l s w e r e a v a i l a b l e .

0 10 20 30 40 50 60 70 80

TIME (MINUTES)

Fig. 1—C h ro m a to g ra m o f c o ffe e a ro m a headspace. S a m p le : C o lle c te d fro m 1 ,0 0 0 m l d is t i l le d w a te r d r ip c o ffe e o n to P o ra p a k Q; In s tru m e n t: V arian A e ro g ra p h 1 2 0 0 w ith fla m e io n iz a t io n d e te c to r ; C o lu m n : 10 m X 3 m m (o .d .) glass c o lu m n . L iq u id ph ase : 3% C a rb o w a x 2 0 M , S o lid

s u p p o r t : 6 0 - 8 0 m esh C h ro m a s o rb G; C o n d it io n s : N itro g e n f lo w : 2 9 .3 m l/m in . H yd ro g e n f lo w : 3 5 .7 m l/m in . A i r f lo w : 3 0 0 m l/m in . C o lu m n te m p : T C / m in fro m 3 0 - 2 3 0 ’ C;

A t te n u a t io n : X 5.


D e s c r i p t i o n o f G L C e f f l u e n t . T h e s a m p l e f o r c h a r a c t e r i z a t i o n o f g a s c h r o m a t o g r a p h i c e f f l u e n t w a s a c o f f e e b r e w e d w i t h d i s t i l l e d w a t e r . T h e s a m p l e s w e r e p r e p a r e d as d e s c r i b e d a b o v e f o r t h e h e a d s p a c e t r a p p i n g s y s t e m , w i t h t h e f o l l o w i n g m i n o r c h a n g e s . V o l a t i l e s w e r e c o l l e c t e d o n t o a 2 2 - c m X 6 - m m ( o . d . ) g la ss c o l u m n . T h i s c o l u m n w a s f i t t e d i n a n A e r o g r a p h 9 0 - P 3 t h e r m a l c o n d u c t i v i t y i n s t r u m e n t . T h e s a m p l e w a s t r a p p e d f o r l 5 m i n o n t o a 3 0 - c m x3 - m m ( o . d . ) g la ss U - t u b e i m m e r s e d in a d r y ice - e t h a n o l b a t h . A f t e r t h e s a m p l e w a s c o l l e c t e d , t h e e n d s o f t h e t u b e w e r e h e a t - s e a l e d a n d t h e t u b e w a s p l a c e d in a - 2 ° C f r e e z e r u n t i l t h e s a m p l e w a s r e q u i r e d f o r a n a l y s i s .

A V a r i a n A e r o g r a p h 2 0 0 i n s t r u m e n t w i t h t h e r m a l c o n d u c t i v i t y d e t e c t o r w a s u s e d f o r s e n s o r y e v a l u a t i o n o f t h e e f f l u e n t . I n j e c t o r t e m p e r a t u r e w a s 2 0 0 ° C , d e t e c t o r t e m p e r a t u r e w a s 2 7 5 ° C , a n d t h e f i l a m e n t s w e r e s e t a t 1 7 5 m a . T h e i n s t r u m e n t w a s e q u i p p e d w i t h t w o 3 . 5 - m x 1 0 - m m ( o . d . ) g la s s c o l u m n s . T h e c o l u m n f r o m w h i c h t h e e f f l u e n t w a s s n i f f e d w a s p a c k e d w i t h 3 % ( w / w ) C a r b o w a x 2 0 M o n 6 0 - 7 0 m e s h C h r o m a s o r b G . H e l i u m w a s t h e c a r r i e r gas , w i t h a f l o w r a t e o f 3 0 m l / m i n . C h r o m a t o g r a p h i c r e s u l t s w e r e r e c o r d e d o n a V a r i a n A e r o g r a p h M o d e l 2 0 r e c o r d e r w i t h a c h a r t s p e e d o f 4 m i n / i n .

T h e f o l l o w i n g m e t h o d w a s u s e d f o r s a m p l e i n j e c t i o n o n t o t h e C a r b o w a x c o l u m n . T h e g la ss U - t u b e c o n t a i n i n g t h e h e a d s p a c e s a m p l e w a s p l a c e d in a d r y i c e - e t h a n o l b a t h , a n d t h e t w o e n d s o f t h e t u b e w e r e b r o k e n o p e n . O n e e n d w a s f i t t e d w i t h a s y r i n g e n e e d l e , a n d t h e o t h e r e n d w a s l i n k e d t o t h e c a r r i e r g a s s o u r c e w i t h t e f l o n t u b i n g . S i m u l t a n e o u s l y , t h e s y r i n g e n e e d l e w a s i n j e c t e d t h r o u g h t h e i n j e c t o r p o r t , a n d a t w o - w a y c a r r i e r g a s v a lv e w a s o p e n e d so t h a t c a r r i e r g a s f l o w e d t h r o u g h t h e U - t u b e . T h e d r y i c e - e t h a n o l b a t h w a s r e m o v e d , a n d t h e U- t u b e w a s h e a t e d w i t h a h o t a i r g u n t o i n j e c t t h e s a m p l e o n t o t h e c o l u m n . T h e c a r r i e r g a s w a s t h e n r e t u r n e d t o n o r m a l f l o w . C h r o m a t o g r a p h i c r u n s w e r e p r o g r a m m e d f r o m 3 0 - 1 0 0 ° C a t 2 ° C / m i n , f r o m 1 0 0 - 1 7 0 ° C a t 4 ° C / m i n a n d t h e n r a p i d l y h e a t e d t o 2 3 0 ° C f o r 1 0 m i n .

F o r s n i f f i n g , t h e e x h a u s t p o r t o f t h e g as c h r o m a t o g r a p h w a s f i t t e d w i t h a 7 0 - m l g lass c u p t o i n t r o d u c e m o i s t e n e d a i r , s i m i l a r t o t h e s y s t e m d e s c r i b e d b y P o t t e r a n d D a y e ( 1 9 7 0 ) . T h e c u p h a d a h o l e i n t h e s id e w h i c h f i t t e d o n t o t h e e x i t p o r t . T y g o r . t u b i n g f r o m a g lass w a s h b o t t l e w a s a t t a c h e d t o t h e s t e m o f t h e c u p . T h e w a s h b o t t l e w a s a p p r o x i m a t e l y o n e - h a l f f i l l e d w i t h d i s t i l l e d w a t e r , a n d a i r f r o m a l a b o r a t o r y l i n e w a s b u b b l e d t h r o u g h t h e s p a r g e r .

T e s t i n g w a s c o n d u c t e d b e t w e e n 7 : 0 0 A M a n d 1 2 n o o n a t a s p e c i f i e d t i m e f o r e a c h p a n e l i s t . T h e t e s t i n g r o o m w a s w e l l v e n t i l a t e d , f r e e f r o m d i s t u r b i n g n o i s e s a n d t h e t e m p e r a t u r e w a s 2 1 ° C . T h e s a m e f ive j u d g e s w h o p a r t i c i p a t e d in t h e d e s c r i p t i v e c h a r a c t e r i z a t i o n o f t h e o d o r o f c o f f e e b r e w s m a d e w i t h v a r i o u s w a t e r s w e r e u s e d in t h i s s t u d y .

J u d g e s s n i f f e d f r o m t h e g la ss c u p a n d r e s p o n d e d b y d e s c r i b i n g t h e o d o r o f t h e e f f l u e n t . B e f o r e t h e s a m p l e w a s i n j e c t e d o n t o t h e c o l u m n , j u d g e s f a m i l i a r i z e d t h e m s e l v e s w i t h t h e b a c k g r o u n d o d o r ( i f a n y ) . J u d g e s w e r e s e a t e d a w a y f r o m t h e r e c o r d e r a n d c o n t r o l o p e r a t i o n s t o b l o c k t h e i r v i e w o f t h e r e c o r d e r r e s p o n s e s . R e s p o n s e s o f t h e p a n e l m e m b e r s w e r e r e c o r d e d d i r e c t l y o n t h e c h r o m a t o g r a m b y t h e e x p e r i m e n t e r a t t h e a p p r o p r i a t e r e t e n t i o n t i m e c o r r e s p o n d i n g t o t h e t i m e r e s p o n s e s w e r e g iv en .

J u d g e s w e r e f a m i l i a r i z e d w i t h t h e e x i t p o r t

s n i f f i n g p r o c e d u r e i n a t r i a l r u n , r e s p o n d i n g t o o d o r s w i t h a n y t e r m s w h i c h o c c u r r e d t o t h e m - b o t h q u a l i t a t i v e d e s c r i p t i v e t e r m s a n d e x a m p l e s o f m a t e r i a l s w h i c h t h e o d o r r e s e m b l e d w e r e a c c e p t a b l e . F o l l o w i n g t h e t r i a l r u n s , t h e r e s p o n s e s w e r e c o m p i l e d a n d g r o u p e d b y t h e e x p e r i m e n t e r a c c o r d i n g t o o d o r q u a l i t i e s . A g r o u p i n g a c c o r d i n g t o r e s p o n s e s g iv e n a t t h e s a m e r e t e n t i o n t i m e w a s a l s o p r e p a r e d , a n d b o t h l i s t i n g s w e r e d i s c u s s e d w i t h t h e j u d g e s .

A f t e r e s t a b l i s h m e n t o f t h e f i n a l s e t o f d e s c r i p t i v e t e r m s , j u d g e s e v a l u a t e d t h e G L C e f f l u e n t o n t h r e e d i f f e r e n t d a y s . T h e li s t o f d e s c r i p t iv e t e r m s w a s c o n s u l t e d a t t h e b e g i n n i n g o f e a c h s e s s i o n , b u t j u d g e s w e r e f r e e t o u s e a d d i t i o n a l t e r m s .

A f t e r t h e e f f l u e n t s n i f f i n g s t u d y , t h e t o t a l h e a d s p a c e w a s c o l l e c t e d a s t h r e e s a m p l e s - v o l a t i l e s w i t h e l u t i o n t e m p e r a t u r e o f 3 0 - 1 0 0 ° C , 101 - 1 7 0 ° C a n d 1 7 1 - 2 3 0 ° C - a n d e v a l u a t e d b y t h e p a n e l t o d e t e r m i n e i f a n y g r o u p in g o f c o m p o u n d s h a d a c o f f e e o d o r . T h e t h i r d s a m p l e o f v o l a t i l e s ( 1 7 1 - 2 3 0 ° C ) w a s a l s o s u b d i v i d e d i n t o c o m p o u n d s w i t h e l u t i o n t e m p e r a t u r e s o f 1 7 1 - 1 9 0 ° C , 1 9 1 - 2 1 0 ° C a n d 2 1 1 - 2 3 0 ° C a n d e v a l u a t e d b y t h e p a n e l . J u d g e s w e r e a s k e d i f a n y o f t h e s a m p l e s p o s s e s s e d a c o f f e e o d o r ; i f s o , t o r a n k s a m p l e s p o s s e s s i n g t h a t o d o r in o r d e r o f i n c r e a s i n g i n t e n s i t y o r s t r e n g t h , a n d t o d e s c r i b e t h e a r o m a o f e a c h s a m p l e .

RESULTSAnalytical study—GLC analysis of coffee headspace

Gas chromatographic analysis of the headspace volatiles of coffee brews made with distilled, hard and soft waters resulted in collection of quantitative data on 44 peaks. A typical chromatogram of this type of analysis is shown in Figure 1.

It was recognized in the integration of these chromatographic data, that probably none of the peaks involved represented the elution of a single component. The complexity of coffee headspace aromagrams has been demonstrated in many detailed studies in published literature. Obviously, even with sophisticated direct on-line computing algorithms, much variability would be anticipated from shifts in retention of individual components that cannot be controlled. With the CRS-104 digital integration, and its simple-peak sensing and baseline tracking system, these integrated areas were intended only to indicate “clusters” of individual components, and to treat each of the peaks thus detected as a single variable. Despite these limitations, of the 44 peaks on which data were collected, 1 2 , 19 and 21 peaks for distilled, hard and soft waters, respectively, had total system coefficients of variance less than 1 0 %.

There are many analytical studies reporting much less variability for model systems with pure components and simple gas standards. However, for a “practical” application of quantitative measurements, the study by Grant and Clarke(1971) clearly showed that long term errors (those over the course of several days, with variables such as temperature

and flow rate) could have coefficients of variation as high as 25%. Even with a model system of five pure components, the effects of six variables and two-factor interactions for individual peak areas showed five out of six factors were significant at p<0.05. These effects were greatly reduced when peak areas were normalized to an internal standard.

Peak 4 was the most reproducible and constant across waters, with coefficients of variance within each water of 3.11%, 0.05% and 2.73% for distilled, hard and soft waters respectively, and across water types equal to 3.73%. This peak was used as an intrinsic internal standard on which normalized areas of other peaks were computed and analyzed statistically. (The normalized area of an individual peak Xjj was equal to the integrated area of peak Xy divided by the integrated area of peak 4jj, where i = type of water and j = the replicate within the ith water type.)

Application of analysis of variance to the normalized average data indicated that eight peaks differed significantly as shown in Table 2, depending on the type of water used in the coffee brewing. These were peaks 1, 2, 3, 9, 19, 20, 21 and 32 as labelled in Figure 1. Each peak differed depending on which brew produced the greatest or fewest volatiles. There was no overall trend relating increasing or decreasing proportions of significant peaks in relation to the water type.Sensory analysis

Triangle test results indicated that the panelists could not differentiate among aromas of coffees made to constant extracted solids nor to a constant formulation (Table 3). Development of 17 terms to describe various coffee odors provided some interesting results concerning the effect of water on coffee aroma. Brewed drip coffees were considered to have the most fully described aromas, often described as burnt and musty. The burnt quality of coffee was characteristic of the grounds themselves. The addition of water to the grounds apparently added a definite musty character to coffee, most prominent in coffee made with soft water. The musty quality of the grounds- water combination remained with the grounds even after drying, greatly decreasing the desirable aroma character of the grounds.

Resulting from sniffing of the GLC effluent showed that twenty descriptive words were used more than three times each. Buttery, musty, burnt, sulfury, and sweet were used 56, 56, 48, 47 and 41 times, respectively. The early portion of the chromatogram (peaks 7—8, 10—14, 18—19) was characterized as buttery then burnt (peaks 12-13, 15-17), with the reappearance of a buttery character (peaks 15—17 and 21—23). Peaks 21—26 were described as musty, peaks 29-32 had a

COFFEE B E VE RAG E HEADSPACE V O L A T IL E S -6 7

n u t t y c h a r a c t e r , p e a k s 3 1 — 3 2 h a d a v e g e t a b l e - l i k e q u a l i t y , a n d p e a k s 3 6 —4 4

w e r e o f a f l o r a l c h a r a c t e r . A c o m p o u n d , o r g r o u p o f c o m p o u n d s , e m e r g i n g a t 2 3 0 ° C ( r e t e n t i o n t i m e 8 5 . 0 m i n ) w a s d e s c r i b e d a t l e a s t o n c e b y a l l j u d g e s ( 1 1 / 1 5 t i m e s t o t a l ) a s r e s e m b l i n g c o f f e e . T h e c o m p o u n d ( s ) m u s t b e p r e s e n t i n v e r y l o w l e v e l s f o r d e t e c t i o n b y f l a m e i o n i z a t i o n d e t e c t o r s , a s t h e G L C c h r o m a t o g r a m s h o w e d a f l a t b a s e l i n e i n t h i s a r e a .

R e s u l t s o f t e m p e r a t u r e g r o u p i n g o f v o l a t i l e s c o r r e s p o n d e d w i t h t h o s e f r o m

s n i f f i n g t h e i n i t i a l e f f l u e n t o f e l u t e d c o m p o u n d s f o r t h e G L C . F o u r o f t h e f i v e

p a n e l i s t s a g r e e d t h a t a c o f f e e a r o m a e x i s t e d i n o n e o f t h e s a m p l e s , n a m e l y t h e g r o u p o f v o l a t i l e s e l u t e d b e t w e e n 1 7 1 —

2 3 0 ° C . W h e n t h i s c l u s t e r o f v o l a t i l e s

w a s b r o k e n i n t o t h r e e s m a l l e r t e m p e r a

t u r e g r o u p s ( 1 7 1 - 1 9 0 ° C , 1 9 1 - 2 1 0 c C

a n d 2 1 1 — 2 3 0 ° C ) , t h e l a s t g r o u p i n g p o s

s e s s e d a c o f f e e a r o m a . T h i s a g r e e d w i t h e a r l i e r r e s u l t s i n d i c a t i n g t h a t s o m e c o m p o u n d , o r g r o u p o f c o m p o u n d s , e l u t i n g

a t 2 3 0 ° C p o s s e s s e s , o r c o n t r i b u t e s t o

a c h a r a c t e r i s t i c c o f f e e a r o m a .

W h e n g r o u p e d t o g e t h e r , l o w - b o i l i n g c o m p o u n d s , i . e . , t h o s e w i t h e l u t i o n t e m

p e r a t u r e s o f 3 0 — 1 0 0 ° C , s e e m e d t o p r o v i d e u n d e r l y i n g q u a l i t i e s o f c o f f e e a r o m a —s o u r , m e l l o w , s l i g h t l y b u r n t . T h e m e d i u m - b o i l i n g c o m p o u n d s a p p e a r e d r e s p o n s i b l e f o r t h e h e a v i e r , m u s t y , r o a s t e d c h a r a c t e r i s t i c s o f c o f f e e , a n d t h e h i g h e r

b o i l e r s ( 1 7 1 —2 3 0 ° C ) r o u n d e d o u t t h e

a r o m a , c r e a t i n g “ c o f f e e . ”

DISCUSSION

A n a l y t i c a l i n v e s t i g a t i o n

V o l a t i l e s c o l l e c t e d f r o m c o f f e e b r e w s u s i n g t h e P o r a p a k h e a d s p a c e m e t h o d o l o g y p o s s e s s e d a s t r o n g c h a r a c t e r i s t i c c o f f e e a r o m a . W h e n c h r o m a t o g r a p h e d ,

q u a n t i t a t i v e d a t a w e r e o b t a i n e d o n 4 4

p e a k s , h a l f o f w h i c h h a d a t o t a l s y s t e m v a r i a b i l i t y o f l e s s t h a n 1 0 % . T h i s d e g r e e o f r e p r o d u c i b i l i t y w a s q u i t e g o o d , c o n s i d

e r i n g t h e c o m p l e x i t y o f t h e e n t i r e c o f f e e

v o l a t i l e s s y s t e m .T h e P o r a p a k m e t h o d o l o g y h a s s e v e r a l

f e a t u r e s t h a t o f f e r a d v a n t a g e s o v e r s o l v e n t e x t r a c t i o n . V o l a t i l e s c o l l e c t e d v i a

t h e P o r a p a k t e c h n i q u e a r e c o n c e n t r a t e d o n t h e b a s i s o f r e l a t i v e v o l a t i l i t y , r a t h e r t h a n o n a f f i n i t y f o r o r s o l u b i l i t y i n a p a r

t i c u l a r s o l v e n t v e r s u s w a t e r . A m a j o r p r o b l e m e n c o u n t e r e d i n p r e p a r i n g a n y v o l a t i l e s a m p l e f o r c h r o m a t o g r a p h i c s e p a r a t i o n i s c o n c e n t r a t i o n o f t h a t s a m p l e w i t h a m i n i m u m l o s s o f v o l a t i l e s a n d a m i n i m u m c h e m i c a l c h a n g e . C o m p a r i s o n o f t h e P o r a p a k d a t a w i t h t h a t i n w h i c h F r e o n 1 1 3 ( R u s s e l l e t a l . , 1 9 7 3 ) w a s u s e d t o e x t r a c t v o l a t i l e s r e v e a l e d l a r g e d i f f e r e n c e s i n t h e u p t a k e o f l o w - b o i l i n g c o m p o u n d s . T h i s w a s d u e t o t h e c l o s e e l u t i o n p r o x i m i t y o f t h e s o l v e n t , t o t h e s e l o w

b o i l e r s . D u r i n g c o n c e n t r a t i o n o f t h e s a m

p l e v o l a t i l e s , m a n y o f t h e s e l o w b o i l e r s

w e r e e v a p o r a t e d a l o n g w i t h t h e s o l v e n t ,

w h e r e a s o t h e r s e l u t e d f r o m t h e c h r o m a t o g r a p h i c c o l u m n s i m u l t a n e o u s l y w i t h t h e s o l v e n t . I n t h e h e a d s p a c e t e c h n i q u e , c o n c e n t r a t i o n o f t h e s a m p l e i n v o l v e s r e m o v a l

o f t h e w a t e r p r e s e n t i n t h e s a m p l e , a n d o c c u r s a l o n g w i t h s a m p l e c o l l e c t i o n .

I n t h i s s t u d y , a n o t h e r a d v a n t a g e o f P o r a p a k a n a l y s i s o v e r s o l v e n t e x t r a c t i o n w a s t h e l a c k o f a s o l v e n t o d o r m a s k i n g l o w - b o i l i n g c o m p o u n d s i n t h e s e n s o r y s t u d y . A f t e r c h r o m a t o g r a p h y o n a t h e r

m a l c o n d u c t i v i t y i n s t r u m e n t , r e c o l l e c t e d P o r a p a k e x t r a c t e d s a m p l e s s t i l l p o s s e s s e d a s t r o n g c h a r a c t e r i s t i c c o f f e e a r o m a .

C o m p a r i s o n o f t h e P o r a p a k m e t h o d o l -

o g y w i t h t h a t d e v e l o p e d b y R h o a d e s

( 1 9 5 8 ; 1 9 6 0 ) r e v e a l s a d v a n t a g e s i n s a m p l e a n a l y s i s . V o l a t i l e s w e r e c o l l e c t e d i n a s i m i l a r m a n n e r i n t h e t w o e x p e r i m e n t s , t h e d i f f e r e n c e s b e i n g i n t h e m e t h o d o f s a m p l e c o n c e n t r a t i o n , i . e . , w a t e r v a p o r

r e m o v a l , a n d t h e a m o u n t o f s a m p l e t r a n s f e r b e f o r e c h r o m a t o g r a p h i c a n a l y s i s . W i t h t h e R h o a d e s t e c h n i q u e , w a t e r w a s r e m o v e d f r o m t h e h e l i u m a n d w a t e r s a m p l e

b y p a s s a g e t h r o u g h i c e w a t e r c o n d e n s e r s , w h i c h p r o b a b l y a l s o r e s u l t e d i n t h e l o s s

o f q u a n t i t i e s o f t h e v o l a t i l e s p r e s e n t . O n c e t r a p p e d o n t h e P o r a p a k c o l u m n , t h e m a j o r v o l a t i l e s r e m a i n e d t h e r e u n t i l j u s t

p r i o r t o i n j e c t i o n o n t o t h e c h r o m a t o g r a p h i c c o l u m n .

Table 2—F ratios and least significant differences (LSD) for significant peak areas present in the chromatographic headspace of coffee made w ith distilled, hard and soft water

Peakno. F Ratios LSD Mean normalized peak area3

1 14.4** 0.9733 Distilled Hard Soft1.3940 0.4166 0.3740

2 10.62* 0.6086 Hard Distilled Soft1.5737 1.3905 0.6323



19 5.98* 0.4075 Hard Soft Distilled2.6875 2.3437 2.1581

20 8.16* 2.9684 Soft Distilled Hard10.5817 8.7503 6.0715

21 7.23* 0.3406 Hard Soft Distilled2.3989 2.3091 1.9623

32 8.15* 0.5894 Soft Distilled Hard0.9689 0.2749 0.2223

3 A n y t w o - m e a n s u n d e r s c o r e d b y t h e s a m e l i n e a r e n o t s i g n i f i c a n t l y

d i f f e r e n t .

* S i g n i f i c a n t a t p < 0 . 0 5

* * S i g n i f i c a n t a t p < 0 . 0 1

Table 3 —Results of triangle testing of aroma o f coffees made w ith distilled, hard and soft waters

Coffees made to constant extracted solids (0.96 ± 0.02%)

ComparisonNumbercorrect

No. necessary for sig. at p < 0.05a

Distilled-hard 8/18 10Distilled-soft 7/18 10Hard-soft 9/18 10

Coffees made to constant form ula (50g coffee/1000 ml water)Distilled-hard 2/13 8Distilled-soft 7/15 9Hard-soft 1/12 8

3 A m e r i n e e t a l . t 1 9 6 5 )

6 8 - JO U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

S e n s o r y i n v e s t i g a t i o n

T h e v o c a b u l a r y d e v e l o p e d t o d e s c r i b e

b r e w a r o m a , w h e n c o m p a r e d w i t h t h a t d e v e l o p e d f o r c o m p o u n d s e l u t i n g f r o m t h e c h r o m a t o g r a p h i c a n a l y s i s o f t h e b r e w s , o f f e r s a p o s s i b l e e x p l a n a t i o n f o r t h e l a c k o f s e n s o r y p e r c e p t i o n o f c h r o m a t o g r a p h i c d i f f e r e n c e s . Q u a l i t i e s a t t r i b u t e d t o t h e q u a n t i t a t i v e l y s i g n i f i c a n t p e a k s w e r e o n e s n o t f o u n d i n t h e c o f f e e b r e w a r o m a , a n d s e v e r a l o f t h e s i g n i f i c a n t p e a k s d i d n o t e v e n p o s s e s s a n a r o m a . I n t h e b l e n d i n g a n d i n t e r a c t i o n o f t h e v a r i o u s c o m p o u n d s m a k i n g u p t h e t o t a l a r o m a c o m p l e x , t h e s e i n d i v i d u a l p e a k q u a l i t i e s a p p e a r e d t o b e s u b o r d i n a t e d ,

w h e r e a s o t h e r s p e r s i s t e d a n d n e w o n e s w e r e c r e a t e d . W h e t h e r t h i s c o m b i n a t i o n o f s o m a n y d i s s i m i l a r , p l e a s a n t , a n d u n p l e a s a n t o d o r s t o c r e a t e a n a r o m a s u c h a s c o f f e e t a k e s p l a c e w i t h i n t h e b e v e r a g e , o r

a t t h e n a s a l r e c e p t o r s r e m a i n s t o b e a n s w e r e d .

I n a d d i t i o n t o t h e v a r i e t y o f o d o r a n t s r e p o r t e d t o b e p r e s e n t i n c o f f e e , t h e q u e s t i o n s o f t h e i r i n d i v i d u a l a n d c u m u l a t i v e

o d o r t h r e s h o l d s a r e i m p o r t a n t a l s o . H e a d - s p a c e s a m p l i n g t e c h n i q u e s a r e u s e f u l i n a n a l y s i s o f l o w b o i l i n g c o m p o u n d s , b u t

n o t a l w a y s w i t h h i g h b o i l e r s . T h e m e t h o d

r e p o r t e d h e r e w a s e f f e c t i v e i n c a p t u r i n g

t h e h i g h e r - b o i l i n g a r o m a c o n t r i b u t o r s a s

e v i d e n c e d b y t h e j u d g e s ’ u n a n i m o u s o p i n i o n t h a t a c o m p o u n d , o r g r o u p o f

c o m p o u n d s , e l u t i n g a t a r e t e n t i o n t i m e o f 8 5 m i n , s t r o n g l y r e s e m b l e d c o f f e e . A l

t h o u g h t h i s c o m p o u n d ( s ) w a s n o t d e t e c t e d b y t h e g a s c h r o m a t o g r a p h , i t w a s s h o w n t o b e p r e s e n t b y s e n s o r y a n a l y s i s , a n d o v e r s h a d o w e d t h e b u t t e r y a n d o t h e r q u a l i t i e s t o c r e a t e t h e c h a r a c t e r i s t i c c o f f e e a r o m a . T h i s c o m p o u n d m u s t h a v e a v e r y l o w o d o r t h r e s h o l d i n r e l a t i o n t o t h e o t h e r c o m p o u n d s w h i c h r e g i s t e r e d s u c h l a r g e c h r o m a t o g r a p h i c r e s p o n s e s . T h i s p h e n o m e n o n h a s b e e n d e s c r i b e d p r e v i o u s

l y b y s u c h w o r k e r s a s G u a d a g n i e t a l .( 1 9 6 6 ) , w h e r e i n t h e m a i n c o m p o u n d r e s p o n s i b l e f o r a c h a r a c t e r i s t i c a r o m a w a s s m a l l i n t e r m s o f d e t e c t o r r e s p o n s e .

REFERENCESAmerine, M.A., Pangborn, R.M. and Roessler,

E.B. 1965. “ Principles of Sensory Evaluation of Food.” Academic Press, New York.

Grant, D.W. and Clarke, A. 1971. A systematic study of the quantitative effects of instrum ent control on analytical precision in flame ionization gas chromatography. Anal. Chem. 43: 1951.

Guadagni, D.G., Okano, S., Buttery, R.G., Burr,H.K. 1966. Correlation of sensory and gas- liquid chromatographic m easurem ents of apple volatiles. Food Technol. 20: 518.

Hollis, O.L. 1966. Separation of gaseous mixtures using porous polyarom atic polymer beads. Anal. Chem. 38: 309.

Hollis, O.L. and Hayes, W.V. 1966. Water analysis by gas chrom atography using porous polymer columns. J. Gas Chrom. 4: 235.

Lockhart, E.E. 1967. The coffee hydrom eter. Pub. #43, The Coffee Brewing Center, New York, N.Y.

Potter, R.H. and Daye, J. 1970. Apparatus to introduce m oisture into effluent gas. The Givaudan Flavorist (2): 8.

Rhoades, J.W. 1958. Sampling m ethod for analysis of coffee volatiles by gas chrom atography. Food Res. 23: 254.

Rhoades, J.W. 1960. Analysis of the volatile constituents of coffee. J. Agric. Food Chem. 8: 136.

Richard, H.M., Russell, G.F. and Jennings, W.G. 1971. The volatile com ponents of black pepper varieties. J. Chrom at. Sci. 9: 560.

Russell, G.F., Pangborn, R.M. and Hsu, C. 197 3. Analysis of coffee beverage. 1. Instrumental evaluation of color and volatile profiles. In preparation.

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Ms received 7 /18 /73 ; revised 9 /4 /73; accepted9/6/73. ____________

Appreciation is extended to Professor R.M. Pangborn for assistance with sensory m ethodology and to the judges for their participation. We thank Mr. John Adinolfi of the Coffee Brewing Center for his patience, guidance and support of this project.

D . J. S E S S A , K . W A R N E R a n d D. H. H O N IG

U S D A N o r th e rn R e g io n a l Research L a b o ra to ry , A R S , P eoria , IL 6 1 6 0 4

SOYBEAN PHOSPHATIDYLCHOLINE DEVELOPS BITTER TASTE ON AUTOXIDATION

INTRODUCTION

R E S E A R C H O N S O Y B E A N f l a v o r h a s b e e n e x t e n s i v e , a n d m a n y p a t e n t s f o r i m

p r o v i n g o r r e d u c i n g f l a v o r o f s o y b e a n

p r o d u c t s h a v e b e e n g r a n t e d . S t i l l , t h i s f a c t o r a d v e r s e l y a f f e c t s t h e a c c e p t a n c e , a n d i n m a n y w a y s l i m i t s t h e u s e , o f s o y p r o d u c t s i n f o o d s . K a l b r e n e r e t a l . ( 1 9 7 1 )

r e p o r t e d t h a t t h e p r e d o m i n a n t b e a n y a n d b i t t e r f l a v o r s o f m a t u r e s o y b e a n s a r e s t i l l

p r e s e n t i n c o m m e r c i a l s o y f l o u r s , c o n c e n t r a t e s a n d i s o l a t e s . O x i d a t i v e d e g r a d a t i o n o f l i p i d s , w h i c h o c c u r s e i t h e r d u r i n g s t o r a g e o r p r o c e s s i n g , i s a c o m m o n c a u s e o f

o b j e c t i o n a b l e f l a v o r s i n f o o d s .B i t t e r t a s t e i s n o t . u s u a l l y a s s o c i a t e d

w i t h l i p i d d e g r a d a t i o n p r o d u c t s . H o w e v e r , W e i s s a n d D i e m a i r ( 1 9 3 9 ) e s t a b l i s h e d t h a t b i t t e r n e s s c a n a r i s e f r o m o x i d a t i o n o f p h o s p h o l i p i d s f r o m l u p i n e a n d r a p e . S e s s a e t a l . ( 1 9 6 9 ) d e m o n s t r a t e d

t h a t o i l - f r e e p h o s p h a t i d e s f r o m s o y b e c o m e i n t e n s e l y b i t t e r a f t e r i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t . S e s s a e t a l . ( 1 9 6 9 ) , R a c k i s e t a l . ( 1 9 7 0 ) a n d M a g a a n d J o h n s o n ( 1 9 7 2 ) h a v e r e p o r t e d o n l i p i d o x i d a t i o n a n d c h a n g e s i n l i p i d c o m p o s i t i o n o f s o y p r o d u c t s a s a f f e c t e d b y p r o c e s s i n g a n d s t o r a g e .

W e h a v e i s o l a t e d a n i n t e n s e l y b i t t e r f r a c t i o n c o n t a i n i n g a m o d i f i e d p h o s p h a t i d y l c h o l i n e a s t h e m a j o r c o n s t i t u e n t

f r o m a u t o x i d i z e d , d e f a t t e d s o y b e a n f l a k e s . T h e b i t t e r t a s t e o f s o y b e a n s m a y i n v o l v e o x i d a t i o n o f p h o s p h o l i p i d s . T h e p u r p o s e o f t h i s i n v e s t i g a t i o n w a s t o p u r i f y s o y p h o s p h a t i d y l c h o l i n e ( S P C ) a n d t o

d e t e r m i n e b y o r g a n o l e p t i c e v a l u a t i o n w h e t h e r a b i t t e r t a s t e d e v e l o p s d u r i n g i t s a u t o x i d a t i o n . H y d r o g e n a t e d S P C w a s p r e

p a r e d f o r a f l a v o r r e f e r e n c e . C h e m i c a l a n d p h y s i c a l a n a l y s e s w e r e u s e d t o f o l l o w t h e c o u r s e o f o x i d a t i o n o f t h e s e c o m p o u n d s .

EXPERIMENTALP repara tion o f SPC and hydrogenated SPC

SPC was purified by co lum n ch rom atography o f 1 -g p o rtio n s o f oil-free refined soy lecith in (N u tritio n a l B iochem icals C orp ., Cleveland, O hio) on 50g ac id -trea ted Florisil (Supel- cosil A T F-061, Supelco Inc., B ellefonte , Pa.). The co lum n (2.1 x 27 cm ) was e lu ted successively w ith : 1 0 0 ml ch lo ro fo rm ; 2 0 0 ml chloro- fo rm im eth an o l (19 :1 v/v); 200 m l (9 :1 ); 400 ml (3 :1 ); 6 0 0 ml (1 :1 ); and 600 ml m ethano l. The m aterial e lu ting w ith ch lo ro fo rm :m eth an o l ( 1 : 1 ) was s tripped o f solvent and then rech ro m atographed on a co lum n o f D EAE cellulose (Selectacel type 40 w ith capacity 1.14 nieq/g from Brow n C o., Berlin, N .H .) according to the p rocedure o f R ouser e t al. (1 9 6 7 ).

To prepare hydrogenated SPC, refined soy lecith in was freed o f residual oil and phospha- tidy le th an o lam in e by the p rocedure o f A neja et al. (1971) and then h y drogenated according to exam ple 22 o f a U.S. p a te n t by Davis (1962). This m ateria l was sub jected to the sam e ch ro m atograph ic p rocedures used to purify SPC. A nalytical m ethods

T he m aterials th a t e lu ted from the D EA E cellulose colum ns w ith ch lo ro fo rm n n e th an o l (9 :1 ) were iden tified as p h o spha tidy lcho lines by their infrared spectra recorded on a Perkin- E lm er Model 621 grating in frared spectro p h o to m eter. These spectra , ob ta in ed by the p rocedure o f M arinetti and S to tz (1 9 5 4 ), show ed strong bands a t the frequencies: 2910; 2850; 1730; 1460; 1375; 1240; 1080, broad d o u b le t; and 970 cm ' 1 . A bso rp tio n bands were iden tica l w ith those o f sy n th e tic p h o sp h a tid y lcholine (L -a-lecith in , /3-7 -d ipalm itoy l, Calbio- chem , San Diego, Calif.).

T h in - la y e r ch rom ato g rap h y o f 300-Mg am o u n ts o f our SPC and hydro g en a ted SPC and 99% pure SPC (T he H orm el In s titu te , A ustin , M inn.) was carried o u t on e ither 0 .25 m m or p reparative p recoated Silica Gel F-254 plates (E. M erck, D arm stad t, G erm any , d is trib u ted by B rinkm an In s tru m en ts , Inc., W aterbury , N.Y.) developed w ith the follow ing solvent system s: ch lo ro fo rm :m eth an o l:w a te r, 7 5 :2 5 :4 .2 (O ette ,1 9 6 5 ) ; c h l o r o f o r m : m e th a n ol: am m onium h y d ro x id e , 1 40 :50 :7 (C hapm an, 1972); chloro- fo rm :m eth an o l:ace tic ac id :w ater 1 7 0 :2 5 :2 5 :4 (N ichols and Jam es, 1964). S po ts w ere visualized by spraying w ith 0.5% potassium d ich ro m ate in 50% su lfu ric acid fo llow ed by heating 30 m in a t 150°C. W ith each sam ple only one spot appeared w hich also gave positive color reaction fo r p h o sp h o ru s w ith m o lybdenum blue reagent (D ittm er and L ester, 1964) and for choline w ith D ragendorff reagen t (W agner et al., 1961). W ith the three so lvent system s the R f values o f our sam ples w ere equal to that o f the purchased SPC (Fig. 1).

SPC and h y d rogenated SPC were saponified separately by refluxing w ith 0.5M potassium hydro x id e in ch lo ro fo rm :m eth an o l (1 :1 ) for 4

A B C

Fig. 1 — T h in - la y e r c h ro m a to g ra p h y o f i-m g a m o u n ts o f so y p h o s p h a

t id y lc h o lin e s ISPC) o n s ilic a ge l 0 .2 5 m m p re c o a te d p la tes . D e v e lo p m e n t: c h lo ro fo rm :m e th a n o l:w a te r 7 5 :2 5 :4 .2 , v /v . A , SPC; B, h y d ro

ge n a te d SPC; C, SPC fro m The H o rm e l In s t itu te .

Volume 3 9 (1 9 7 4 )-J O U R N A L OF FOOD S C IE N C E -6 9

7 0 - J O U R N A L OF FOOD S C IE N C E -V o /u m e 3 9 (1974)

hr. A fte r ac id ifica tion and e x trac tio n w ith e th e r , the com bined e th e r e x trac ts w ere back- ex tra c te d w ith w ater and trea ted w ith d iazom eth an e to convert the fa tty acids to m ethy l esters. F a tty acid com position o f b o th were d e te rm in e d by gas-liquid ch rom atography (Table 1). H ydrogenation o f SPC reduced the am o u n t o f 18:2 and 18:3 fa tty acids by ab o u t 90%.

T he freeze-dried aqueous layers o f the sapo n ifica tio n m ix tu re o f each SPC w ere analyzed fo r am ine co m p ounds by ascending paper ch rom atography on W hatm an N o. 1 paper developed w ith « -p ropano l: am m onium h ydro x- id e :w ate r (6 0 :3 0 :1 0 ) . C hrom atogram s show ed only one spo t th a t gave a positive color reaction fo r cho line and no reaction w ith n in h y d rin reagen t. T he R f value and color reaction o f this co m p o n en t w ere iden tica l to those o f choline chloride (C alb iochem , San D iego, Calif.).

M icroanalyses fo r carbon , hydrogen , n itro gen (D um as), phosphorus and choline (Kush- ner, 1956) are show n in T able 2. O n the basis o f p h o sp h o ru s c o n te n t, SPC and hydrogenated SPC are 95% pure.A u to x id a tio n reactions

P hospholip ids w ere dispersed in a ir-sa tu ra ted , deionized-distilled w ater in e ither the presence o r absence o f cupric su lfate to prepare 0.3% suspensions o f phospha tid y lch o lin e . SPC readily d ispersed in w ater by gently swirling the flask. H ydrogenated SPC w as dispersed in a V irtis 45 hom ogenizer a t full speed fo r 1 m in. These suspensions w ere stored in a con stan t tem p era tu re b a th a t 25°C for up to 4 wk.

Suspension changesA ny changes in co m position o f the suspen

sions during the 4-w k storage w ere observed by th in-layer ch rom ato g rap h y . A B eckm an DK2A record ing sp e c tro p h o to m ete r recorded u ltra v io let abso rp tio n spectra over the w avelength region 2 2 0 - 2 6 0 nm . A bsorbance a t 232 nm (diene con jugation) w as read on a G ilfo rd M odel 240 sp e c tro p h o to m ete r . W hen necessary, suspensions were d ilu ted w ith abso lu te e th an o l. A bsorbance readings per m g/m l are expressed as

cm • w ay, resu lts w ere com pared onan equal w eight basis.

T he e x te n t o f o x id a tio n during storage was assayed w ith TBA . T o m easure relative am oun ts o f TB A -reactive substances, 1.0 m l o f the phosp ho lip id suspension or a d ilu tion o f it was m ixed w ith 4 .0 m l 0.02M TBA (in 90% acetic acid) in g lass-stoppered tes t tubes. A cetic acid preven ted tu rb id ity in the reac tio n m ix tu re . A fte r heating in a boiling w ater b a th fo r 40 m in , the tubes w ere rapid ly coo led ; then absorbance w as m easured a t 5 3 2 nm w ith a Gilfo rd sp e c tro p h o to m ete r .O rganoleptic evaluation

Seven experienced tasters scored sam ples fo r b itte rn e ss in tensity on a scale o f 0 , none; 1 , w eak; 2, m od era te ; 3, strong. T he average score o f all the responses is then expressed as BIV (b itte r in ten sity value). A 0.05% aqueous so lutio n o f caffeine w ith a m odera te b itte r taste (BIV o f 2.0) served as a reference standard .

Sam ples con ta in ing 0.1% phospholip id dispersed in carbon-filtered tap w ater w ere sub

m itted to the panel. A ran dom ized p resen ta tio n o f fo u r 7-m l sam ples consisted o f: 0 .05% caffeine as refe rence s ta n d ard , carbo n -filte red tap w ater, and suspensions o f SPC and h y d ro g en a ted SPC. O th er sam ples tes ted fo r th e ir BIV w ere sy n th e tic p h o sp h a tid y lch o lin e , lysophos- phatidy lcho line (Pierce C hem ical C o ., R o c k fo rd , 1 1 1 .) and choline.

T he b itte r d e tec tio n th resho ld o f au to x i- dized SPC was d e term ined by 10 experienced tasters. M ore taste rs w ere used in these tests to increase th e reliab ility o f the th resh o ld d e te rm in a tio n . S o lu tions ranging in co n cen tra tio n from 0 .001 to 0 .025% w ere p re sen ted along w ith one w ater sam ple. E ach ta s te r w as in structed to record w hich sam ples w ere b itte r . The b itte r d e tec tio n th resho ld is defined as th a t co n cen tra tio n a t w hich 50% o f th e panel gave a positive response.


C o m p o s i t i o n a l c h a n g e s d u r i n g a u t o x i d a t i o n

A 0 . 3 % s u s p e n s i o n o f S P C w i t h o u t a d d e d C u + + w a s t u r b i d a n d g a v e a p H o f

3 . 8 . D u r i n g 4 w k a t 2 5 ° C , t u r b i d i t y g r a d u a l l y c l e a r e d a n d p H d r o p p e d t o 3 . 0 .

F a t t y a c i d a n a l y s i s s h o w e d d e c r e a s e s i n d i e n e c o n t e n t f r o m 6 2 . 0 t o 4 8 . 8 % a n d i n

t r i e n e c o n t e n t f r o m 6 . 5 t o 3 . 2 % . H y d r o p e r o x i d e s w e r e d e t e c t e d o n t h i n - l a y e r

c h r o m a t o g r a m s o f u n h y d r o g e n a t e d S P C s u s p e n s i o n s s t o r e d a t 2 5 C b y s p r a y i n g

A B C D E

F ig . 2 —T h in - la y e r c h ro m a to g ra p h y o f 3 0 0 -¡ig a m o u n ts o f S PC w h ic h w ere d isp e rse d in w a te r a n d s to re d a t 2 5 ° C u p to 4 w k . Pre c o a te d , p re p a ra tiv e s ilic a g e l p la tes . D e v e lo p m e n t: c h lo ro fo rm :m e th a n o l:w a te r 7 5 :2 5 :4 .2 , v /v . A , SPC, 0 t im e ; B , 1 w k ; C, 2 w k ; D , 4 w k ; E, h y d ro g e n a te d SPC, 4 w k .

Table 1—Fatty acid composition21 o f soy phosphatidylcholine (SPC) and hydrogenated SPC

Fatty acidb SPC Hydrogenated SPC

16:0 16.8 13.418:0 5.1 67.418:1 9.4 11.718:2 62.0 7.018:3 6.5 0.4

b

C a l c u l a t e d a s p e r c e n t a g e o f t o t a l g a s - l i q u i d

c h r o m a t o g r a p h i c p e a k a r e a , a s m e t h y l e s t e r s .

G l a s s c o l u m n ( 6 f t X % i n . o . d . ) p a c k e d w i t h

1 5 % C a r b o w a x 2 0 M o n 8 0 — 1 0 0 m e s h C h r o -

m o s o r b W / A W . C a r r i e r g a s - h e l i u m 4 0 m l / m i n ;

o v e n - t e m p e r a t u r e p r o g r a m m e d 1 5 0 — 2 2 0 C

a t 1 . 5 ° / m i n . I n l e t , 2 1 0 ° C ; d e t e c t o r 2 1 0 ° C .

C o d e : n u m b e r b e f o r e c o l o n = c h a i n l e n g t h ;

n u m b e r a f t e r c o l o n = n u m b e r o f d o u b l e

b o n d s .

Table 2 - Analysis of SPC and hydrogenated SPC

Constituent1%)

SPCaCalculated Found

Hydrogenated SPCb Calculated Found

C 65.80 62.68 65.40 64.42H 10.29 10.40 10.87 11.11N 1.77 1.88 1.76 1.79P 3.92 3.73 3.89 3.69C ho lin e 15.32 14.15 15.20 14.67

3 ^ 4 3 . 3 ^ 8 0 . 5 ^ ^ 9 ^ m o l e c u l a r w e i g h t 7 9 0 . 3 . M o l e c u l a r w e i g h t c a l c u l a t i o n w a s b a s e d o n f a t t y a c i d a n a l y s i s ,

k C 4 3 . 4 H 8 5 . « n 0 9 P - m o l e c u l a r w e i g h t 7 6 9 . 9 .

BITTERNESS FROM S O Y PHOSPHAT!D Y L C H O U N E -T [

F ig . 3 —Changes in abso rba nce , exp ressed as

f j c * o f a q ueou s suspensions o f SPC a n d h y d ro g e n a te d SPC. D ie n e c o n ju g a t io n w ith

f j j * a t 2 3 2 n m : e, S PC ; • , h y d ro g e n a te d SPC. T h io b a rb itu r ic a c id IT B A I- re a c t iv e substances

w ith a f 5 3 2 n m : a, S PC ; A, h y d ro g e n a te dSPC.

F ig . 4 —Changes in ab sorbance , exp ressed as o f aqueou s suspensions o f SPC c o n ta in

in g 0 .2 5 p p m C u ++ . D ie n e c o n ju g a tio n w ith

E] c m a t 2 3 2 r im : 0, T B A -re a c tiv e substancesw ith e t 5 3 2 n m : a.

1 c m

t h e d e v e l o p e d p l a t e s w i t h s t a r c h - p o t a s s i u m i o d i d e r e a g e n t ( O e t t e , 1 9 6 5 ) . T h e r e f o r e , p a r t o f t h e d i s a p p e a r a n c e o f d i e n e

a n d t r i e n e f a t t y a c i d s m a y b e a t t r i b u t e d

t o a u t o x i d a t i o n . T h e d e c r e a s e i n p e r c e n t

a g e o f d i e n e s a n d t r i e n e s w a s p r o p o r t i o n a l t o a n i n c r e a s e i n s a t u r a t e d a n d m o n o e n e

f a t t y a c i d s : 1 6 : 0 f r o m 1 6 . 8 t o 2 6 . 5 % ; 1 8 : 0 f r o m 5 . 1 t o 7 . 8 % ; 1 8 : 1 f r o m 9 . 4 t o

1 1 . 9 % . T w o a d d i t i o n a l u n i d e n t i f i e d p e a k s w i t h r e t e n t i o n t i m e s f o r m e t h y l e s t e r s o f

l a u r i c a n d m y r i s t i c a c i d s a p p e a r e d o n t h e c h r o m a t o g r a m .

S u s p e n s i o n s o f h y d r o g e n a t e d S P C w i t h o u t a d d e d C u + + g a v e a p H o f 4 . 0 . T h e h o m o g e n i z e d s u s p e n s i o n w a s n o t s t a b l e a n d a g e l - l i k e m a s s s e t t l e d o n s t a n d

i n g . H o w e v e r , t h i s m a t e r i a l w a s r e a d i l y r e d i s p e r s e d b y s w i r l i n g t h e f l a s k b e f o r e e a c h t e s t . A f t e r s t o r a g e f o r 4 w k , t h e p H o f t h i s s u s p e n s i o n d r o p p e d t o 3 . 4 .

T h i n - l a y e r c h r o m a t o g r a p h y ( F i g . 2 ) s h o w e d d e g r a d a t i o n o f S P C a t 1 , 2 , a n d 4 w k a n d d e g r a d a t i o n o f h y d r o g e n a t e d S P C o n l y a t 4 w k o f s t o r a g e a t 2 5 ° C . S P C w i t h R f = 0 . 2 5 ( c h r o m a t o g r a m A ) a n d t h e c o m p o n e n t s w i t h l o w e r m o b i l i t y g a v e

p o s i t i v e c o l o r r e a c t i o n s f o r p h o s p h o r u s .

I n B , C a n d D , t h e c o m p o n e n t w i t h m o b i l i t y g r e a t e r t h a n t h a t o f S P C g a v e a

p o s i t i v e c o l o r r e a c t i o n f o r c a r b o n y l c o m p o u n d s w i t h 2 % 2 , 4 - d i n i t r o p h e n y l h y d r a - z i n e i n h y d r o c h l o r i c a c i d . E x t e n s i v e t r a i l i n g o f t h i s s p o t i n c h r o m a t o g r a m D i n d i c a t e d d e c o m p o s i t i o n o f t h i s c a r b o n y l

c o m p o u n d . I n E , h y d r o g e n a t e d S P C d e g r a d e d t o a s l o w e r m o v i n g c o m p o n e n t

t h a t g a v e p o s i t i v e c o l o r r e a c t i o n s f o r p h o s p h o r u s a n d c h o l i n e a n d p o s s e s s e d a n R f v a l u e e q u a l t o t h a t o f l y s o p h o s p h a - t i d y l c h o l i n e ( R f = 0 . 1 5 ) r u n a s a s t a n d a r d f o r c o m p a r i s o n . T h e f a s t e r m o v i n g c o m p o n e n t g a v e n o c o l o r r e a c t i o n w i t h2 , 4 - d i n i t r o p h e n y l h y d r a z i n e a n d s h o w e d n o t r a i l i n g o n c h a r r i n g .

D i e n e c o n j u g a t i o n a n d T B A - r e a c t i v e s u b s t a n c e s

L i t t l e u l t r a v i o l e t a b s o r b a n c e , e x c e p t f o r m i n o r p e a k s a t 2 3 2 a n d 2 7 4 n m , w a s o b s e r v e d i n f r e s h l y p r e p a r e d s u s p e n s i o n s ( w i t h o u t a d d e d C u + + ) o f : S P C i n a i r -

s a t u r a t e d w a t e r ; S P C i n n i t r o g e n - s a t u r a t e d w a t e r ; a n d h y d r o g e n a t e d S P C i n a i r - s a t u r a t e d w a t e r . S i n c e t h e l a s t t w o s h o w e d n o i n c r e a s e s o n s t o r a g e , t h e i n

Table 3—Effect o f storage on bitterness intensity values (BIV) ofSPCa and hydrogenated SPCa

Time (hr at 25°C)No Cu++ 1.0 ppm Cu++

pO.1%M cm

232 nm

of SPC 532 nm SPC

Bl V cHydrogenated SPC

0 — 0.3 0.1 0.7 0.8- 180 13.7 2.5 1.6 -240 — 14.0 2.5 2.0 0.9- 432 5.1 2.9 3.0 0.8672 - 7.0 2.9 3.0 0.8

a T a s t e d a t 0 . 1 % c o n c e n t r a t i o n

b D e f i n e d i n t e x t

c S t a n d a r d e r r o r o f B I V m e a n o v e r r a n g e 0 . 4 t o 2 . 0 = 0 . 3 .

Time, Hour at 25°C

F ig . 5 —Changes in a b so rba nce , exp ressed as

f ?'cm- aq ueou s suspensions o f SPC a n d h y d ro g e n a te d SPC b o th c o n ta in in g 1 .0 p p m

C u++. D ie n e c o n ju g a tio n w ith a t 2 3 2 n m :

i>, S PC ; • , h y d ro g e n a te d SPC. T B A -re a c tiv e s u b stances w ith a t 5 3 2 n m : a, SPC; * , h y d ro g e n a te d SPC.

c r e a s e i n a b s o r b a n c e a t 2 3 2 n m o f S P C d i s p e r s e d i n a i r - s a t u r a t e d w a t e r r e s u l t s f r o m a u t o x i d a t i o n o f t h e u n s a t u r a t e d f a t t y a c i d s . A s s h o w n i n F i g u r e 3 , c o n

j u g a t e d d i e n e s f o r m e d r a p i d l y d u r i n g t h e f i r s t 2 4 0 h r . A f t e r 2 4 0 h r , a b s o r b a n c e v a l u e s a t 2 3 2 n m d e c l i n e d a n d t h e p e a k a t t h i s w a v e l e n g t h g r a d u a l l y s h i f t e d t o 2 2 2 n m . S c h a u e n s t e i n ( 1 9 6 7 ) r e p o r t e d t h a t a u t o x i d a t i o n o f p o l y u n s a t u r a t e d e s t e r s i n w a t e r g i v e s r i s e t o w a t e r - s o l u b l e s u b

s t a n c e s t h a t a b s o r b a t 2 2 2 n m . W h e t h e r w e a r e o b s e r v i n g c o m p o u n d s o f s i m i l a r n a t u r e h a s y e t t o b e d e t e r m i n e d .

T h e f o r m a t i o n o f T B A - r e a c t i v e s u b

s t a n c e s f r o m S P C p a r a l l e l e d t h e f o r m a t i o n o f c o n j u g a t e d d i e n e s d u r i n g t h e i n i t i a l s t a g e s o f o x i d a t i o n . W h e n t h e a b s o r b a n c e v a l u e s a t 2 3 2 n m d e c l i n e d , t h o s e a t 5 3 2 n m c o n t i n u e d t o i n c r e a s e u n t i l a m a x i m u m w a s r e a c h e d a t 5 8 0 h r o f s t o r a g e . T h e T B A a s s a y m e a s u r e s n o t o n l y t h e a m o u n t o f h y d r o p e r o x i d e s t h a t

a r e d e g r a d e d t o T B A - r e a c t i v e s u b s t a n c e s , b u t a l s o t h e a m o u n t o f s e c o n d a r y o x i d a t i o n p r o d u c t s t h a t r e s u l t f r o m t h e h y d r o p e r o x i d e d e g r a d a t i o n . S u s p e n s i o n s o f h y d r o g e n a t e d S P C s h o w e d n o i n c r e a s e i n a m o u n t s o f T B A - r e a c t i v e s u b s t a n c e s .

C o p p e r - c a t a l y z e d o x i d a t i o n

W i t h s o m e o f o u r S P C p r e p a r a t i o n s , t h e r a t e o f o x i d a t i o n v a r i e d g r e a t l y f r o m t h a t r e c o r d e d i n F i g u r e 3 . M o r i t a a n d F u j i m a k i ( 1 9 7 2 ) d e m o n s t r a t e d t h a t C u + + i n i t i a t e s o x i d a t i o n o f S P C a n d a l s o c a t a l y z e s t h e b r e a k d o w n o f t h e h y d r o p e r o x i d e s . T h e r e f o r e , C u + + w a s u s e d i n o u r s y s t e m s t o o v e r c o m e s o m e o f t h e i n h e r

e n t b u t u n k n o w n f a c t o r s t h a t a f f e c t r a t e o f o x i d a t i o n .

C u + + a d d e d t o s u s p e n s i o n s o f S P C a t t h e 0 . 2 5 p p m l e v e l h a d l i t t l e e f f e c t ( F i g .4 ) . A b s o r b a n c e v a l u e s d e n o t i n g f o r m a t i o n

7 2 - JO U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

o f c o n j u g a t e d d i e n e s a n d T B A - r e a c t i v e s u b s t a n c e s a r e s i m i l a r t o t h o s e r e p o r t e d i n

F i g u r e 3 .T h e r a t e o f o x i d a t i o n l e a d i n g t o d e

c r e a s e d a b s o r b a n c e a t 2 3 2 n m i s a c c e l e r a t e d w h e n s u s p e n s i o n s o f S P C c o n t a i n 1 . 0 p p m C u + + . I n F i g u r e 5 , f o r m a t i o n o f c o n j u g a t e d d i e n e s r e a c h e d a m a x i m u m

( E J ' c' J = 1 3 . 7 ) i n 1 8 0 h r r a t h e r t h a n 2 4 0 h r . I n b o t h s u s p e n s i o n s t h e e x t e n t o f o x i d a t i o n i s a b o u t t h e s a m e . T h e 2 3 2 - n m

a b s o r b i n g p e a k s h i f t e d t o o n e a b s o r b i n g a t 2 2 2 n m w i t h i n 4 3 2 h r r a t h e r t h a n 6 7 2 h r . T h e s u s p e n s i o n c l e a r e d a n d p H

d r o p p e d t o 2 . 9 . T h e T B A - r e a c t i v e s u b s t a n c e s a b s o r b i n g a t 5 3 2 n m c o n t i n u e d t o f o r m a f t e r a b s o r b a n c e d u e t o d i e n e - c o n j u g a t i o n r e a c h e d i t s m a x i m u m . I n t h i s s u s p e n s i o n t h e a m o u n t s o f T B A - r e a c t i v e s u b s t a n c e s w e r e a t a m a x i m u m ( E ^ - * % =

3 . 6 ) a t a b o u t 3 4 0 h r . S i n c e l i t t l e d e c r e a s e i n a m o u n t s o f T B A - r e a c t i v e s u b s t a n c e s w a s o b s e r v e d i n t h e o t h e r t w o s y s t e m s o v e r t h e e n t i r e 4 w k , t h e a d d i t i o n a l C u + +

m u s t h a v e c a u s e d f u r t h e r d e g r a d a t i o n o f t h e s e c o n d a r y o x i d a t i o n p r o d u c t s . N e w p r e p a r a t i o n s o f S P C t r e a t e d a c c o r d i n g t o t h e s a m e p r o c e d u r e w i t h 1 . 0 p p m C u + + y i e l d e d r e p r o d u c i b l e r e s u l t s . O n t h e b a s i s o f f o u r r e p l i c a t e s f o r S P C w i t h 1 . 0 p p m C u + + , t h e r e l a t i v e s t a n d a r d d e v i a t i o n f o r e a c h p o i n t o n t h e p l o t s ( F i g . 5 ) i s 6 . 4 % f o r m e a s u r e m e n t s a t w a v e l e n g t h 2 3 2 n m a n d 9 . 8 % f o r m e a s u r e m e n t s a t w a v e l e n g t h

5 3 2 n m .A d d i t i o n o f C u + + t o h y d r o g e n a t e d

S P C d i s p e r s e d i n w a t e r h a d v i r t u a l l y n o e f f e c t e i t h e r o n a b s o r b a n c e a t 2 3 2 n m o r

5 3 2 n m .

Bitter tasteO u r s a m p l e s w e r e e v a l u a t e d o n l y f o r

b i t t e r t a s t e e v e n t h o u g h r a n c i d f l a v o r s a l s o d e v e l o p e d w i t h S P C d u r i n g s t o r a g e . I n T a b l e 3 , 0 . 1 % s u s p e n s i o n s o f S P C a n d h y d r o g e n a t e d S P C b o t h w e r e r a t e d w e a k l y b i t t e r a t z e r o t i m e . H o w e v e r , s y n t h e t i c

p h o s p h a t i d y l c h o l i n e , c o n t a i n i n g s a t u r a t e d p a l m i t i c f a t t y a c i d s , w h e n t a s t e d a t t h i s l e v e l , g a v e a B I V o f o n l y 0 . 3 . S i n c e b o t h

S P C a n d h y d r o g e n a t e d S P C b r o k e d o w n w h e n d i s p e r s e d i n w a t e r ( s e e F i g . 2 ) , t h e i r s u s p e n s i o n s m a y c o n t a i n s o m e l y s o p h o s - p h a t i d y l c h o l i n e a n d c h o l i n e . T h e s e , w h e n t a s t e d a t t h e 0 . 1 % l e v e l , p o s s e s s e d o n l y a t r a c e o f b i t t e r n e s s : l y s o p h o s p h a t i d y l c h o - l i n e B I V = 0 . 4 a n d c h o l i n e B I V = 0 . 3 . T h e r e f o r e , t h e y d o n o t c a u s e t h e w e a k b i t t e r t a s t e . T h e i n i t i a l w e a k b i t t e r t a s t e i n s u s p e n s i o n s o f S P C a n d h y d r o g e n a t e d S P C m u s t b e d u e t o s o m e o x i d a t i o n o f t h e c o n s t i t u e n t u n s a t u r a t e d f a t t y a c i d s t h a t o c c u r r e d e i t h e r d u r i n g h a n d l i n g o r s a m p l e p r e p a r a t i o n .

S i n c e t h e r e l a t i o n s h i p b e t w e e n o u r

c h e m i c a l a n d p h y s i c a l t e s t s v e r s u s f l a v o r s c o r e s i s n o t k n o w n , t h e s e t e s t s w e r e u s e d s o l e l y t o e s t a b l i s h a c o m m o n t i m e f o r t a s t i n g t h e s a m p l e s . F o r e x a m p l e , w h e n

t h e c o n j u g a t e d d i e n e c o n t e n t o f a s u s p e n -

Table 4—Bitter response to SPC autoxidized432 hr w ith 1.0 ppm Cu++

Concentration<%)a

B itter response (%)b

0.025 1000.010 630.005 440.003 400.001 0

a P e r c e n t b y w e i g h t i n c a r b o n - f i l t e r e d t a p w a t e r

b P e r c e n t a g e o f p a n e l i s t s g i v i n g p o s i t i v e r e -

s p o n s e

s i o n o f S P C e i t h e r w i t h 1 . 0 p p m C u + + o r n o n e r e a c h e d a m a x i m u m E ^ - ^ o f a b o u t 1 4 , t h e B I V o f b o t h s u s p e n s i o n s w a s 1 . 6 a n d 2 . 0 , r e s p e c t i v e l y . T h e s c o r e s o f t h e s e

t w o s y s t e m s d i d n o t v a r y s i g n i f i c a n t l y . B I V o f S P C i n c r e a s e d a l m o s t t h r e e f o l d f r o m t h e v a l u e a t z e r o t i m e .

U p o n f u r t h e r o x i d a t i o n , t h e c o n j u g a t

e d d i e n e c o n t e n t o f S P C d e c r e a s e d . S u s p e n s i o n s o f S P C w e r e n e x t e v a l u a t e d w h e n t h e y b e c a m e c l e a r a n d d r o p p e d t o a b o u t p H 3 . 0 . T h i s d r o p o c c u r r e d a t 4 3 2 h r f o r S P C s y s t e m w i t h 1 . 0 p p m C u + +

a n d a t 6 7 2 h r f o r a n S P C s y s t e m w i t h o u t C u + + . E x t e n t o f o x i d a t i o n a s m e a s u r e d b y T B A a s s a y w a s t h e s a m e i n b o t h . T h e S P C t a s t e d s t r o n g l y b i t t e r . H o w e v e r

h y d r o g e n a t e d S P C , t a s t e d a f t e r s t o r a g e f o r 6 7 2 h r , g a v e a B I V o f 0 . 8 , i d e n t i c a l t o t h a t c f t h e f r e s h l y d i s p e r s e d s a m p l e .

F r o m d a t a i n T a b l e 4 , t h e a v e r a g e t h r e s h o l d f r o m t h r e e t r i a l s f o r d e t e c t i n g b i t t e r n e s s i n a u t o x i d i z e d S P C w a s a t t h e 0 . 0 0 6 % c o n c e n t r a t i o n .

A c c o r d i n g t o o u r r e s u l t s , S P C d e f i n i t e l y b e c o m e s b i t t e r d u r i n g s t o r a g e . A u t o x i -

d a t i o n o f t h e u n s a t u r a t e d f a t t y a c i d s a t t a c h e d t o S P C i s i n d i c a t e d b e c a u s e t h e h y d r o g e n a t e d S P C t r e a t e d s i m i l a r l y d i d n o t i n c r e a s e i n B I V . W e i s s a n d D i e m a i r( 1 9 3 9 ) , E v a n s e t a l . ( 1 9 6 0 ) a n d K a l b r e n e r e t a l . ( 1 9 7 3 ) r e p o r t e d t h a t b i t t e r n e s s c a n a r i s e f r o m a u t o x i d a t i o n o f u n s a t u r a t e d f a t t y a c i d s o r t h e i r m e t h y l e s t e r s . O u r

p r e s e n t s t u d i e s s h o w t h a t t h e b i t t e r f a c t o r o f S P C , o x i d i z e d f o r 4 w k u n d e r t h e c o n d i t i o n s u s e d i n t h i s i n v e s t i g a t i o n , r e s i d e s w i t h t h e c o m p o n e n t s i n c h r o m a t o g r a m D ( F i g . 2 ) t h a t p o s s e s s R f l e s s t h a n 0 . 2 5 a n d g i v e p o s i t i v e c o l o r r e a c t i o n s f o r p h o s p h o r u s . T h e r e f o r e , a m o d i f i e d f o r m o f S P C

m a y b e a s o u r c e o f b i t t e r n e s s i n d e f a t t e d s o y b e a n m e a l , e s p e c i a l l y s i n c e c o n c e n t r a t e s o f t h e b i t t e r p r i n c i p l e f r o m t h i s

m e a l s h o w e d c h r o m a t o g r a p h i c e q u i v a l e n c e t o t h e a u t o x i d i z e d S P C . F u t u r e

w o r k w i l l i n c l u d e f u r t h e r c h a r a c t e r i z a t i o n o f t h e s o y b i t t e r p r i n c i p l e a s i t i s r e l a t e d t o a u t o x i d i z e d S P C a n d e v a l u a t i o n o f t h e e f f e c t t h a t s o y p r o t e i n h a s o n o x i d a t i o n

o f S P C . W e f o u n d t h a t o x i d a t i o n i s c a t a l y z e d b y a c r u d e s o y b e a n l i p o x y g e n a s e . R a c k i s e t a l . ( 1 9 7 2 ) d e m o n s t r a t e d t h a t t h e t h r e e - t o f o u r f o l d i n c r e a s e i n b i t t e r

n e s s o f s o y b e a n s d u r i n g m a t u r a t i o n c o r

r e l a t e d w i t h a n i n c r e a s e i n l i p o x y g e n a s e

a c t i v i t y . W h e t h e r a b i t t e r t a s t e d e v e l o p s i n e n z y m a t i c a l l y o x i d i z e d S P C i s y e t t o

b e d e t e r m i n e d .

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Chapman, L.R. 1972. Analysis of phospholipids by thin-layer chrom atography and X-ray emission spectrom etry. J. Chrom atogr. 66: 303.

Davis, P.F. 1962. Process for the hydrogenation of phosphatides. U.S. Patent 3 ,026,341.

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Kalbrener, J.E ., Eldridge, A.C., Moser, H.A. and Wolf, W.J. 1971. Sensory evaluation of com mercial soy flours, concentrates and isolates. Cereal Chem. 48: 595.

Kalbrener, J.E ., Warner, K. and Eldridge, A.C.1973. Flavors derived from linoleic and lino- lenic acid hydroperoxides. Cereal Chem. (in press).

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Maga, J.A. and Johnson, J.A. 197 2. Effect of processing and storage conditions on lipid com position of soy products. Cereal Chem. 49: 79.

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Morita, M. and Fujimaki, M. 1972. The effects of copper and EDTA on the au toxidation of phospholipid emulsions. Agr. Biol. Chem. 36: 1163.

Nichols, B.W. and James, A.T. 1964. The lipids of plant storage tissues. Fette Seifen Anstrichm. 66: 1003.

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Ms received 6/28/73; revised 8 /21 /73 ; accepted 8/24/73.______________


The m ention of firm names or trade products does not imply th a t they are endorsed or recommended by the U.S. Dept, o f Agriculture over o ther firms or similar p roducts no t m entioned.

C. H. M A N L E Y , P. P. V A L L O N a n d R. E. E R IC K S O N

G ivau dan C o rp o ra t io n , 100 D e la w a n n a A v e ., C li f to n , N J 0 7 0 1 4

SOME AROMA COMPONENTS OF ROASTED SESAME SEED (Sesamum indicum L.)

INTRODUCTION

S E S A M E (Sesamum indicum L . ) i s o n e o f t h e o l d e s t o i l s e e d c r o p s k n o w n t o m a n . I t s o i l h a s a m i l d t a s t e a n d i s h i g h i n u n s a t u r a t e d f a t s ( c a . 8 5 % ) . I t s m e a l i s o f i n t e r e s t m a i n l y b e c a u s e o f i t s h i g h p r o t e i n

c o n t e n t w i t h a g o o d a m i n o a c i d b a l a n c e . I n t h e U n i t e d S t a t e s , s e s a m e i n t h e f o r m o f t h e r o a s t e d s e e d s i s p o p u l a r o n b r e a d

a n d b a k e r y g o o d s . T h e r o a s t i n g p r o c e s s r e n d e r s a f l a v o r o f u n i q u e q u a l i t y c h a r a c t e r i z e d a s n u t t y a n d m e a t - l i k e . P a r t l y b e c a u s e o f t h e c h a r a c t e r o f i t s f l a v o r , t h e u s e o f s e s a m e s e e d , w h i c h i s m a i n l y i m p o r t e d f r o m t h e t e m p e r a t e a r e a s o f t h e w o r l d , h a s n e a r l y d o u b l e d i n t h e l a s t d e c a d e , a c c o r d i n g t o A g r i c u l t u r a l S t a t i s

t i c s ( 1 9 7 0 ) . A n u m b e r o f g o o d r e v i e w s o n t h e c o m p o s i t i o n o f t h e m e a l a n d t h e o i l

h a v e b e e n p u b l i s h e d b y B u d o w s k i a n d M a r k l e y ( 1 9 5 1 ) , J o h n s o n a n d R a y m o n d

( 1 9 6 4 ) a n d L y o n ( 1 9 7 2 ) . S o m e o f t h e a r o m a c o m p o n e n t s o f t h e o i l h a v e b e e n s t u d i e d b y Y a m a n i s h i e t a l . ( 1 9 6 7 ) a n d Y o k o e t a l . ( 1 9 6 9 ) . O t h e r s t u d i e s o n

b r o i l e d s e s a m e d e m o n s t r a t e d t h a t c y s t i n e

a n d g l u c o s e m a y p l a y i m p o r t a n t r o l e s a s p r e c u r s o r s t o t h e o v e r a l l f l a v o r ( Y a m a n i s h i e t a l . , 1 9 6 0 ) . H o w e v e r , s i n c e n o

s t u d y t o d a t e h a s i n v e s t i g a t e d t h e r o a s t e d s e s a m e s e e d a r o m a , w e a r e r e p o r t i n g o n o u r i n i t i a l r e s u l t s w i t h t h i s m a t e r i a l .

EXPERIMENTALP r e p a r a t i o n o f s a m p l e

A 250-g sam ple o f “ G olden R oast Sesame S eed” (H.B. T ay lo r C o., Chicago, 111.) was b lended in a W aring B lendor for 1 m in w ith 1 lite r o f d istilled w ater. T he resulting slurry was p laced in a 2 lite r round b o tto m flask and d istilled in an e x tra c to r sim ilar in design to the d istilla tio n -ex trac tio n app ara tu s developed by N ickerson and L ikens (1966). T he distilla tion was co n tin u ed fo r 5 h r w ith isopen tane (99+ m ol %, M atheson C hrom ato Q uality ) as the ex trac tin g solvent. T his p rocedure was repeated four tim es, and the isopen tane ex trac ts were p oo led , dried over an h y d ro u s sodium sulfate and co n cen tra ted u nder a flow o f d ry n itrogen a t room tem pera tu re to ab o u t 1 m l. This concen tra te was frac tio n a ted by separa tion acco rd ing to func tio n a l g roups and by gas-liquid

Table 1—Basic components of roasted sesame seed aroma

Name

Identification

Positive Tentative

1. Methylpyrazine X2. 2,6-Dimethylpyrazine X3. 2,5-Dimethylpyrazine X4. Ethylpyrazine X5. 2,3-D ¡methylpyrazine X6. 2-Ethyl-6-methylpyrazine X7. 2-Ethyl-5-methylpyrazine X8. Trim ethylpyrazine X9. 2-Ethyl-3-methylpyrazine X

10. 2-Ethyl-3,6-dimethylpyrazine X11. 2-Ethyl-3,5-dimethylpyrazine X12. 2-Ethyl-5,6-dimethylpyrazine X13. 2,3-Diethyl-5-methylpyrazine X14. D iethylmethylpyrazine X15. D iethyld im ethylpy razine X16. Isopropenylpyrazine X17. Acetylpyrazine X18. 5-Methyl-6,7-dihydro-5H-cyclopentapyrazine X

19. 6,7-Dihydro-5H-cyclopentapyrazine X20. 2(3)5-D imethyl-6,7-dihydro-5H-cyclopentapy razine X21. 6-Acetyl-2-methylpy razine X22. 2-Methyl-6,7-dihydro-5H-cyclopentapy razine X23. 5,6,7,8-Tetrahydroquinoxaline X24. 2(2' Furyl)-3-methylpy razine X25. 2(2'Furyl)pyrazine X

ch rom ato g rap h y . C o m p o n en ts w ere iden tified by GLC re te n tio n tim e (K ovats Index) and m ass spectroscopy .F u n c t i o n a l g r o u p i s o l a t i o n

C o n cen tra tes p repared as n o ted above were d ilu ted w ith m eth y len e chloride and th en fractio n a ted by e x tra c tio n o f the d ilu ted m aterial w ith the follow ing sequence:

(1) 10 x 3 m l o f 5% HC1 fo r bases;(2) 10 x 3 m l o f 5% N a2C 0 3 fo r strong

acids;(3) 10 x 3 m l o f 5% NaOH for w eak acids.

T he rem aining co n cen tra te was d ried and defined as the n eu tra l m aterial.

Each frac tio n 1 — 3 was n eu tra lized and ex trac ted w ith m eth y len e chloride. T he individual ex trac ts w ere dried and co n cen tra ted as described above fo r fu rth e r use. Sam ples were d ilu ted w ith m ethy lene chloride and stored a t 5°C un til needed.G a s c h r o m a t o g r a p h y

G as ch rom atograph ic separa tions w ere carried o u t on 2% SF 96 on G as C hrom Q and 2% C arbow ax 20M on Gas C hrom Q packed in 18 f t X 1/8 in. copper colum ns. T he co lum ns were tem p era tu re program m ed from 70°C to 230° C a t 4 °C /m in w ith a linear flow ra te o f 6 cm /sec.

K ovats indices fo r the co m p o n en ts w ere calcu la ted by co m p u ter according to the Kovats- W ehrli m eth o d s using e th y l esters as in ternal s tandards fo r the C arbow ax co lum n and h y d ro carbons fo r the SF 96 co lum n (K ovats and W ehrli, 1959). C om parison o f know n data , b o th for GLC and MS, was handled partly by com p u ter.

A lso used in th is study was a 250 f t x 0 .03 in . O V-101 capillary co lum n w ith 5% Igepal. This co lum n was tem p era tu re p rogram m ed fro m 70°C to 200°C a t 2 °C /m in w ith a linear helium velocity o f 24 cm /sec.G a s c h r o m a t o g r a p h y / M a s s s p e c t r o s c o p y

Mass spectral d a ta on indiv idual com po n en ts w ere ob ta in ed on a P erkin E lm er M odel 270 double-focusing m ass spec tro m eter (reso lu tion = 800) in terfaced w ith a gas liquid ch ro m ato graph th ro u g h a W atson-B iem ann S eparato r (W atson and B iem ann, 1965). The co nd itions fo r the mass spectral scans w ere: ion source p ressure, 1 0 '6 to rr ; ion source tem p era tu re , 1 7 5 ° ;eV , 70.


A T Y P I C A L g a s l i q u i d c h r o m a t o g r a p h i c s c a n o f t h e t o t a l e x t r a c t i s s h o w n i n F i g u r e 1 . C o m p o n e n t s w e r e s e p a r a t e d i n t o f u n c t i o n a l g r o u p s i n o r d e r t h a t m o r e s i m p l e a n d l e s s c r o w d e d g a s c h r o m a t o g r a p h i c s e p a r a t i o n s c o u l d b e m a d e . R e s u l t s o f o u r i d e n t i f i c a t i o n o f t h e v a r i o u s p e a k s i n t h e b a s i c f r a c t i o n a r e f o u n d i n

Volume 3 3 (1 9 7 4 )-J O U R N A L OF FOOD S C IE N C E -7 3

7 4 —JO U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

T a b l e 1 . T h e c o m p o n e n t s a r e l i s t e d a c c o r d i n g t o t h e i r o r d e r o f e l u t i o n o n C a r -

b o w a x . T h e t a b u l a t i o n n o t e s t h e c e r t a i n

t y o f i d e n t i f i c a t i o n .T h e p o s i t i v e i d e n t i f i c a t i o n o f c o m -

p o u n d s a s n o t e d i n t h e t a b u l a t i o n r e p r e s e n t s a g r e e m e n t o f K o v a t s i n d i c e s ( ± 5 u n i t s ) o n t h e C a r b o w a x 2 0 M c o l u m n a n d

t h e S F 9 6 c o l u m n . T h e a g r e e m e n t i s w i t h

r e t e n t i o n d a t a o f s t a n d a r d k n o w n c o m -

Fig . 1 —C a p illa ry G L C scan o f to ta l sesame o d o r.

W " X XR2 R2^ N R1 H' ^ V h N

CH-,

R'v

R1 AND R2 = H , CHj ,CH2 CHj

F ig . 2 —F o rm a tio n o f a k ly l p y ra z in e s .

SUGARS0 OH

OH

1 0 0 OH

L h2 ¿ ^ oh *R2 ^ CH

0 0

R2 ♦ AMINO ACID ♦ [OJ

• N .^ R1 " 2

Rl or 2

R=H.CHj

Fig . 4 —F o rm a tio n o f c y c lo p e n ta p y ra z in e s .

0 0 HO OH WITH AMINOACIDS

, M , * H X 0

Lou R- fN ^ r

0

R = H or ALKYL

Fig. 3—Formation of acetyl pyrazines.

p o u n d s a n d m a s s s p e c t r a l d a t a o f t h o s e

s t a n d a r d s . A t e n t a t i v e a s s i g n m e n t i n d i c a t e s a m a t c h w i t h l i t e r a t u r e d a t a o r i n t e r p r e t a t i o n o f G C a n d M S d a t a .

B a s e s

A n u m b e r o f a l k y l p y r a z i n e s h a v e b e e n

i s o l a t e d i n v a r i o u s f o o d p r o d u c t s w h i c h h a v e u n d e r g o n e h i g h t e m p e r a t u r e p r o c e s s

i n g . T h e s e i n c l u d e c o f f e e ( B o n d a r o v i c h e t

a l . , 1 9 6 7 ; G o l d m a n e t a l . , 1 9 6 7 ) , c o c o a

( M a r i o n e t a l . , 1 9 6 7 ; R i z z i , 1 9 6 7 ; V a n

P r a a g e t a l . , 1 9 6 8 ) , d e e p f a t - f r i e d s o y b e a n s ( W i l k e n s a n d L i n , 1 9 7 0 ) , s o y p r o t e i n h y d r o l y s a t e ( M a n l e y a n d F a g e r s o n

1 9 7 0 a , b ) , f i l b e r t s ( S h e l d o n e t a l . , 1 9 7 2 ) , b e e f ( W a t a n a b e a n d S a t o , 1 9 7 1 ) a n d p o p

c o r n ( W a l r a d t e t a l . , 1 9 7 0 ) . A r e v i e w b y M a g a a n d S i z e r ( 1 9 7 3 ) o f p y r a z i n e s i n f o o d s h a s b e e n p u b l i s h e d w h i c h c o v e r s t h e f o o d s i n w h i c h p y r a z i n e s h a v e b e e n

n o t e d .H y p o t h e s e s f o r t h e f o r m a t i o n o f t h e s e

s i m p l e a l k y l p y r a z i n e s m a i n l y i n v o l v e c o n

d e n s a t i o n r e a c t i o n s b e t w e e n a m i n o a c i d s a n d s u g a r s ( D a w e s a n d E d w a r d , 1 9 6 6 ; M a s o n e t a l . , 1 9 6 6 ) . A g e n e r a l s u m m a r y o f t h e p o s s i b l e m e c h a n i s m s h a s b e e n p u b l i s h e d b y H o d g e e t a l . ( 1 9 7 2 ) a n d M a g a a n d S i z e r ( 1 9 7 3 ) .

T h e m a j o r p y r a z i n e s f o u n d i n s e s a m e

a r e 2 , 5 - a n d 2 , 6 - d i m e t h y l p y r a z i n e s w h i c h p r o b a b l y o c c u r b y t h e c o n d e n s a t i o n o f p y r u v a l d e h y d e w i t h a n a m i n o a c i d ( s e e

F i g . 2 ) . T h i s p r o d u c t c a n u n d e r g o S t r e c k -

e r d e g r a d a t i o n t o f o r m a n a m i n o r e d u c - t o n e w h i c h i n s u b s e q u e n t s t e p s o f s e l f c o n d e n s a t i o n a n d o x i d a t i o n c a n f o r m 2 ,

5 - a n d 2 , 6 - d i m e t h y l p y r a z i n e s . T h e 2 , 5 - d i - m e t h y l p y r a z i n e s h o u l d b e p r o d u c e d r e g a r d l e s s o f t h e t y p e o f a m i n o a c i d e m p l o y e d a s t h e n i t r o g e n s o u r c e ( W a n g

e t a l . , 1 9 6 9 ) . F o r m a t i o n o f s o m e o f

t h e o t h e r a l k y l p y r a z i n e s i s p r o b a b l y b y

t h e s a m e m e c h a n i s m . F o r e x a m p l e , i t h a s

b e e n s u g g e s t e d ( B o n d a r o v i c h e t a l . , 1 9 6 7 ;

F l a m e n t e t a l . , 1 9 6 7 ; G o l d m a n e t a l . , 1 9 6 7 ) t h a t t h e e t h y l a n d d i e t h y l p y r a

z i n e s a r e f o r m e d f r o m a - d i k e t o n e p r e c u r s o r s .

T h e f o r m a t i o n o f a c e t y l p y r a z i n e m a y

b e t h e r e s u l t o f t h e c o n d e n s a t i o n o f C - m e t h y l r e d u c t o n e ( a k n o w n b r o w n i n g r e a c t i o n p r o d u c t ) w i t h g l y o x a l a n d a n

a m i n o a c i d ( F i g . 3 ) . T h e c y c l o p e n t a p y r a z i n e s m a y r e s u l t v i a t h e s a m e m e c h a n i s m f r o m t h e c o n d e n s a t i o n o f 2 - h y d r o x y -

3 - m e t h y l - 2 - c y c l o p e n t e n - l - o n e ( a k n o w n p r o d u c t o f b r o w n i n g r e a c t i o n s ) w i t h g l y o x a l o r p y r u v a l d e h y d e a n d a m i n o a c i d s ( s e e F i g . 4 ) ( W a l r a d t e t a l . , 1 9 7 0 ) .

T h e f o r m a t i o n o f 2 ( 2 , f u r y l ) p y r a z i n e s m a y c o m e f r o m t h e c o n d e n s a t i o n o f a k n o w n M a i l l a r d b r o w n i n g p r o d u c t , 1 ( 2 f u r y l ) - l , 2 - p r o p a n e d i o n e ( F e r r e t t i a n d F l a n a g a n , 1 9 7 1 ; G i a n t u r c o e t a l . , 1 9 6 4 ) , w i t h g l y o x a l a n d a n a m i n o a c i d a s n o t e d i n F i g . 5 .

T h e a r o m a o f t h e b a s i c f r a c t i o n o f s e s a m e i s d o m i n a t e d m a i n l y b e g r e e n

AROMA COMPONENTS OF SESAME SEED-75

0 0

H K R1

AMINOACIDS

COJ

Fig . 5 —F o rm a tio n o f f u r y / p y raz ines .

FERULIC ACID VINYL GUAIACOL

[ 0 ]

GUAIACOL

F ig . 6 —F o rm a tio n o f gua iaco ls .

5-METHYL-2-PHENYL-2-HEXENAL

F ig . 7 —F o rm a tio n o f p h e n y l a lk e n y l a lde hyde s.

p y r a z in e n o te s . T h e b a s ic f r a c t io n a lo n e d o e s n o t p ro v id e th e fu ll a ro m a r e c o g n iz e d as r o a s te d se s a m e . A n a ro m a g ra m o f t h e b a s ic f r a c t io n in d ic a te d th a t m o s t o f t h e c o m p o n e n ts h a d “ p e a n u t - l ik e ,” “ g r e e n ” o r “ r o a s t e d ” n o t e d . O n io n - l ik e o d o r s a p p e a r e d t o b e a s s o c ia te d w ith th e fu r y l p y ra z in e s a n d a p o p c o r n o d o r w ith t h e a c e t y lp y r a z in e . T h e p o p c o r n a ro m a an d a c e ty lp y r a z in e h a v e b e e n r e p o r te d b y Y o k o e t a l. ( 1 9 6 9 ) in se s a m e o il an d are b e lie v e d t o b e m a jo r c o n t r ib u to r s to th e a r o m a o f se s a m e o il .

W e a k a c id s

O n ly a fe w m a jo r p h e n o ls w e re is o la t e d ; th e y a re p h e n o l an d g u a ia c o l (2 -m e t h - o x y p h e n o l ) a n d i ts is o m e r s (s e e T a b le 2 ) w ith g u a ia c o l b e in g t h e m a jo r c o m p o n e n t o f th e w e a k a c id f r a c t io n . P re s u m a b ly th e s e c o m p o u n d s a rise m a in ly th r o u g h t h e d e g r a d a t io n o f t h e n a tu r a l p h e n o lic c a r b o x y l ic a c id s fo u n d in th e se s a m e se e d . F ig u r e 6 su g g e sts t h e p o s s ib le p r o d u c t io n o f g u a ia c o l an d re la te d c o m p o u n d s . F u k u s h im a an d Y o k o t s u k a( 1 9 6 7 ) h av e n o te d lig n in as a s o u r c e o f t h e p h e n o lic m a te r ia ls fo u n d in s o y sa u c e . A sa o an d Y o k o t s u k a ( 1 9 5 8 ) a lso n o te d t h a t g ly c o s id e s m a y a c t as p r e c u rs o rs . T h e a ro m a s o f t h e p h e n o ls , p a r t ic u la r ly th e g u a ia c o l a n d v in y l g u a ia c o l, a re a v e ry in te n s e “ s m o k e t y p e ” ( F id d le r e t a l . , 1 9 6 7 ) . T h e s e c o m p o n e n ts h av e b e e n fo u n d in a n u m b e r o f r o a s te d p r o d u c ts (D ie t r ic h e t a l . , 1 9 6 4 ; F e r r e t t i a n d F la n

a g a n , 1 9 7 1 ; M a n le y an d F a g e r s o n , 1 9 7 0 a , b ; 1 9 7 1 ; W a lra d t e t a l ., 1 9 7 0 ) an d p r o b a b ly c o n t r ib u te a g re a t d e a l t o th e ir o v e r a ll a r o m a .

Table 2—Weakly acidic components of roasted sesame seed aroma

NamePositive

identification

1. Phenol X2. Guaiacol X3. p-Methoxyphenol X4. m-Methoxy phenol X5. Methoxymethylphenol6. Vinylguaiacol X

Table 3—Neutral components sesame seed aroma

of roasted

PositiveName identification

1. Benzaldehyde X2. 5-M ethylfurfural X3. 2-Phenyl-2-butenal X4. 4-Methyl-2-phenyl-2-pentenal X5. 5-Methyl-2-phenyl-2-hexenal X6. 2-Phenyl-2-pentenal X7. 2-Phenyl-2-hexenal X

N e u tr a ls a n d s tro n g a c id s

T h e n e u tr a l a n d s tr o n g ly a c id ic m a te r i a ls fo u n d in s e s a m e se e d a re o f g re a t sign i f i c a n c e t o t h e a r o m a , an d f u r t h e r w o rk is b e in g d o n e o n th e s e f r a c t io n s . P r e lim in a ry r e s u lts o n t h e n e u tr a l f r a c t io n are s h o w n in T a b le 3 .

T h e 5 - m e t h y l- 2 -p h e n y l- 2 -h e x e n a l is p re s u m a b ly fo r m e d f r o m t h e c o n d e n s a t io n o f p h e n y la c e ta ld e h y d e an d iso v a le r - a ld e h y d e . T h e s e p r e c u r s o r m a te r ia ls a re k n o w n t o b e fo r m e d f r o m t h e S t r e c k e r d e g r a d a t io n o f t h e a m in o a c id s p h e n y la la n in e an d le u c in e , r e s p e c t iv e ly (s e e F ig . 7 ) . M a te r ia ls s im ila r t o th e s e h a v e b e e n fo u n d in a n u m b e r o f r o a s te d p r o d u c ts in c lu d in g c o c o a b e a n s an d h av e b e e n n o te d t o h av e a s tr o n g c o c o a a r o m a (V a n P ra a g e t a l ., 1 9 7 1 ; V a n P ra a g an d S t e in , 1 9 6 8 ) .

I t w o u ld a p p e a r t h a t a ll o f th e c o m p o n e n ts is o la te d an d id e n t i f ie d in th is p r e lim in a r y s tu d y a re o f im p o r ta n c e to th e t o t a l a ro m a o f th e r o a s te d se s a m e se e d s . C u rre n t w o rk is p ro g re ss in g w h ic h w ill id e n t i f y c o m p o n e n ts o f f u r t h e r sig n i f i c a n c e t o r o a s te d se s a m e .

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G. M . E V A N C H O a n d D . H . A S H T O N

C a m p b e l l I n s t i t u t e f o r F o o d R e s e a r c h , C a m d e n , N J 0 8 1 0 1

a n d A . A . Z I V A R U N

J o h n s t o n L a b o r a to r ie s , I n c . , C o c k e y s v i i l e , M D 2 1 0 3 0

USE OF A RADIOMETRIC TECHNIQUE FOR THE RAPID DETECTION OF GROWTH OF CLOSTRIDIAL SPECIES

INTRODUCTIONT H E D E T E C T I O N o f g r o w t h o f a n

a e r o b i c s p o r e f o r m e r s c a n b e a s l o w a n d l e n g t h y p r o c e s s , p a r t i c u l a r l y i f t h e i n o c u l u m i s l o w n u m b e r s o f h e a t - s t r e s s e d

s p o r e s . C u r r e n t l y a c c e p t e d m e t h o d o l o g y f o r a s s e s s i n g t h e s t e r i l i t y o f c a n n e d f o o d s c a n r e q u i r e f r o m 1 d a y t o s e v e r a l w e e k s ( S h a r f , 1 9 6 6 ) . L i k e w i s e , i n s t u d y i n g t h e t h e r m a l r e s i s t a n c e o f s p o r e s u s p e n s i o n s , s e v e r a l w e e k s a r e o f t e n r e q u i r e d t o o b t a i n g r o w t h f r o m t h e m o s t s e v e r e l y h e a t - s t r e s s e d s p o r e s . C o n s i d e r a b l e e r r o r i n t h e

h e a t r e s i s t a n c e o f a n o r g a n i s m c a n b e o b t a i n e d i f m a x i m a l r e c o v e r y o f s u r v i v i n g s p o r e s i s n o t o b t a i n e d . I n o b t a i n i n g g r o w t h f r o m a n a e r o b i c s p o r e f o r m e r s ,

m e d i u m c o m p o n e n t s h a v e b e e n s h o w n t o i n f l u e n c e r e c o v e r y s i g n i f i c a n t l y ( A s h t o n ,

1 9 7 1 ) . U s i n g t h e b e s t m e d i u m f o r o p t i

m a l r e c o v e r y , h o w e v e r , s t i l l o f t e n r e q u i r e s l e n g t h y i n c u b a t i o n .

R e c e n t p u b l i c a t i o n s h a v e d e m o n s t r a t e d r a p i d d e t e c t i o n o f b a c t e r i a i n c l i n i c a l s p e c i m e n s ( b l o o d a n d u r i n e s a m p l e s )

w i t h i n a f e w h o u r s ( D e B l a n c e t a l . , 1 9 7 1 ; D e L a n d a n d W a g n e r , 1 9 7 0 ; G a l l a n d

J o h n s o n , 1 9 7 3 : K i l b o u r n a n d B r a m h a l l , 1 9 7 3 ; R a n d a l l , 1 9 7 2 , 1 9 7 3 ; W a t e r s ,

1 9 7 2 ) u s i n g a r a d i o m e t r i c t e c h n i q u e . D e L a n d a n d W a g n e r ( 1 9 7 0 ) r e p o r t e d t h e

a v e r a g e t i m e r e q u i r e d t o d e t e c t t h e p r e s e n c e o f b a c t e r i a w a s l e s s t h a n 4 h r , a n d t h e m a x i m u m t i m e w a s 6 h r . N o f a l s e

n e g a t i v e o r f a l s e p o s i t i v e r e s u l t s w e r e f o u n d w h e n c o m p a r e d t o r o u t i n e b a c t e r i o l o g i c a l p r o c e d u r e s . D e B l a n c e t a l .( 1 9 7 1 ) r e p o r t e d t h e r a d i o m e t r i c m e t h o d w a s f a s t e r t h a n c o n v e n t i o n a l t e c h n i q u e s a n d c o m p a r a b l e i n a c c u r a c y . 7 5 % o f p o s i t i v e s a m p l e s w e r e d e t e c t e d f i r s t b y t h e r a d i o m e t r i c m e t h o d , 6 5 % w e r e p o s i t i v e o n t h e d a y o f i n o c u l a t i o n . T h e d e t e c t i o n t i m e , h o w e v e r , h a s b e e n s h o w n t o b e p r o p o r t i o n a l t o t h e l o g a r i t h m o f t h e i n i t i a l i n o c u l u m ( W a t e r s , 1 9 7 2 ) . H i g h s e n s i t i v i t y ( d e t e c t i o n o f a s i n g l e v i a b l e c e l l ) a l s o h a s b e e n d e m o n s t r a t e d .

P r e v i t e ( 1 9 7 2 , 1 9 7 3 ) r e c e n t l y a p p l i e d t h e r a d i o m e t r i c t e c h n i q u e t o t h e d e t e c

t i o n o f f o o d - b o r n e b a c t e r i a a n d s u g g e s t e d t h e a p p l i c a t i o n o f t h i s t e c h n i q u e f o r s t e r i l i t y t e s t i n g o f f o o d s . H o w e v e r , h e r e c o g n i z e d t h e n e e d f o r f u r t h e r s t u d i e s w i t h a v a r i e t y o f a e r o b e s a n d a n a e r o b e s f r o m

d i f f e r e n t s o u r c e s , a n d t h e i m p o r t a n c e o f o p t i m i z i n g t h e m e d i a u s e d f o r t h e i r d e t e c t i o n . T h e p r e s e n t s t u d y w a s u n d e r t a k e n t o e v a l u a t e t h e r a d i o m e t r i c m e t h o d a s a

p o s s i b l e r a p i d m e t h o d f o r d e t e c t i n g g r o w t h o f c l o s t r i d i a l s p e c i e s a n d t o o p t i

m i z e t h e m e d i u m a n d c u l t u r a l c o n d i t i o n s n e c e s s a r y f o r r a p i d g r o w t h .

EXPERIMENTALC u l t u r e s

I n o c u l a w e r e p r e p a r e d b y g r o w i n g t h e o r g a n i s m s in C o o k e d M e a t M e d i u m ( C M M )

( D i f c o ) a n d a l l o w i n g t h e m t o s p o r u l a t e . A l l c u l t u r e s w e r e i n c u b a t e d a t 3 5 ° C f o r 5 d a y s . C u l t u r e s u s e d in t h i s s t u d y w e r e C lo s t r id iu m b i fe r - m e n ta n s A T C C 1 9 2 9 9 , C lo s t r id iu m b o tu l i n u m t y p e A ( 6 2 A a n d 1 0 9 A ) , C . b o t u l i n u m t y p e B ( A T C C 7 9 4 9 , 2 B a n d B L a m a n n a ) , C . b o t u l i n u m t y p e 4 6 8 C , C . b o t u l i n u m t y p e 4 3 0 F , C lo s t r id iu m b u t y r i c u m A T C C 1 9 3 9 8 , C lo s t r id iu m c h a u v o e i A T C C 1 9 3 9 9 , C lo s t r id iu m h i s t o l y t i c u m A T C C 1 9 4 0 1 , C lo s t r id iu m n o v y i A T C C 1 9 4 0 2 , C lo s t r id iu m p e r f r in g e n s ( A T C C 1 3 1 2 4 a n d B P 6 K ) , C lo s t r id iu m r p o r o g e n e s ( A T C C 1 9 4 0 4 , N C A 3 6 7 9 a n d 5 5 - 1 2 3 , a n d

C D C 4 4 3 9 ) , C lo s t r id iu m s e p t i c u m A T C C 6 C 0 8 , a n d C lo s t r id iu m t e r t i u m A T C C 1 9 ^ 0 5 . A f t e r s p o r u l a t i o n , c u l t u r e s w e r e s t o r e d a t 4 ° C u n t i l n e e d e d .

A m i x e d i n o c u l u m o f v e g e t a t i v e c e l l s a n d s p o r e s w a s o b t a i n e d b y m a k i n g a n a p p r o p r i a t e d i l u t i o n o f t h e C M M s u s p e n s i o n in 0 .1 M p h o s

p h a t e b u f f e r , p H 7 . 0 . S p o r e i n o c u k w e r e p r e p a r e d b y h e a t i n g a n a p p r o p r i a t e d i l a t i o n o f a C M M s u s p e n s i o n e i t h e r a t 1 0 0 ° C f o r 1 0 m i n (C . b o t u l i n u m a n d C. s p o r o g e n e s ) o r a t 8 3 ° C f o r 1 0 m i n ( r e m a i n i n g s p e c i e s ) , a n d c o o l i n g i m m e d i a t e l y in i c e w a t e r .

M e d ia

J L I A n a e r o b i c C u l t u r e M e d i u m 7 A w a s p u r c h a s e d f r o m J o h n s t o n L a b o r a t o r i e s , I n c . , C o c k e y s v i l l e , M d . T h e m e d i u m w a s s u p p l i e d p r e r e d u c e d ( a m i n i m u m r e d o x p o t e n t i a l o f — 1 5 0 m v ) i n 5 0 m l s e r u m v ia l s , 3 0 m l o f m e d i u m p e r v ia l . T h e a t m o s p h e r e i n t h e v i a l s w a s c o m p o s e d o f 9 0 % N 2 a n d 1 0 % C 0 2 . T h e m e d i u m c o n t a i n e d 1 .7 % T r y p t i c a s e , 0 .3 % P h y t c n e , 0 .5 % y e a s t e x t r a c t , 0 .5 % N a C l , 0 .2 5 % K 2 H P 0 4 , 5 p p m h e m i n ( x f a c t o r ) , 0 . 5 p p m v i t a m i n K ( m e n a d i o n e ) , 0 . 0 5 % L - c y s t e i n e , 0 . 0 2 5 % s o d i u m p o l y a n e t h o l s u l f o n a t e a n d 1 .5 p C i o f 14 C - l a b e l e d s u b s t r a t e s o f h i g h s p e c i f i c a c t i v i t y p e r v ia l .

B e c a u s e o f d i f f i c u l t y e n c o u n t e r e d in o b t a i n in g g r o w t h o f s e v e r a l s p e c i e s i n M e d i u m 7 A , t h e m e d i u m w a s s u p p l e m e n t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f T r y p t i c a s e , P h y t o n e o r g l u c o s e . S e v e r a l m o d i f i c a t i o n s a l s o w e r e m a d e t o t h e

c o m m e r c i a l l y a v a i l a b l e m e d i u m . H e m i n , v i t a

m i n K a n d s o d i u m p o l y a n e t h o l s u l f o n a t e w e r e e l i m i n a t e d f r o m t h e m e d i u m s in c e t h e s e w e r e i n c l u d e d t o e n c o u r a g e t h e g r o w t h o f f a s t i d i o u s

o r g a n i s m s f r o m b l o o d c u l t u r e s . T h e b a s a l m e d i u m , d e s i g n a t e d M e d i u m A , c o n t a i n e d 1 .7 %

T y p t i c a s e , 0 .3 % P h y t o n e , 0 .5 % y e a s t e x t r a c t , 0 . 5 % N a C l , 0 . 2 5 % K 2 H P 0 4 a n d 1 .7 5 p C i o f 14 C - l a b e l e d s u b s t r a t e s p e r v i a l . T h e b a s a l m e d i u m w a s s u p p l e m e n t e d a s f o l l o w s :

M e d i u m C o m p o s i t i o n

A a s a b o v e

B M e d i u m A + 0 . 1 7 m g /1 u n l a b e l e d g l u c o s e ( 1 0 X a m t . o f 1 4 C -g lu - c o s e )

C M e d i u m A + 0 .5 m g /1 u n l a b e l e d g l u c o s e ( 3 0 x a m t . o f 1 4 C - g lu - c o s e )

D M e d i u m A + 1 .7 m g /1 u n l a b e l e d g l u c o s e (1 0 0 x a m t . o f 14 C - g lu - c o s e )

E M e d i u m A + 3 .3 g / l T r y p t i c a s e

F M e d i u m A + 1 6 . 5 g / l T r y p t i c a s eG M e d i u m A + 3 .3 g / l T h i o t o n eH M e d i u m A + 1 .7 m g /1 u n l a b e l e d

g l u c o s e + 1 6 . 5 g / l T r y p t i c a s e + 3 .3 g / l T h i o t o n e

I n a d d i t i o n t o t h e a b o v e m e d i a , M e d i u m J ,

c o m p o s e d o f 2 % T h i o t o n e p e p t o n e , 0 .5 % N a C l , 0 .2 5 % K 2 H P O , a n d 1 .7 5 p C i o f 1 4 C - l a b e l e d s u b s t r a t e s p e r v i a l , w a s t e s t e d .

A l l m e d i a w e r e d i s p e n s e d in 3 0 - m l q u a n t i t i e s i n 5 0 - m l s e r u m v ia l s a n d s e a l e d w i t h a n

a l u m i n u m c a p o v e r a r u b b e r s e a l . V ia l s o f m e d i a A t h r o u g h J w e r e p r e p a r e d w i t h a n d w i t h o u t m a g n e t i c s t i r r i n g b a r s ( 2 . 7 c m ) t o e v a l u a t e t h e e f f e c t o f a g i t a t i o n d u r i n g i n c u b a t i o n . A l l v ia l s w e r e f l u s h e d o n c e w i t h 8 5 % N 2 - 1 0 % C 0 2 -5 % H 2 g a s , f o l l o w e d b y a s e c o n d f l u s h i n g w i t h 9 0 % N 2 - 5 % C 0 2 - 5 % H 2 g a s , a n d a t h i r d f l u s h i n g w i t h

8 5 % N 2 - 1 0 % C 0 2 -5 % H 2 g a s . M e d i a w e r e a u t o c l a v e d f o r 1 5 m i n a t 1 2 1 ° C .

V ia l s o f m e d i a w e r e i n o c u l a t e d w i t h e i t h e r a m i x t u r e o f v e g e t a t i v e c e l l s a n d s p o r e s o r h e a t - s t r e s s e d s p o r e s o f t h e v a r i o u s c l o s t r i d i a l s p e c i e s a t l e v e l s r a n g i n g f r o m 1 t o 1 0 7 o r g a n i s m s . A n i n o c u l u m v o l u m e o f 0 .1 m l w a s p l a c e d i n t o e a c h v ia l u s i n g a t u b e r c u l i n s y r i n g e a n d a 2 5 g a u g e n e e d l e . A l l v ia l s w e r e i n c u b a t e d a t 3 7 ° C .

l n o c u l a c o u n t s w e r e p e r f o r m e d in C M M u s i n g t h e t h r e e t u b e M P N t e c h n i q u e . C M M w a s a l s o u s e d f o r g r o w t h o f o r g a n i s m s a n d t h e r e s u l t s c o m p a r e d t o r e c o v e r i e s o b t a i n e d u s i n g t h e r a d i o m e t r i c t e c h n i q u e . T h e C M M t u b e s w e r e o v e r l a y e d w i t h 2 m l o f 2 % s t e r i l e a g a r a n d r e c o v e r i e s w e r e c o n s i d e r e d p o s i t i v e a t t h e f i r s t s ig n o f g a s b u b b l e s .

R a d i o m e t r i c d e t e c t i o n s y s t e m

B a c t e r i a l g r o w t h w a s d e t e c t e d w i t h a B a c t e c

Volume 3 9 ( 1974)—JO U R N A L OF FOOD S C IE N C E -11

7 8 —JO U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

Table 1—E ffe c t o f m edium on the recovery tim e to the de tec tion o f g row th o f various anaerobic organisms

T im e to de tec tion o f g row th (hr)

M ediumC. b o tu lin u m

typ e 62 AC. b o tu lin u m

typ e 109AC. b o tu lin u m typ e 7949B

C. b o tu lin u m typ e 2B

C. b o tu lin u m type 4304 F

C .sporogenes PA 19404

A 13a (47b )(9 8 c)14d 1 1 (2 7 )0 8 )1 2 7 (3 4 )0 8 )8 1 1 (2 8 )0 8 )1 3 1 0 (3 5 )0 8 )1 1 4 (3 9 )0 8 )5

B 1 3 (2 6 )0 8 )1 4 1 1 (2 1 )0 8 )1 3 7 (4 2 )0 8 )8 1 1 (2 8 )0 8 )1 3 1 0 (3 6 )0 8 )1 1 4 (3 1 )0 8 )5

C 1 3 (2 5 )0 8 )1 4 1 1 (2 5 )0 8 )1 2 7 (3 3 )0 8 )8 1 0 (3 2 )0 8 )1 1 1 0 (3 6 )0 8 )1 1 4 (3 3 )0 8 )5

D 1 3 (3 0 )0 8 )1 4 1 1 (2 2 )0 8 )1 3 7 (3 6 )0 8 )8 9 (2 5 )0 8 )1 1 1 0 (3 6 )0 8 )1 1 4 ( 4 6 )0 8 )5

E 1 3 (2 3 )0 8 )1 5 1 2 (6 7 )0 8 )1 3 7 (4 6 )0 8 )8 1 0 (4 3 )0 8 )1 1 1 0 (3 0 )0 8 )1 1 4 ( 4 2 )0 8 )5

F 1 5 (6 8 )0 8 )1 6 1 3 (3 2 )0 8 )1 4 7 (2 7 )0 8 )8 1 0 (2 9 )0 8 )1 2 1 0 (2 1 )0 8 )1 2 4 (4 2 )0 8 )5

G 1 3 (2 5 )0 8 )1 4 1 1 (2 7 )0 8 )1 2 7 (7 1 )0 8 )8 9 (3 8 )0 8 )1 0 1 0 0 5 )0 8 )1 1 4 (5 4 )0 8 )5

H 1 5 (2 2 )0 8 )1 7 1 3 (3 1 )0 8 )1 4 9 (2 1 )0 8 )1 1 9 (2 1 )0 8 )1 1 1 0 (3 1 )0 8 )1 1 4 (4 5 )0 8 )5

a Incub a tion t im e to in it ia l d e tec tion (h r)b G ro w th Index (% o f fu ll scale) w hen f ir s t detected as pos itive c M a x im u m G ro w th Index a tta inedd Inc u b a tio n t im e to reach m a x im u m G ro w th Index (h r)

m o d e l 2 2 5 ( J o h n s t o n L a b o r a t o r i e s , I n c . ) . T h e

t e c h n i q u e is b a s e d o n t h e r e l e a s e a n d d e t e c t i o n o f 1 4 C 0 2 p r o d u c e d b y b a c t e r i a l m e t a b o l i s m o f

' 4 C - l a b e l e d s u b s t r a t e s in t h e c a p p e d s e r u m v ia ls . T h e v ia l s w e r e i n o c u l a t e d a n d l o a d e d i n t o

a s a m p l e t r a y h a v i n g a c a p a c i t y o f 2 5 v ia l s . T h e t r a y w a s p l a c e d in t h e B a c t e c 2 2 5 a n d t h e ‘s t a r t ’ b u t t o n p r e s s e d i n i t i a t i n g a f u l l y a u t o

m a t e d t e s t c y c l e . C o n t r o l l e d i n c u b a t i o n a n d a g i t a t i o n ( w h e n s t u d i e d ) w e r e p r o v i d e d f o r e a c h v ia l b y t h e i n s t r u m e n t . P r i o r t o t h e s a m p l i n g

n e e d l e s e n t e r i n g t h e v ia l s , v ia l t o p s a n d n e e d l e s w e r e s t e r i l i z e d a u t o m a t i c a l l y t o p r e v e n t c r o s s

c o n t a m i n a t i o n . T h e v ia l s w e r e t e s t e d i n i t i a l l y t o o b t a i n a b a c k g r o u n d r e a d i n g a n d a g a in a t c i t h e r

1 o r 4 - h r i n t e r v a l s u n t i l s a m p l e s t e s t e d p o s i t i v e o r t h e e x p e r i m e n t w a s t e r m i n a t e d . T h e r e s u l t s w e r e p r i n t e d o n a p a p e r t a p e s o t h a t c o n t i n u o u s o b s e r v a t i o n o f s a m p l e s w a s n o t n e c e s s a r y f o r d e t e c t i o n o f p o s i t i v e s a m p l e s . A r e a d i n g o f 2 0 % o f f u l l s c a l e ( G r o w t h I n d e x ) a s r e c o m m e n d e d b y t h e m a n u f a c t u r e r , w a s c o n s i d e r e d a s p o s i t i v e e v i d e n c e o f m e t a b o l i s m in t h e c u l t u r e v ia l . B a c k g r o u n d r e a d i n g s a b o v e 6 % o f s c a l e w e r e n o t o b s e r v e d w i t h a n y o f t h e s a m p l e s .

RESULTS & DISCUSSIONR A P I D G R O W T H w a s o b t a i n e d i n t h e J L I A n a e r o b i c C u l t u r e M e d i u m 7 A w i t h

m o s t o f t h e 2 0 a n a e r o b i c s p e c i e s t e s t e d w i t h t h e e x c e p t i o n o f C. b o t u l i n u m s t r a i n s 6 2 A , 1 0 9 A , 2 B a n d 4 6 8 C , C. c h a u v o e i , C. n o v y i a n d C. s e p t i c u m . S u p p l e m e n t a t i o n o f M e d i u m 7 A w i t h O . O l g o f T r y p t i c a s e p e r m l , 0 . 0 2 g o f P h y t o n e

p e r m l o r O . O l g o f g l u c o s e p e r m l r e s u l t e d i n e x c e l l e n t g r o w t h b y t h e s e o r g a n i s m s . I n s e p a r a t e e x p e r i m e n t s i t w a s d e t e r m i n e d t h a t t h e m i n i m u m a m o u n t o f g l u c o s e n e e d e d t o b e a d d e d t o t h e m e d i u m w a s 1 8 /Ug o r 3 . 3 x 1 0 ' 6 M f i n a l c o n c e n t r a t i o n ( 3 3 t i m e s t h e l e v e l p r e s e n t i n M e d i u m 7 A ) . T h e m e d i u m a s s u p p l i e d i s 1 0 " 7 M w i t h r e s p e c t t o g l u c o s e ( a l l ' 4 - C - l a b e l e d ) . T h e a d d i t i o n o f t h i s q u a n t i t y o f u n l a b e l e d g l u c o s e t o t h e m e d i u m ,

h o w e v e r , d i d n o t s e r i o u s l y a f f e c t t h e d e t e c t i o n t i m e .

E x p e r i m e n t s w e r e c o n d u c t e d t o d e t e r m i n e i f t h e f a i l u r e o f t h e s e v e n o r g a n i s m s

( a t i n o c u l u m l e v e l s u p t o 1 0 7 o r g a n i s m s

p e r v i a l ) t o i n i t i a t e g r o w t h i n t h e J L I A n a e r o b i c M e d i u m 7 A w a s d u e t o t h e a b

s e n c e o f s u f f i c i e n t n u t r i e n t s o r t o t h e p r e s e n c e o f i n h i b i t o r s . S t u d i e s w i t h m e d i

u m c o m p o n e n t s i n d i c a t e d t h a t h e m i n w a s r e s p o n s i b l e f o r t h e i n h i b i t i o n o f g r o w t h o f t h e s e v e n o r g a n i s m s . M e d i u m 7 A p r e p a r e d w i t h o u t h e m i n r e s u l t e d i n r a p i d

g r o w t h b y a l l s p e c i e s . E x p e r i m e n t s s t u d y i n g t h e i n h i b i t o r y e f f e c t o f h e m i n s h o w e d t h a t a c o n c e n t r a t i o n o f 0 . 0 0 5 m g o r h i g h e r p e r m l o f m e d i u m w o u l d i n h i b i t t h e g r o w t h o f C. b o t u l i n u m t y p e 6 2 A r e g a r d l e s s o f w h e t h e r t h e i n o c u l u m w a s 2 4 - h r o l d v e g e t a t i v e c e l l s o r h e a t - s t r e s s e d s p o r e s . T h e r e i s g o o d i n d i c a t i o n t h a t g l u c o s e r e l i e v e s t h i s i n h i b i t i o n b u t t h e m e c h

a n i s m o f t h i s r e l i e f h a s n o t b e e n d e t e r

m i n e d . T h e f a c t t h a t P h y t o n e c o n t a i n s 3 7 % c a r b o h y d r a t e c o u l d p o s s i b l y e x p l a i n t h e r e l i e f o f h e m i n i n h i b i t i o n b y P h y t o n e .

B a c t e c A n a e r o b i c C u l t u r e M e d i u m 7 A

w a s m o d i f i e d t o e l i m i n a t e h e m i n , v i t a m i n K a n d s o d i u m p o l y a n e t h o l s u l f o n a t e s i n c e

t h e s e w e r e a d d e d p r i m a r i l y f o r b l o o d c u l t u r e w o r k . T h e b a s a l m e d i u m , d e s i g n a t e d

M e d i u m A , w a s s u p p l e m e n t e d a s d e s c r i b e d t o e v a l u a t e m e d i u m c o m p o s i t i o n o n t h e r e c o v e r y t i m e s e n c o u n t e r e d f o r t h e d e t e c t i o n o f g r o w t h o f t h e c l o s t r i d i a l

s p e c i e s . T h e e f f e c t o f m e d i u m c o m p o s i t i o n o n t h e g r o w t h o f s i x o f t h e a n a e r o b i c o r g a n i s m s s t u d i e d i s p r e s e n t e d i n T a b l e 1. A l l v i a l s o f m e d i a w e r e i n o c u l a t e d w i t h a s u s p e n s i o n o f t h e o r g a n i s m b e i n g s t u d i e d a t a n i n o c u l u m l e v e l o f a p p r o x i m a t e l y

1 0 4 o r g a n i s m s p e r v i a l . I n c u b a t i o n w a s a t 3 7 ° C w i t h a g i t a t i o n . T h e r e s u l t s s h o w t h a t , d e p e n d i n g o n t h e o r g a n i s m s a n d t h e

m e d i u m u s e d , d e t e c t i o n c a n b e a s r a p i d a s 4 h r . T h e m e d i u m w h i c h a p p e a r s b e s t

f r o m t h e s e r e s u l t s i s M e d i u m G ( i t s h o w e d a s h o r t e r i n c u b a t i o n t i m e t o d e t e c t i o n o r a h i g h e r G r o w t h I n d e x w h e n f i r s t d e t e c t e d p o s i t i v e , t h a n m o s t o t h e r m e d i a ) . N o s i g n i f i c a n t d i f f e r e n c e i n d e t e c t i o n t i m e w a s n o t e d b e t w e e n M e d i u m A

a n d M e d i a B , C o r D i n s p i t e o f t h e i n

c r e a s e d u n l a b e l e d g l u c o s e c o n t e n t o f M e d i a B , C a n d D . I n o n e i n s t a n c e , w i t h C. b o t u l i n u m t y p e 2 B ( n o n p r o t e o l y t i c ) ,

d e t e c t i o n t i m e w a s s h o r t e n e d b y i n c r e a s i n g t h e a m o u n t o f u n l a b e l e d g l u c o s e i n

t h e m e d i u m .M e d i u m G w a s o b t a i n e d b y s u p p l e

m e n t i n g M e d i u m A w i t h T h i o t o n e p e p

t o n e a n d t o d e t e r m i n e a n y b e n e f i c i a l e f f e c t o f T h i o t o n e a l o n e , M e d i u m J w a s

e v a l u a t e d . A t t h e s a m e t i m e , t h e e f f e c t o f

a g i t a t i o n d u r i n g i n c u b a t i o n w a s d e t e r m i n e d . V i a l s o f M e d i u m G a n d J w e r e

p r e p a r e d w i t h a n d w i t h o u t m a g n e t i c s t i r r i n g b a r s ( t h e B a c t e c 2 2 5 e m p l o y s a

m a g n e t i c s t i r r i n g d e v i c e w h i c h a g i t a t e s

t h e m e d i u m i n t h e v i a l s d u r i n g i n c u b a t i o n ) . A c o m p a r i s o n o f M e d i u m G a n d J ,

a n d t h e e f f e c t o f a g i t a t i o n d u r i n g i n c u b a

t i o n , i s p r e s e n t e d i n T a b l e 2 . T h e i n o c u l u m c o n s i s t e d o f a m i x t u r e o f v e g e t a t i v e

c e l l s a n d s p o r e s a t a l e v e l o f a p p r o x i m a t e l y 1 0 4 o r g a n i s m s p e r v i a l .

I n a l l c a s e s , t h e e f f e c t o f a g i t a t i o n w a s b e n e f i c i a l , s h o w i n g a t i m e a d v a n t a g e o f f r o m 2 - 4 h r w i t h t h e G m e d i u m a n d f r o m 1 — 2 h r w i t h t h e J m e d i u m . W i t h

t h r e e o f t h e f i v e o r g a n i s m s t e s t e d , M e d i u m J w i t h a g i t a t i o n s h o w e d a t i m e a d v a n t a g e o f f r o m 1 - 3 h r , w h i l e w i t h t h e r e m a i n i n g t w o o r g a n i s m s , t h e d e t e c t i o n t i m e w a s i d e n t i c a l w i t h b o t h m e d i a . I n m o s t c a s e s w h e r e a g i t a t i o n w a s n o t e m p l o y e d , t h e m a x i m u m G r o w t h I n d e x a t

t a i n e d w a s l e s s t h a n t h e m a x i m u m a t t a i n e d w i t h a g i t a t i o n . W i t h C. b o t u l i n u m t y p e 6 2 A i n c u b a t e d s t a t i s t i c a l l y i n M e d i u m G , t h e G r o w t h I n d e x n e v e r e x c e e d e d 2 1 , t h e G r o w t h I n d e x w h e n f i r s t d e t e c t e d a s p o s i t i v e .

H e a t s t r e s s e d s p o r e s u s p e n s i o n s ( h e a t e d a t 1 0 0 ° C f o r 1 0 m i n ) o f C . b o t u l i n u m t y p e 6 2 A w e r e c o u n t e d b y a 3 - t u b e

M P N t e c h n i q u e . A c o m p a r i s o n o f c o u n t s a n d d e t e c t i o n t i m e s w a s m a d e b e t w e e n B a c t e c w i t h A n a e r o b i c M e d i u m J a n d

t u b e s o f C M M . I n c u b a t i o n w a s a t 3 7 ° C ;

GROWTH DETECTION OF CLOSTRIDIAL SPECIES-79

Table 2 —E ffe c t o f T h io ton e and ag ita tion on the recovery tim e to de tec tion o f g row th o f various anaerobic organisms

T im e to de tec tion o f g row th (h r)

M ediumC. b o tu lin u m

typ e 62 AC. b o tu lin u m

type 109AC. b o tu lin u m typ e 7949B

C. bo tu lin u m typ e 2B

C. b o tu lin u m ty p e 4304F

C.sporogenes PA 19404

G, agitated G, static J, agitated J, static

14a(49)b(98)c15d16(21)13(27)198)1514(20X84)16

10(24X98)12 13(481(98)14 7(20X98)9

, 9(20X82)11

8(22X98)1010(26X98)128(31X98)109(23X89)11

9(29X98)1013(46X98)149(23X59)1111(30X42)12

9(20X98)1111(21X98)137(24X98)89(68X94)10

3(27 ) (98)4 5(27)(98)6

_e_e

a Inc u b a tio n t im e to in it ia l d e te c tio n (h r)b G ro w th Index (% o f fu l l scale) w hen f ir s t detected as pos itive c M a x im u m G ro w th Index a tta inedd In c u b a tio n t im e to reach m a x im u m G ro w th Index (h r) e E x p e rim e n t n o t pe rfo rm ed

B a c t e c m e d i u m w a s a g i t a t e d b y s t i r r i n g b a r s w h i l e C M M t u b e s r e m a i n e d s t a t i c d u r i n g i n c u b a t i o n . C M M t u b e s w e r e o b

s e r v e d v i s u a l l y w h i l e t h e J m e d i u m w a s e x a m i n e d r a d i o m e t r i c a l l y w i t h B a c t e c . T y p i c a l r e s u l t s p r e s e n t e d i n T a b l e 3 i n d i

c a t e t h a t a h i g h e r c o u n t ( 4 6 , 0 0 0 , 0 0 0 v s . 1 5 , 0 0 0 , 0 0 0 ) a n d m o r e r a p i d d e t e c t i o n o f g r o w t h w e r e o b t a i n e d u s i n g t h e B a c t e c

s y s t e m . F o r d e t e c t i o n o f v i a b l e s p o r e s ( g e r m i n a t i o n a n d o u t g r o w t h ) t h e t i m e a d

v a n t a g e g a i n e d b y u s i n g t h e B a c t e c s y s t e m c a n b e s u b s t a n t i a l ( u p t o 1 5 2 h r i n t h i s i n s t a n c e ) .

R e s u l t s p r e s e n t e d i n t h i s p a p e r c o n f l i c t w i t h r e s u l t s r e c e n t l y p u b l i s h e d b y P r e v i t e

( 1 9 7 3 ) . O n t h e b a s i s o f h i s r e s u l t s , h e c o n c l u d e d t h a t t h e a d d i t i o n o f u n l a b e l e d g l u c o s e s i g n i f i c a n t l y i n c r e a s e d t h e t i m e t o

d e t e c t i o n o f g r o w t h b u t t h a t t h e a d d i t i o n

o f l a b e l e d g l u c o s e i n a m o u n t s g r e a t e r t h a n 0 . 0 8 3 3 ¿1 C i / m l d i d n o t s e e m t o d e

c r e a s e t h e t i m e t o d e t e c t i o n . O u r r e s u l t s i n d i c a t e t h a t t h e a d d i t i o n o f u n l a b e l e d g l u c o s e u p t o 1 0 0 t i m e s t h e a m o u n t o f I 4 C - l a b e l e d g l u c o s e d o e s n o t a l t e r t h e

s e n s i t i v i t y o f d e t e c t i o n , i n o t h e r w o r d s , i n c r e a s e d e t e c t i o n t i m e . T h i s d i s c r e p a n c y m a y p o s s i b l y b e e x p l a i n e d b y t h e f a c t

t h a t t h e t e c h n i q u e u s e d b y P r e v i t e d i f f e r s f r o m t h a t e m p l o y e d i n t h i s s t u d y . P r e v i t e i n c u b a t e d h i s c u l t u r e s w i t h o u t a g i t a t i o n ,

w h e r e a s i n t h i s s t u d y , m e d i a i n v i a l s w e r e c o n s t a n t l y s t i r r e d w i t h a m a g n e t i c s t i r r i n g r o d . W e h a v e e v i d e n c e t h a t a g i t a t i o n o f t h e c u l t u r e b r o t h d u r i n g i n c u b a t i o n i s c r i t i c a l , a n d n o t o n l y d e c r e a s e s t h e t i m e t o d e t e c t i o n , b u t i n c r e a s e s t h e m a x i

m u m a m o u n t o f 1 4 C 0 2 d e t e c t e d d u r i n g g r o w t h ( i n T a b l e 2 , s o m e n o n a g i t a t e d c u l

t u r e s n e v e r a c h i e v e d t h e m a x i m u m G r o w t h I n d e x o f 9 8 ) . D e B l a n c e t a l .

( 1 9 7 1 ) r e p o r t e d t h a t t h e s i z e o f t h e m a g n e t a n d t h e a m o u n t o f a g i t a t i o n p r o d u c e d d u r i n g i n c u b a t i o n h a d a p r o f o u n d e f f e c t o n t h e d e t e c t i o n t i m e . E v i d e n c e c o l l e c t e d i n o u r l a b o r a t o r y , h o w e v e r , i n d i c a t e s t h a t

w i t h i n E m i t s , t h e t y p e a n d s p e e d o f a g i t a t i o n h a s l i t t l e e f f e c t o n t h e t i m e t o d e t e c t i o n .

T h e r e a s o n f o r t h e d e c r e a s e i n t i m e t o

Table 3 —T im e fo r MPN cou n t o f a heat-stressed spore suspension o f C. b o tu lin u m 62 A using Cooked Meat M edium and Bactec A naerob ic M edium J

T im e to de tec tion o f g row th (hr)

SuspensionD ilu tio n

Cooked Meat M edium A naerob ic M edium J-Bactec

Tube 1 Tube 2 Tube 3 V ia l 1 V ia l 2 V ia l 3

-4 34 34 34 14 14 16-5 36 36 36 16 18 20- 6 36 101 101 18 20 22-7 101 196 24 32 40- 8 175 44Count = 15,000,000 spores/ml Count =46,000,000 spores/ml

d e t e c t i o n n o t e d w i t h s a m p l e s a g i t a t e d

d u r i n g i n c u b a t i o n i s n o t c l e a r l y u n d e r s t o o d . W h e t h e r t h e g r o w t h r a t e o f t h e

o r g a n i s m i s i n c r e a s e d b y a g i t a t i o n , t n u s c a u s i n g a m o r e r a p i d p r o d u c t i o n o f 1 4 C 0 2 d u e t o i n c r e a s e d r a t e o f m e t a b o l i s m , o r w h e t h e r t h e r a t e o f m e t a b o l i s m

r e m a i n s c o n s t a n t a n d a g i t a t i o n c a u s e s a f a s t e r r e l e a s e o f 1 4 C 0 2 f r o m t h e m e d i u m a n d t h u s m o r e r a p i d d e t e c t i o n h a s n o t b e e n d e t e r m i n e d . W i t h v e g e t a t i v e c e l l s , t h e d i f f e r e n c e i n d e t e c t i o n t i m e b e t w e e n a g i t a t e d a n d n o n a g i t a t e d s a m p l e s i s o n l y 1 — 2 h r . W i t h s p o r e s , h o w e v e r , r e d u c t i o n i n d e t e c t i o n t i m e i s d r a m a t i c i n d i c a t i n g a g i t a t i o n d o e s a f f e c t g e r m i n a t i o n a n d o u t g r o w t h .

T h e B a c t e c s y s t e m o f f e r s a s i g n i f i c a n t t i m e a d v a n t a g e i n d e t e c t i n g g r o w t h o f a n a e r o b i c s p o r e f o r m e r s w h e n c o m p a r e d t o t h e c o n v e n t i o n a l m e t h o d s o f s u b c u l t u r e . A t i m e a d v a n t a g e r a n g i n g f r o m 2 0 — 1 5 2 h r w a s o b t a i n e d w h e n c o u n t i n g C. bctu- linum h e a t - s t r e s s e d s p o r e s b y t h e M o s t P r o b a b l e N u m b e r t e c h n i q u e u s i n g B a c t e c a s c o m p a r e d t o a n M P N d o n e i n t u b e s o f C o o k e d M e a t M e d i u m .

REFERENCESAshton, D.H. 1971. Identification of a pea

component stimulating for heat-stressed

Putrefactive Anaerobe 59-123 spores. Appl. Microbiol. 21: 38.

DeBlanc, H.J. Jr., DeLand, F. and Wagner, H.N. Jr. 1971. Automated radiometric detection of bacteria in 2,967 blood cultures. Appl. Microbiol. 22: 846.

DeLand, F. and Wagner, H.N. Jr. 1970. Automated radiometric detection of bacterial growth in blood cultures. J. Lab. Clin. Med. 75: 529.

Gall, L.S. and Johnson, G. 1973. Radiometric method for rapid detection of Bacteriuria. Abstracts of the Annual Meeting of the American Society for Microbiology, p. 99.

Kilbourn, J.P. and Bramhall, J.L. 1973. Rapid radiometric quantitation and antibiotic sensitivity testing of urine specimens. Abstracts of the Annual Meeting of the American Society for Microbiology, p. 99.

Previte, J.J. 1972. Radiometric detection of some food-borne bacteria. Appl. Microbiol. 24: 535.

Previte, J.J. 1973. Improvement of media for radiometric detection of food-borne bacteria. Abstracts of the Annual Meeting of the American Society for Microbiology, p.10.

Randall, E.L. 1972. Comparison of a radiometric method with conventional cultural methods for detection of bacteremia. Abstracts of the Annual Meeting of the American Society for Microbiology, p. 87.

Randall, E.L. 1973. Further studies on a radiometric method for determining bacteremia. Abstracts of the Annual Meeting of the American Society for Microbiology, p. 82.

Sharf, J.M. 1966. “Recommended Methods for the Microbiological Examination of Foods,” 2nd ed., p. 35. American Public Health Assoc., Inc. New York.

Waters, J.R. 1972. Sensitivity of the 14C02 radiometric method for bacterial detection. Appl. Microbiol. 23: 198.


W . J. S T A M P E R a n d G . J. B A N W A R T

D e p t , o f M ic r o b io lo g y , T h e O h io S t a t e U n iv e r s i ty , C o lu m b u s , O H 4 3 2 1 0

EFFECT OF VARIOUS PEPTONES IN THE GROWTH MEDIUM ON THE AGGLUTINATION OF TEN Salmonella SPECIES

WITH POOLED SPICER-EDWARDS ANTISERA

INTRODUCTIONF O O D I N F E C T I O N r e s u l t i n g f r o m t h e i n

g e s t i o n o f p r o d u c t s c o n t a m i n a t e d w i t h S a l m o n e l l a i s a w e l l - r e c o g n i z e d p r o b l e m .

S a l m o n e l l o s i s a f f e c t s t h e p u b l i c h e a l t h a n d r e s u l t s i n s e r i o u s e c o n o m i c l o s s e s .

O n e o f t h e p r o b l e m s c o n f r o n t i n g t h e f o o d i n d u s t r y t o d a y i s t h e l o n g t i m e

n e e d e d t o d e t e r m i n e w h e t h e r o r n o t a

p r o d u c t i s f r e e o f S a l m o n e l l a c o n t a m i n a t i o n . P r e s e n t d e t e c t i o n s y s t e m s a r e t i m e c o n s u m i n g , e x p e n s i v e a n d n o t e n t i r e l y

r e l i a b l e .A p r o c e d u r e f o r t e s t i n g S a l m o n e l l a f o r

t h e f l a g e l l a r H a n t i g e n w a s d e s c r i b e d b y H a j n a a n d D a m o n ( 1 9 5 0 ) . T h e y d e v i s e d a s p e c i a l H b r o t h t o d e v e l o p t h e f l a g e l l a r a n t i g e n s d u r i n g g r o w t h . T h e r e a s o n s f o r

i n c l u d i n g a n y p a r t i c u l a r i n g r e d i e n t i n t h e b r o t h w e r e n o t e x p l a i n e d . M o r e r e c e n t l y , S p e r b e r a n d D e i b e l ( 1 9 6 9 ) d e v i s e d a m e d i u m t e r m e d M b r o t h w h i c h a c c o r d i n g t o t h e m g a v e b e t t e r r e s u l t s a s a g r o w t h m e d i u m f o r t h e o r g a n i s m s p r i o r t o t e s t i n g w i t h H a n t i s e r u m t h a n d i d b r a i n h e a r t i n f u s i o n b r o t h . O t h e r w o r k e r s h a v e s u g g e s t e d t h a t d i f f e r e n t i m p r o v e d f l a g e l l a r b r o t h s b e s u b s t i t u t e d i n t h e r e c o m m e n d e d A O A C 2 4 - h r S a l m o n e l l a f l a g e l l a r t e s t . P o e l m a

a n d R o m e r o ( 1 9 7 1 ) s t a t e d t h a t a d i s a d v a n t a g e o f r e c o m m e n d e d m e t h o d s o f g r o w i n g c u l t u r e s f o r s e r o l o g i c a l t e s t i n g is p o o r a n t i g e n d e v e l o p m e n t , r e s u l t i n g i n

a f a l s e n e g a t i v e H a g g l u t i n a t i o n t e s t . B a n w a r t a n d K r e i t z e r ( 1 9 7 2 ) r e p o r t e d

t h e e f f e c t s o f v a r i o u s c a r b o h y d r a t e s i n t h e g r o w t h m e d i u m o n t h e a g g l u t i n a t i o n

r e a c t i o n . P r e v i o u s l y u n r e p o r t e d w o r k b y t h e s e a u t h o r s s h o w e d t h a t n e i t h e r B H I n o r t h e M b r o t h o f S p e r b e r a n d D e i b e l w e r e a s a c c e p t a b l e a s w a s d u l c i t o l i n p u r p l e b r o t h b a s e f o r t h e g r o w t h m e d i u m . I t i s e v i d e n t t h a t m o r e r a p i d a n d r e l i a b l e d e t e c t i o n p r o c e d u r e s a r e n e e d e d .

T h i s s t u d y w a s u n d e r t a k e n t o d e t e r m i n e i f t h e a d d i t i o n o f v a r i o u s p e p t o n e s t o t h e g r o w t h m e d i u m r e s u l t e d i n i m p r o v e d f l a g e l l a r d e v e l o p m e n t f o r t h e S a l m o n e l l a f l a g e l l a r H t e s t .

EXPERIMENTALC U L T U R E S w e r e o b t a i n e d f r o m t h e s t o c k c o l l e c t i o n a t T h e O h i o S t a t e U n i v e r s i t y . T h e t e n S a lm o n e lla u s e d in t h i s s t u d y i n c l u d e d : S a lm o nella st. pa u l, S. d e r b y , S. read ing , S. in fa n t is, S. m u e n c h e n , S . e n te r itid is , S . a n a tu m , S . m elea-

grid is, S. n e w in g to n a n d S. gam inara . T h e s e o r g a n i s m s w e r e s e l e c t e d t o r e p r e s e n t d i f f e r e n t s o m a t i c 0 g r o u p s . B e f o r e u s e , t h e s t o c k c u l t u r e s w e r e t e s t e d b i o c h e m i c a l l y a n d s e r o l o g i c a l ly t o d e t e r m i n e i f t h e S a lm o n e lla w e r e p u r e c u l t u r e s a n d i f p h a s e v a r i a t i o n o f t h e H a n t i g e n s h a d o c c u r r e d . T h e r e s u l t s m a t c h e d t h e r e f e r e n c e d a t a w i t h a l l s t r a i n s t e s t e d . T h e c u l t u r e s w e r e r e c h e c k e d a f t e r t h e e x p e r i m e n t s a n d t h e s a m e r e s u l t s w e r e o b t a i n e d .

T h e e l e v e n p e p t o n e s u s e d in t h i s s t u d y i n c l u d e d : c a s i t o n e , b e e f e x t r a c t , p e p t o n e , p e p t o n i z e d m i l k , p r o t e o s e p e p t o n e # 2 , p r o t o n e , s o y t o n e , t r y p t o n e a n d y e a s t e x t r a c t f r o m D if -

c o , a n d g e l y s a t e a n d t r y p t i c a s e f r o m B B L . T h e p e p t o n e s w e r e s e l e c t e d t o i n c l u d e a c i d a n d e n

z y m i c h y d r o l y s a t e s o f p l a n t a n d a n i m a l t i s s u e s .A b a s a l m e d i u m w a s p r e p a r e d a c c o r d i n g t o

t h e f o l l o w i n g f o r m u l a : 2 . 5 g d e x t r o s e , 5 g N a C l , 5 g K 2 H P 0 4 a n d 1 l i t e r d i s t i l l e d w a t e r . T o t h i s b a s a l m e d i u m t h e p e p t o n e s w e r e a d d e d a t t h e r a t e s o f 5 , 1 0 a n d 2 0 g p e r l i t e r . T h e m e d i a w e r e d i s p e n s e d i n t o b o t t l e s i n 9 9 - n i l q u a n t i t i e s a n d

w e r e a u t o c l a v e d a t 1 2 1 ° C f o r 1 8 m i n .T h e m e d i a f o r c o m p a r i s o n t e s t i n g i n c l u d e d :

b r a i n h e a r t i n f u s i o n ( B H I ) b r o t h ( D i f c o ) , H b r o t h ( D i f c o ) , M b r o t h ( S p e r b e r a n d D e i b e l , 1 9 6 8 ) , a n d t r y p t i c a s e s o y t r y p t o s e b r o t h

( E w i n g a n d D a v is , 1 9 7 0 ) . T h e s e m e d i a w e r e p r e p a r e d a c c o r d i n g t o t h e m a n u f a c t u r e r s ’ o r a u t h o r s ’ d i r e c t i o n s a n d w e r e d i s p e n s e d a n d

a u t o c l a v e d a s d e s c r i b e d a b o v e .T h e o r g a n i s m s w e r e i n o c u l a t e d i n t o B H I

b r o t h a n d i n c u b a t e d a t 3 7 ° C f o r 2 4 h r . D i l u t i o n s o f g r o w t h a f t e r i n c u b a t i o n w e r e m a d e in 0 .1 % p e p t o n e w a t e r . E a c h t e s t m e d i u m w a s t h e n i n o c u l a t e d w i t h a p p r o x i m a t e l y t h e s a m e

n u m b e r o f c e l l s , 1 m l o f t h e 1 0 ~ 7 d i l u t i o n . T h e n u m b e r s o f c e l l s i n o c u l a t e d w e r e d e t e r m i n e d b y p l a t i n g a p p r o p r i a t e d i l u t i o n s o n p l a t e c o u n t a g a r a n d c o u n t i n g t h e p l a t e s a f t e r i n c u b a t i o n a t 3 7 ° C f o r 4 8 h r .

T h e i n o c u l a t e d b o t t l e s o f t e s t b r o t h s w e r e i n c u b a t e d a t 3 7 ° C f o r 2 4 h r a n d t h e n s a m p l e d f o r t o t a l p l a t e c o u n t a n d t h e H a g g l u t i n a t i o n t e s t . T h e t u b e a g g l u t i n a t i o n t e s t s w e r e c o n d u c t e d a s o u t l i n e d b y D i f c o u s i n g p o o l e d B a c - to -S a lm o n e lla H A n t i s e r u m S p i c e r - E d w a r d s ( 1 ,

2 , 3 , 4 a n d c o m p l e x e s I , E N a n d L ) d i l u t e d 1 : 1 0 0 0 . T h e a g g l u t i n a t i o n t u b e s w e r e r e m o v e d f r o m t h e 5 0 ° C w a t e r b a t h a n d o b s e r v e d f o r a g

g l u t i n a t i o n a t 1 a n d 2 h r .

T h e a g g l u t i n a t i o n t e s t w a s g iv e n a v a l u e o f 0 i f n o v i s i b l e r e a c t i o n w a s o b s e r v e d , 1 f o r a s l i g h t r e a c t i o n ( v i s ib l e f i b e r s ) , 2 f o r a r e a c t i o n h a v i n g a s m a l l v i s i b l e c l u m p ( e a s i l y b r o k e n i f m i x e d g e n t l y ) a n d w i t h a c l o u d y s u p e r n a t e , 3 f o r a r e a c t i o n h a v i n g a l a r g e v i s ib l e c l u m p ( n o t b r o

k e n i f m i x e d g e n t l y ) a n d w i t h a c l o u d y s u p e r n a t e , a n d 4 i f t o t a l a g g l u t i n a t i o n o c c u r r e d ( c l e a r s u p e r n a t e w i t h a l a r g e f l o c c u l a r c l u m p ) .

C o n t r o l t u b e s y i e l d e d n o s p o n t a n e o u s a g

g l u t i n a t i o n s .F o r s t a t i s t i c a l a n a l y s i s , a m u l t i p l e c o m p a r i

s o n p r o c e d u r e s e t u p f o r m u l t i n o m i a l p o p u l a

t i o n s ( G a b r i e l , 1 9 6 6 ) w a s u s e d t o d e t e r m i n e t h e s i g n i f i c a n c e o f e x p e r i m e n t a l d i f f e r e n c e s .

RESULTS & DISCUSSIONT H E T O T A L P L A T E c o u n t s o f t h e t e n S a l m o n e l l a s p e c i e s i n e a c h p e p t o n e b r o t h o b t a i n e d i n t h r e e r e p l i c a t i o n s w e r e n o t

Table 1—E ffe c t o f tim e o f observation on the H agg lu tina tion reaction observed fo r ten Sal-m onella grow n in eleven peptone b ro ths

Peptone b ro th 0 a 1b Organism 0 1

1 . Casitone 29 61 1. S a lm o n e l la s t . p a u l 11 882. Gelysate 9 81 2. S. d e r b y 19 803. Beef extract 5 85 3. S. r e a d in g 17 824. Peptone 21 69 4. S. i n f a n t i s 20 795. Peptonized milk 12 78 5. S . m u e n c h e n 15 846 . Proteose

Peptone #2 19 71 6 . S . e n t e r i t i d i s 23 767. Protone 13 77 7. S . a n a tu m 20 798 . Soytone 16 74 8. S. m e le a g r id is 30 699. Trypticase 36 54 9. S . n e w i n g to n 21 78

1 0 . Tryptone 34 56 1 0 . S . g a m in a r a 24 7511. Yeast extract 6 84a Num bers o f tubes in w h ich there was no d iffe rence between 1- and 2 -h r observa tions based on

scaled observations rated fro m 0 to 4h Num bers o f tubes in w h ich the re was a d iffe rence between 1- and 2 -h r observa tions based on

scaled observations rated fro m 0 to 4


H AGGLUTINATION TEST, EFFECT OF PEPTONES-8 1

Table 2 —Com parison o f peptone con cen tra tion e ffec t on the num ber and degree o f H agg lu tina tions observed at 1 and 2 hr a fte r grow th in three concentra tions o f the peptone b ro ths3

Peptone Cone g /lite r 0 1

Score

2 3 4

1-hr Observations

5 26 150 139 15 010 10 112 175 33 020 4 61 222 41 2

2-hr Observations

5 5 20 190 98 1710 1 7 126 171 2520 1 2 68 228 31

a Values represent sum o f observations fo r all peptones and all organisms fo r three replications.

Table 3 —E ffe c t o f peptone. 1-hr observations o f the num ber and degree o f H agg lu tina tions produced by the ten Salm onella a fte r g row th in various peptone b ro ths3

G ro w th media

Peptone added

Num bers o f tubes Score

1 2 3

s i g n i f i c a n t l y d i f f e r e n t ( 5 % l e v e l ) . T h e

n u m b e r s o f o r g a n i s m s i n e a c h c o n c e n t r a t i o n o f e a c h b r o t h w e r e g r e a t e r t h a n t h e m i n i m u m ( 2 x 1 0 s p e r m l ) r e q u i r e d f o r t h e f l a g e l l a r H a g g l u t i n a t i o n t e s t . G e n e r a l

l y , t h e c o u n t s i n c r e a s e d s l i g h t l y a s t h e c o n c e n t r a t i o n s o f t h e p e p t o n e s i n c r e a s e d .

T h e t e n Salmonella s p e c i e s u s e d i n t h i s

s t u d y d i d n o t b i a s t h e r e s u l t s i n f a v o r o f

a n y p a r t i c u l a r p e p t o n e . F r o m t h e 3 3 t e s t s

p e r f o r m e d f o r e a c h s p e c i e s i n e a c h o f t h e

t h r e e r e p l i c a t i o n s , t h e d i s t r i b u t i o n o f r e s u l t s f o r e a c h o r g a n i s m w a s c o n s i s t e n t i n

a l l t h e p e p t o n e s t e s t e d . N o c o r r e l a t i o n

w a s f o u n d b e t w e e n t h e n u m b e r s a n d

t y p e s f l a g e l l a r a n t i g e n s o f e a c h o r g a n i s m

a n d t h e a g g l u t i n a t i o n s c o r e s w i t h t h e

p o o l e d H a n t i s e r a . T h a t i s , t h e a n t i s e r a d i d n o t b i a s t h e r e s u l t s b y c o n t a i n i n g

m o r e s p e c i f i c f a c t o r s f o r a p a r t i c u l a r o r g a n i s m .

T h e e x t e n t o f a g g l u t i n a t i o n o b s e r v e d a f t e r t h e t u b e s w e r e i n a 5 0 ° C w a t e r b a t h

f o r 2 h r w a s s i g n i f i c a n t l y g r e a t e r t h a n t h e r e s u l t s o b s e r v e d a f t e r 1 h r ( T a b l e 1 ) . I n

9 9 0 H t e s t s , 7 9 0 t u b e s e x h i b i t e d a n i n c r e a s e d r e a c t i o n a f t e r 2 h r .

1. Casitone 0 3 73 12 2T a b l e 2 d e m o n s t r a t e s t h e e f f e c t o f

p e p t o n e c o n c e n t r a t i o n . I n t h e 1 - h r o b s e r -2. Gelysate 5 37 43 3 0 v a t i o n s , t h e n u m b e r a n d d e g r e e o f a g g l u t i -3. Beef ex trac t 0 55 31 4 0 n a t i o n s i n c r e a s e d w i t h i n c r e a s i n g p e p t o n e4. Peptone 4 25 56 5 0 c o n c e n t r a t i o n . A t t h e 2 - h r o b s e r v a t i o n5. Peptonized

m ilk 4 27 53 6 0t i m e , t h e r e s u l t s f r o m t h e 1 0 a n d 2 0 g p e r l i t e r c o n c e n t r a t i o n s w e r e s i g n i f i c a n t l y

6. ProteoseP eptone#2 1 0 63 26 0

g r e a t e r t h a n t h o s e r e s u l t s o b t a i n e d f r o m o r g a n i s m s g r o w n i n a 5 g p e r l i t e r c o n c e n -

7. Protone 19 60 10 1 0 t r a t i o n o f t h e p e p t o n e b r o t h . T h e r e w a s a8. S oytone 1 21 59 9 0 s l i g h t b u t n o t s i g n i f i c a n t d i f f e r e n c e b e -9. Tryp ticase 1 39 41 9 0 t w e e n t h e 1 0 a n d 2 0 g p e r l i t e r c o n c e n t r a -

10. T ryp to n e 0 5 73 12 0 t i o n s .11. Yeast ex trac t 5 51 32 2 0 T h e r e w e r e 3 0 t e s t s p e r f o r m e d f o r

a Values represent sum o f observations fo r three concentra tions and three rep lications.

e a c h p e p t o n e i n e a c h o f t h e t h r e e r e p l i c a t i o n s , f o r a t o t a l o f 9 0 t e s t s . T h e 1 - h r

Table 4 —E ffe c t o f peptone. 2-hr observations o f the num ber and degree o f H agglu tina tions produced by the ten Salm onella a fte r grow th in various peptone b ro thsa

G row th media

Peptone added 0

Num bers o f tubes Score

1 2 3 4

1. Casitone 0 0 22 59 92. Gelysate 0 5 32 47 63. Beef ex trac t 0 0 48 35 74. Peptone 1 0 44 37 85. Peptonized

m ilk 0 4 33 46 76 . Proteose

Peptone # 2 0 0 2 79 97. Protone 6 15 53 15 18. S oytone 0 1 27 55 79. Trypticase 0 3 27 56 1

10. T ryp ton e 0 0 27 56 711. Yeast ex trac t 0 1 31 47 11

3 Values represent sum o f observations fo r three concentra tions andthree replications.

Table 5 —N um ber and degree o f H agglu tina tions produced by the ten Salm onella a fte r g row th in com parison b ro ths3

G row th m edium 0 1

N um ber o f tubes Score

2 3 4

1-hr Observations

Brain Heart in fus ion 8 19 3 0 0M B roth 0 4 26 0 0H B roth Trypticase-Soy

3 7 17 3 0

T ryp tose bro th 1 14 12 3 0

2-hr ObservationsBrain heart in fus ion 0 9 18 3 0M B roth 0 1 11 15 3H B roth Trypticase-Soy

0 6 5 16 3

Tryp tose bro th 0 3 14 10 3

a Values represent sum of observations for all the organisms and threereplications.

8 2 - J O U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

o b s e r v a t i o n s o f t h e n u m b e r a n d d e g r e e o f

a g g l u t i n a t i o n s p r o d u c e d b y t h e t e n Salmonella a f t e r g r o w t h i n v a r i o u s p e p t o n e

b r o t h s a r e l i s t e d i n T a b l e 3 . P r o t e o s e p e p

t o n e # 2 y i e l d e d t h e h i g h e s t d i s t r i b u t i o n

o f r e s u l t s . C a s i t o n e a n d p e p t o n e h a d a

s i m i l a r d i s t r i b u t i o n o f r e s u l t s w h i c h w a s s i g n i f i c a n t l y g r e a t e r t h a n t h o s e o f t h e r e

m a i n i n g p e p t o n e s . P r o t o n e y i e l d e d t h e l e a s t n u m b e r a n d d e g r e e o f p o s i t i v e a g

g l u t i n a t i o n s .

P r o t e o s e p e p t o n e # 2 a l s o h a d t h e h i g h e s t d i s t r i b u t i o n o f r e s u l t s a t t h e 2 - h r o b

s e r v a t i o n t i m e ( T a b l e 4 ) . C a s i t o n e , t r y p t o n e , s o y t o n e a n d y e a s t e x t r a c t a l l h a d a s i m i l a r d i s t r i b u t i o n o f r e s u l t s w h i c h

w a s s i g n i f i c a n t l y d i f f e r e n t ( 5 % l e v e l ) t h a n t h a t o f p r o t e o s e p e p t o n e # 2 . P r o t o n e a g a i n y i e l d e d t h e l o w e s t n u m b e r a n d d e g r e e o f a g g l u t i n a t i o n s .

T h e n u m b e r a n d d e g r e e o f a g g l u t i n a t i o n o b s e r v e d a t 1 a n d 2 h r f o r e a c h c o m p a r i s o n b r o t h i n t h r e e r e p l i c a t i o n s a r e

l i s t e d i n T a b l e 5 . T h e r e w e r e t e n t e s t s p e r f o r m e d f o r e a c h b r o t h i n e a c h o f t h e t h r e e r e p l i c a t i o n s . T h e t o t a l p l a t e c o u n t s w e r e o b t a i n e d f r o m t h e s e c o m p a r i s o n b r o t h s ( a p p r o x i m a t e l y 1 0 9 p e r m l ) . O b

s e r v a t i o n s a t 1 h r s h o w e d n o v i s i b l e r e a c t i o n i n 1 2 o f t h e c o m p a r i s o n b r o t h t u b e s .

M b r o t h y i e l d e d t h e h i g h e s t n u m b e r o f

p o s i t i v e r e a c t i o n s a f t e r 1 h r . O b s e r v a t i o n s a t 2 h r s h o w e d a l l p o s i t i v e r e a c t i o n s i n a l l t h e c o m p a r i s o n b r o t h s . A f t e r 2 h r , t h e r e

w a s l e s s d i f f e r e n c e i n t h e r e s u l t s o f M , H

a n d T S T b r o t h s . B H I h a d t h e l o w e s t d i s t r i b u t i o n o f r e s u l t s .

T h e e l e v e n p e p t o n e s w e r e t e s t e d i n a

b a s a l m e d i u m c o n t a i n i n g g l u c o s e a s t h e

c a r b o n s o u r c e . P r e v i o u s w o r k h a s s h o w n

t h a t d u l c i t o l i n t h e g r o w t h b r o t h e n

h a n c e s t h e H a g g l u t i n a t i o n t e s t m o r e t h a n g l u c o s e . P e r h a p s a c o m b i n a t i o n o f p r o t e o s e p e p t o n e # 2 a n d d u l c i t o l i n t h e

g r o w t h b r o t h w o u l d h a v e a n a d d i t i v e e f

f e c t o n t h e H a g g l u t i n a t i o n t e s t .

REFERENCESBanwart, G.J. and Kreitzer, M.J. 1972. Effects

of carbohydrates in the growth medium on the agglutination of several species of Salmonella with polyvalent H antiserum. Appl. Microbiol. 23: 62.

Ewing, W.H. and Davis, B.R. 1970. Media and tests for the differentiation of Enterobac- teriaceae, p. 18. National Communicable Disease Center, Public Health Service. Atlanta, Ga.

Gabriel, K.R. 1966. Simultaneous test procedures for multiple comparisons on categorical data. J.A.S.A. 61: 1081.

Hajna, A.A. and Damon, S.R. 1950. Polyvalent Salmonella “H” agglutination as a rapid screening test for Salmonella organisms. Public Health Repts. 65: 116.

Poelma, P.L. and Romero, A. 1971. Media to improve flagellar development for Salmonella flagellar (H) test. J. Assoc. Off. Ag. Chem. 54: 745.

Sperber, W.H. and Deibel, R.H. 1969. Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures and serological reactions. Appl. Microbiol. 17: 533.


B A S A N T K . DI/VI V E D I a n d J O H N E . K I N S E L L A

D e p t , o f F o o d S c ie n c e , C o r n e l l U n iv e r s i ty , I th a c a , N Y 1 4 8 5 0

CARBONYL PRODUCTION FROM LIPOLYZED MILK FAT BY THE CONTINUOUS MYCELIAL CULTURE OF Pénicillium roqueforti

A 2 - l i t e r w i d e m o u t h r e a g e n t b o t t l e w i t h a n o u t l e t a t h a l f - h e i g h t w a s u s e d a s t h e f e r m e n t a

t i o n v e s s e l ( F i g . 1 ) . A l l g l a s s w a r e a n d t u b i n g w e r e a u t o c l a v e d f o r 1 h r a t 1 2 1 ° C a n d c o n n e c t e d a s e p t i c a l l y a f t e r c o o l i n g . T h e P. r o q u e f o r t i m y c e l i u m w a s g r o w n c o n t i n u o u s l y o n a

s t e r i l i z e d c o r n - s t e e p l i q u o r ( l g / 1 0 0 m l , 5 2 % t o t a l s o l i d s , C P C , I n t e r n a t i o n a l ) a n d s u c r o s e ( l g / 1 0 0 m l ) m e d i u m a t 2 5 ° C , p H 4 - 5 . 5 , a e r a t i o n r a t e 1 l i t e r a i r / m i n / l i t e r m e d i u m . T h e d i l u t i o n r a t e w a s a t 0 . 0 5 h r ' 1 t h u s p r o v i d i n g a d o u b l i n g t i m e o f 2 0 h r f o r m o l d g r o w t h . A f t e r 4 d a y s o f c o n t i n u o u s g r o w t h , m o l d m y c e l i u m w a s c o l l e c t e d in a s t e r i l i z e d f l a s k a n d u s e d f o r c a r b o n y l p r o d u c t i o n . D i r e c t m i c r o s c o p i c e x a m i n a t i o n r e v e a l e d n o s p o r u l a t i o n i n m o l d c u l t u r e a n d c o n s i s t e d o f o n l y m o l d m y c e l i u m ( F i g . 2 ) .

B u f f e r e d l i p o l y z e d c r e a m u s e d f o r c a r b o n y l p r o d u c t i o n w a s p r e p a r e d b y a d d i n g 5 g S t e a p s i n ( N u t r i t i o n a l B i o c h e m . C o r p . ) / 1 0 0 0 m l h e a v y c r e a m d i l u t e d t o 1 6 % f a t c o n t e n t . T h e c o n t e n t s w e r e t h o r o u g h l y m i x e d a n d l e f t a t 2 5 ° C f o r 5 d a y s . A p p r o x i m a t e l y 8 0 % o f t r i g l y c e r i d e s w e r e h y d r o l y z e d .

F r e s h o r 2 4 h r a g e d P. r o q u e f o r t i m y c e l i u m w a s i n c u b a t e d in b u f f e r e d ( 0 .1 M p h o s p h a t e ) l i p o l y z e d c r e a m a t 2 5 ° C w i t h s h a k i n g . T h e i n i t i a l f a t t y a c i d c o n t e n t , p H o f f e r m e n t a t i o n a n d

t h e c o n c e n t r a t i o n o f m y c e l i u m ( d r y w t ) w e r e

2 0 - 8 0 / u m o l e / m l , 6 . 8 a n d 1 .5 m g / m l r e s p e c t i v e

ly . T h e e f f e c t o f N a C l o n t h e p r o d u c t i o n o f m e t h y l k e t o n e s b y P. r o q u e f o r t i m y c e l i u m w a s

s t u d i e d b y a d d i n g 2 % N a C l t o b u f f e r e d l i p o l y z e d c r e a m a n d m o l d m y c e l i u m s o l u t i o n j u s t b e f o r e i n c u b a t i o n .

T h e f r e e - f a t t y a c i d ( F F A ) c o n c e n t r a t i o n s o f f e r m e n t e d s a m p l e s a n d l i p o l y z e d c r e a m w e r e d e t e r m i n e d b y a c o l o r i m e t r i c m e t h o d ( A n d e r s o n a n d M c C a r t y , 1 9 7 2 ) . T h e f a t t y a c i d c o m p o s i t i o n w a s a n a l y z e d a s b u t y l e s t e r s b y g a s - l i q u i d

c h r o m a t o g r a p h y ( G L C ) u s i n g f l a m e i o n i z a t i o n d e t e c t o r u n d e r t h e f o l l o w i n g c o n d i t i o n s :

C o l u m n : 5 f t g l a s s , p a c k e d w i t h 1 5 % D E G S o n G C h r o m P 8 0 / 1 0 0

C o l u m n t e m p : I s o t h e r m a l 7 0 ° C f o r 1 0 m i n t h e n p r o g r a m m e d t o 2 1 0 ° C a t 6 ° / m i n

I n j e c t i o n p o r t t e m p : 3 2 5 ° C , c a r r i e r g a s f l o w r a t e 4 0 m l / m i n .

T h e t o t a l c a r b o n y l c o n c e n t r a t i o n o f f e r

m e n t e d s a m p l e s w a s d e t e r m i n e d b y p r e p a r i n g2 , 4 - d i n i t r o p h e n y l ( D N P ) h y d r a z o n e s o f c a r b o n y l c o m p o u n d s b y t h e m e t h o d o f L a w r e n c e( 1 9 6 5 ) a n d m e a s u r i n g t h e o p t i c a l d e n s i t y a t 3 4 0 n m . T h e m o l a r e x t i n c t i o n c o e f f i c i e n t , E = 2 2 , 5 0 0 w a s u s e d f o r q u a n t i f i c a t i o n o f c a r b o n y l s ( S c h w a r t z e t a l . , 1 9 6 8 ) . S i n c e m e t h y l k e t o n e s a c c o u n t e d f o r m o r e t h a n 9 0 % o f t o t a l c a r b o n y l s in s o l u t i o n s , s e p a r a t i o n o f c a r b o n y l s

INTRODUCTIONPenicillium roqueforti a n d c e r t a i n o t h e r

f u n g i m e t a b o l i z e f a t t y a c i d s i n t o C 0 2 a n d c a r b o n y l c o m p o u n d s , m a i n l y m e t h y l k e t o n e s ( S t a r k l e , 1 9 2 4 ; G e h r i g a n d K n i g h t ,

1 9 6 3 ; L a w r e n c e , 1 9 6 6 ; L a w r e n c e a n d H a w k e , 1 9 6 8 ; D a r t e y a n d K i n s e l l a ,1 9 7 3 ) . T h e m e t h y l k e t o n e s o f i n t e r m e d i a t e c h a i n l e n g t h ( C 5 —C 1 3 ) m a k e a n i m p o r t a n t c o n t r i b u t i o n t o t h e “ t y p i c a l ” f l a v o r o f m o l d - r i p e n e d c h e e s e ( P a t t o n , 1 9 5 0 ; A n d e r s o n a n d D a y , 1 9 6 6 ; D a r t e y a n d K i n s e l l a , 1 9 7 1 ) . T h e o x i d a t i o n o f

f a t t y a c i d s b y s p o r e s o f P. roqueforti h a s b e e n s t u d i e d b y G e h r i g a n d K n i g h t

( 1 9 6 3 ) , L a w r e n c e ( 1 9 6 6 ) a n d D a r t e y a n d K i n s e l l a ( 1 9 7 3 ) . L a w r e n c e a n d H a w k e( 1 9 6 8 ) s t u d i e d t h e c o n v e r s i o n o f f r e e f a t t y a c i d s t o c o r r e s p o n d i n g m e t h y l k e

t o n e s w i t h o n e l e s s c a r b o n a t o m , u s i n g t h e m y c e l i u m o f P. roqueforti. T h e e f f e c t

o f f a t t y a c i d s u p o n o x y g e n u p t a k e b y a g i v e n w e i g h t o f m y c e l i u m , a n d t h e n a t u r e

o f t h e p r o d u c t s o f o x i d a t i o n , w e r e d e p e n d e n t o n t h e c o n c e n t r a t i o n a n d c h a i n l e n g t h o f t h e s u b s t r a t e f a t t y a c i d a n d p H v a l u e o f t h e m e d i u m . T h e C 9 —C 12 f a t t y

a c i d s w e r e m o r e t o x i c t h a n s h o r t c h a i n a c i d s . T h e t o x i c e f f e c t a s s o c i a t e d w i t h

s o m e f a t t y a c i d s w a s l e s s p r o n o u n c e d w h e n m y c e l i u m w a s s h a k e n i n p h o s p h a t e b u f f e r o v e r a n e x t e n d e d p e r i o d ( L a w

r e n c e a n d H a w k e , 1 9 6 8 ) . I t w a s s u g g e s t e d t h a t t h e c e l l u l a r r e g u l a t i o n o f f a t t y a c i d o x i d a t i o n a n d m e t h y l k e t o n e f o r m a t i o n i n v o l v e s d e a c y l a t i o n o f j 3 - o x o a c y l t h i o - e s t e r w h i c h p r o v i d e s a n a l t e r n a t i v e m e a n s

o f r e c y c l i n g c o e n z y m e A , w h e n o x i d a t i o n o f a c e t y l - C o A i s i m p a i r e d ( L a w r e n c e a n d H a w k e , 1 9 6 8 ) . W e h a v e i n v e s t i g a t e d t h e e f f e c t o f v a r i o u s f a c t o r s , i . e . , f r e e f a t t y a c i d c o n c e n t r a t i o n , a g e o f m y c e l i u m , s a l t c o n c e n t r a t i o n , i n i t i a l p H o f g r o w t h m e d i u m o n t h e c o n v e r s i o n o f f a t t y a c i d s f r o m l i p o l y z e d m i l k f a t t o m e t h y l k e t o n e s . B a s e d o n t h i s s t u d y , a c o n t i n u o u s p r o c e s s f o r t h e p r o d u c t i o n o f B l u e - t y p e c h e e s e f l a v o r w a s d e v e l o p e d ( D w i v e d i a n d K i n

s e l l a , 1 9 7 4 ) .

EXPERIMENTALT H E I N O C U L U M o f P. r o q u e f o r t i m y c e l i u m f o r t h e c o n t i n u o u s c u l t i v a t i o n o f m o l d w a s

p r e p a r e d b y i n o c u l a t i n g 5 0 0 m l s t e r i l i z e d ( 1 2 1 ° C / 1 5 m i n ) m a l t m e d i u m ( J a c k s o n a n d H u s s o n g , 1 9 5 8 ) w i t h a b o u t 2 0 0 m g P. r o q u e f o r t i s p o r e p o w d e r ( M i d w e s t B l u e M o ld C o . , S t i l l w a t e r , M i n n . ) . T h e m e d i u m w a s i n c u b a t e d a t 2 5 ° C f o r 4 d a y s w i t h s t i r r i n g .

1, Growth medium vessel.2. Culture vessel,

Fig. 1—F l o w d ia g r a m o f t h e c o n t i n u o u s p r o c e s s u s e d in t h e p r o d u c t i o n

o f P e n ic i l l iu m r o q u e f o r t i m y c e l iu m .

Volume 3 9 ( 1974)—JO U RN AL OF FOOD SCIENCE—S3

8 4 - J O U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

i n t o c l a s s e s b y c o l u m n c h r o m a t o g r a p h y w a s u n n e c e s s a r y . T h e t o t a l D N P h y d r a z o n e s w e r e c o n c e n t r a t e d a n d i n d i v i d u a l m e t h y l k e t o n e s s e p a r a t e d b y t h i n l a y e r c h r o m a t o g r a p h y ( S c h w a r t z

e t a l . , 1 9 6 8 ) . A s t a n d a r d m i x t u r e o f h o m o l o g o u s m e t h y l k e t o n e D N P h y d r a z o n e s ( C 3 - C , 3 )

w a s r u n c o n c u r r e n t l y f o r t h e i d e n t i f i c a t i o n o f m e t h y l k e t o n e s o n T L C p l a t e s . A f t e r d e v e l o p i n g t h e p l a t e s , s p o t s c o r r e s p o n d i n g t o C s , C 7 , C , a n d C , , m e t h y l k e t o n e D N P h y d r a z o n e s w e r e r e c o v e r e d a n d k e t o n e s s c r a p e d a n d e l u t e d w i t h t w o 3 m l v o l u m e s o f d i e t h y l e t h e r . T h e

d i e t h y l e t h e r w a s e v a p o r a t e d a n d t h e D N P h y d r a z o n e s w e r e d i s s o l v e d in n - h e x a n e a n d

t h e i r c o n c e n t r a t i o n d e t e r m i n e d f r o m t h e i r r e s p e c t i v e o p t i c a l d e n s i t y a t 3 4 0 n m ( S c h w a r t z

a n d P a r k s , 1 9 6 3 ) .

RESULTS & DISCUSSIONE f f e c t o f f r e e - f a t t y a c i d l e v e l

o n c a r b o n y l p r o d u c t i o n

T h e c a r b o n y l c o n c e n t r a t i o n s i n m e d i a

c o n t a i n i n g d i f f e r e n t l e v e l s o f f a t t y a c i d s

( 1 3 , 2 6 , 3 9 a n d 5 2 M m o l e f r e e - f a t t y a c i d / m g a g e d m o l d m y c e l i u m , d r y w t ) a t d i f f e r e n t s t a g e s o f i n c u b a t i o n a r e s h o w n i n

F i g u r e 3 . M e d i a c o n t a i n i n g 1 3 a n d 2 6 M m o l e f a t t y a c i d s / m g m y c e l i u m s t a r t e d m e t a b o l i z i n g f a t t y a c i d s i n t o c a r b o n y l c o m p o u n d s a f t e r a b r i e f l a g t i m e ( a b o u t 6

h r ) a n d m a x i m u m p r o d u c t i o n w a s a t t a i n e d i n a b o u t 2 4 h r . S a m p l e s c o n t a i n i n g 3 9 a n d 5 2 M m o l e f a t t y a c i d s / m g m y c e l i u m d i d n o t p r o d u c e a n y s i g n i f i c a n t a m o u n t o f c a r b o n y l c o m p o u n d s i n t h e f i r s t 1 2 h r . S i n c e g r e a t e r q u a n t i t i e s o f t o t a l c a r b o n y l s w e r e p r o d u c e d a f t e r 4 8 h r o f i n c u b a t i o n w i t h 3 9 a n d 5 2 M m o l e f a t t y

a c i d s / m g m y c e l i u m , i t i s a p p a r e n t t h a t t h e h i g h e r c o n c e n t r a t i o n o f f a t t y a c i d h a d o n l y t e m p o r a r y i n h i b i t o r y e f f e c t f o r t h e

e n z y m e s y s t e m m e t a b o l i z i n g f a t t y a c i d s .T h e m a j o r c a r b o n y l s p r o d u c e d b y t h e

m y c e l i u m o f P. roqueforti f r o m l i p o l y z e d c r e a m w e r e 2 - p e n t a n o n e , 2 - h e p t a n o n e , 2 - n o n a n o n e a n d 2 - u n d e c a n o n e . T h e c o n

c e n t r a t i o n o f m e t h y l k e t o n e s a t d i f f e r e n t s t a g e s o f i n c u b a t i o n a n d t h e e f f e c t o f d i f

f e r e n t f a t t y a c i d l e v e l s i s s h o w n ( T a b l e 1 ) . T h e s e d a t a a r e c o n s i s t e n t w i t h t h e p a t -

Fig. 2 —P h o to m ic r o g r a p h o f P é n ic i l l iu m r o q u e f o r t i c u l tu r e , s h o w in g p r e d o m in a n c e o f m y c e l ia l f o r m ; g r o w n o n c o r n s t e e p l i q u o r + s u c r o s e m e d i u m a t p H 5 . 8 (m a g n i f i c a t io n : 1 9 0 x ) .

Table 1—The e ffec t o f free fa t ty acid concen tra tion on m e thy l ke tone p ro d u c tio n by aged and fresh m yce lium o f P. ro q u e fo rti

M e thy l ketone concen tra tion pm ole /m g m yce lium

' im ° Fa tty acid level pm o le /m g aged m yce lium F a tty acid level pm ole /m g fresh m yce liumincuba tion M e thy l -------------------------------------------------------------------- ---------------------------------------------------------------------

(hr) ketone 13 26 39 52 13 26 39 52

6 c, Tr Tr Tr Tr Tr Tr Tr T r

c7 0.05 T r T r Tr Tr Tr Tr Tr

c, 0.08 Tr Tr T r T r Tr T r T r

c , , Tr T r T r T r Tr Tr Tr Tr12 C s 0.06 0.02 Tr Tr Tr Tr Tr Tr

C 7 0.29 0.09 T r Tr 0 .1 1 0.07 T r T r

C , 0.51 0.09 Tr Tr 0.20 0.06 Tr TrC , , 0.08 Tr Tr Tr Tr Tr Tr Tr

18 Cs 0.14 0.10 T r Tr Tr Tr Tr Trc7 0.31 0.41 0.16 0.11 0.15 0.60 0.17 TrC 9 0.53 0.45 0.19 0.10 0.30 0.59 0.19 TrC , , 0.18 Tr T r Tr Tr Tr Tr Tr

24 Cs 0.17 0.24 0.14 0.11 0.05 0.21 Tr T rC 7 0.30 0.54 0.59 0.45 0.21 0.68 0.67 T r

C , 0.51 0.84 0.97 0.43 0.46 0.96 0.60 TrC , , 0.20 0.10 Tr Tr 0.07 0.12 Tr Tr

48 Cs 0.12 0.29 0.20 0.31 0.06 0.25 0.29 0.58C 7 0.22 0.55 1.14 1.22 0.23 0.64 1.06 1.38C , 0.38 0.85 1.82 1.57 0.39 1.13 1.56 1.14C . , 0.15 0.14 0.53 Tr 0.11 0.17 T r T r

CARBONYL PRODUCTION FROM MILK F A T - 8 5

t e r n s o f t o t a l c a r b o n y l p r o d u c t i o n ( F i g

u r e 3 ) . T h e m e t h y l k e t o n e s a p p e a r e d w i t h i n 1 2 h r i n m e d i a c o n t a i n i n g 1 3 a n d

2 6 / i m o l e s f a t t y a c i d s / m g m y c e l i u m . I n m e d i a c o n t a i n i n g 3 9 a n d 5 2 j u m o l e f a t t y

a c i d s / m g m y c e l i u m , m e t h y l k e t o n e s w e r e n o t p r o d u c e d i n a n y s i g n i f i c a n t a m o u n t

u n t i l a b o u t 1 8 h r o f i n c u b a t i o n . T h e f a t t y a c i d c o n c e n t r a t i o n a l s o i n f l u e n c e d t h e r e l

a t i v e p r o p o r t i o n o f m e t h y l k e t o n e s . T h e

p r o d u c t i o n o f C 5 a n d C j i m e t h y l k e t o n e s w a s d e p e n d e n t o n t h e c o n c e n t r a t i o n o f f a t t y a c i d s a n d t h e s e m e t h y l k e -

t o n e s a p p e a r e d o n l y w h e n C g a n d C 10 f a t t y a c i d c o n c e n t r a t i o n d e c r e a s e d s u b

s t a n t i a l l y ( T a b l e s 1 a n d 2 ) . L a w r e n c e a n d

H a w k e ( 1 9 6 8 ) r e p o r t e d t h a t C9- C 12, f a t t y a c i d s s h o w e d a n i n h i b i t o r y e f f e c t o n m y c e l i u m c o m p a r e d t o C6 — C8 f a t t y

a c i d s . O u r d a t a , h o w e v e r , i n d i c a t e t h a t o c t a n o i c a n d d e c a n o i c a c i d s a r e p r e f e r e n

t i a l l y u t i l i z e d b y t h e m o l d m y c e l i u m c o m p a r e d t o h e x a n o i c a n d d o d e c a n o i c a c i d s . T h e r e l a t i v e p r o p o r t i o n o f i n d i v i d u a l m e t h y l k e t o n e s v a r i e d w i t h t h e t i m e o f i n c u b a t i o n a n d t h e f a t t y a c i d

c o n c e n t r a t i o n ( T a b l e 3 ) .

Effect of the age of mycelium on carbonyl production

L a w r e n c e a n d H a w k e ( 1 9 6 8 ) r e p o r t e d

t h a t t h e a g e a t w h i c h m y c e l i u m w a s h a r

v e s t e d m a r k e d l y a f f e c t e d t h e a b i l i t y o f m y c e l i u m t o o x i d i z e f a t t y a c i d s . O u r d a t a

s h o w t h a t b o t h f r e s h a n d a g e d m y c e l i u m w e r e e q u a l l y c a p a b l e o f f o r m i n g m e t h y l

k e t o n e s a f t e r a n i n i t i a l l a g p h a s e . H o w

e v e r , t h e i n i t i a l l a g p h a s e w a s l o n g e r w i t h f r e s h m y c e l i u m c o m p a r e d t o a g e d m y c e l i u m ( F i g . 3 a n d 4 ) .

T h e c o n c e n t r a t i o n o f m e t h y l k e t o n e s

p r o d u c e d b y m y c e l i u m a t d i f f e r e n t s t a g e s o f i n c u b a t i o n a n d t h e e f f e c t o f d i f f e r e n t

l e v e l s o f f a t t y a c i d s w a s s i m i l a r i n f r e s h

a n d a g e d ( 2 4 h r ) m y c e l i u m ( T a b l e 1 ) .

O c t a n o i c a n d d e c a n o i c a c i d s ( C 8 a n d

C 1 0 ) w e r e p r e f e r e n t i a l l y u t i l i z e d a n d c o n

v e r t e d t o m e t h y l k e t o n e s ( C 7 a n d C 9 ) ( T a b l e s 1 a n d 2 ) . F r e s h m y c e l i u m i n c u b a t e d w i t h 3 9 a n d 5 2 / i m o l e s f a t t y a c i d s / m g m y c e l i u m d i d n o t p r o d u c e s i g n i f i c a n t

a m o u n t o f C j j m e t h y l k e t o n e .

Effect of salt on carbonyl production

C a r b o n y l p r o d u c t i o n b y P. roqueforti s p o r e s i s r e p o r t e d l y e n h a n c e d b y t h e a d

d i t i o n o f s a l t t o l i p o l y z e d m i l k f a t m e d i u m ( N e l s o n , 1 9 7 0 ) . T h e a d d i t i o n o f s a l t d i d n o t s h o w s u c h e n h a n c e m e n t o f c a r b o n y l p r o d u c t i o n ; i n s t e a d a p r o l o n g e d l a g p h a s e b e f o r e c a r b o n y l p r o d u c t i o n w a s o b s e r v e d ( T a b l e 4 ) . A f t e r 4 8 - h r i n c u b a t i o n t h e c o n c e n t r a t i o n o f c a r b o n y l s i n b o t h

s a l t e d a n d u n s a l t e d s o l u t i o n s c o n t a i n i n g 2 0 / i m o l e f a t t y a c i d s / m g m o l d w e r e s i m i

l a r . T h e c o n c e n t r a t i o n o f t o t a l c a r b o n y l s i n s a l t e d m e d i a c o n t a i n i n g 4 0 / i m o l e f a t t y a c i d / m g m o l d w e r e s i g n i f i c a n t l y l o w e r

c o m p a r e d t o c o r r e s p o n d i n g u n s a l t e d m e d i a . T h i s m a y b e d u e t o t h e c o m b i n e d

Table 2—Changes in fatty acids during production of carbonyls bymycelium of P. roqueforti

FFApmole/mg

Time of incubation

Relative disappearance of specific fatty acids3 from medium

Fresh mycelium Aged myceliummycelium (hr) c f C,„ ^1 2 c. c , „ Cl 2

13 0 0.10 0.25 0.42 0.07 0.25 0.426 - 0.37 0.34 - 0.18 0.45

12 - 0.09 0.35 - 0.06 0.3918 - 0.10 0.46 - 0.02 0.2924 - 0.06 0.36 - - 0.2948 - 0.03 0.37 - - 0.29

26 0 0.10 0.29 0.36 0.08 0.29 0.366 0.10 0.29 0.40 0.07 0.28 0.31

12 0.04 0.32 0.42 0.07 0.22 0.3718 0.01 0.21 0.40 0.02 0.18 0.3824 0.01 0.06 0.38 - - -48 - 0.05 0.44 - - 0.41

39 0 0.13 0.37 0.38 0.13 0.37 0.386 - - — 0.14 0.37 0.38

12 0.16 0.38 0.43 0.13 0.28 0.3918 0.10 0.23 0.36 0.11 0.28 0.4524 0.06 0.21 0.45 0.07 0.21 0.4548 - 0.08 0.41 - 0.02 0.37

a C14:0 = 1.0

Fig. 3 - E f f e c t o f d i f fe re n t f re e - fa t ty a c id leve ls o n th e c a rb o n y l p ro d u c - F ig. 4 - E f f e c t o f d i f fe r e n t f re e - fa t ty a c id leve ls o n the c a rb o n y l p ro d u c

t io n b y aged P é n ic il l iu m r o q u e fo r t i m y c e liu m . t io n b y fre s h ly h a rve s te d P é n ic il l iu m r o q u e fo r t i m y c e liu m .

8 6 - J O U R N A L OF FOOD S C IE N C E- Vo lu m e 3 9 (1974)

Table 3—The effect of free fatty acid concentration on the ratio of methyl ketones produced by aged and fresh mycelium of P. roqueforti

Methyl ketone ratio C, = 1

Time of incubation

hrMethylketone

Fatty acid level jumole/mg fresh mycelium

13 26 39 52

Fatty acid level pmole/mg aged mycelium

13 26 39 52

6 Cs _ _ _ _ — — — —

C7 - — — — 0.62 - - -c 9 - - - - 1.00 - -

12'-'l 1Cs _ _ — _ 0.12 0.22 — —C7 0.55 1.17 - - 0.57 1.00 - -C9 1.00 1.00 - — 1.00 1.00 - -C , , - — - - 0.16 - - -

18 C5 - - — — 0.26 0.22 - -c 7 0.50 1.02 0.90 - 0.58 0.91 0.85 1.10C, 1.00 1.00 1.00 - 1.00 1.00 1.00 1.00CM - - - - 0.34 - - -

24 c 5 0.11 0.22 — — 0.33 0.29 0.14 0.26C 7 0.46 0.71 1.12 - 0.59 0.64 0.61 1.00c 9 1.00 1.00 1.00 - 1.00 1.00 1.00 1.00Cm 0.15 0.12 - — 0.39 0.24 - -

48 C5 0.15 0.22 0.20 0.51 0.32 0.34 0.11 0.20C7 0.59 0.57 0.68 1.21 0.58 0.65 0.63 0.78c 9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00Cm 0.28 0.15 - - 0.40 0.16 0.29 -

e f f e c t o f s a l t a n d h i g h e r f a t t y a c i d c o n

c e n t r a t i o n . T h e r e l a t i v e c o n c e n t r a t i o n s o f m a j o r m e t h y l k e t o n e s i n s a l t e d a n d u n

s a l t e d s o l u t i o n s w a s n o t s i g n i f i c a n t l y d i f

f e r e n t ( T a b l e 5 ) .

Effect of initial pH of growth medium on carbonyl production

L a w r e n c e a n d H a w k e ( 1 9 6 8 ) r e p o r t e d

t h a t t h e P. roqueforti m y c e l i u m g r o w n a t h i g h e r p H ( 6 . 5 ) m e t a b o l i z e d f a t t y a c i d s

m u c h f a s t e r c o m p a r e d t o m y c e l i u m g r o w n a t l o w e r p H ( 4 . 0 ) . O u r s t u d y s h o w e d a s i m i l a r e f f e c t ; h o w e v e r , t h i s e f

f e c t w a s t e m p o r a r y ( T a b l e 4 ) . T h e r e l a t i v e p r o p o r t i o n o f t h e m a j o r m e t h y l k e

t o n e s w a s s i m i l a r a t a l l p H v a l u e s ( 4 . 0 ,

5 . 8 , 7 . 4 ) ( T a b l e 5 ) .T h e p H o f m o l d m y c e l i u m p l u s l i p o -

l y z e d c r e a m m e d i u m w h e n k e p t b e t w e e n6 — 7 d i d n o t s h o w a n y s i g n i f i c a n t d i f f e r e n c e i n c a r b o n y l p r o d u c t i o n .

Metabolism of free fatty acidsD a t a i n T a b l e 2 s h o w t h a t s h o r t c h a i n

f a t t y a c i d s w e r e p r e f e r e n t i a l l y m e t a b o

l i z e d c o m p a r e d t o l o n g c h a i n a c i d s . C a r b o n y l p r o d u c t i o n w a s m i n i m a l i n i n i t i a l

s t a g e s o f i n c u b a t i o n ( F i g . 3 a n d 4 ) a n d i n c r e a s e d r a p i d l y a f t e r s o m e t i m e . T h e s e

d a t a s h o w t h a t t h e d i s a p p e a r a n c e o f

m e d i u m c h a i n f a t t y a c i d s ( C 6 —C ! 2 ) i s r e l a t e d t o a p p e a r a n c e o f c a r b o n y l c o m

p o u n d s . F r e s h m y c e l i u m s h o w a l e n g t h y l a g p h a s e c o m p a r e d t o a g e d m y c e l i u m

( F i g . 3 a n d 4 ) . S i m i l a r l y , f r e s h m y c e l i u m m e t a b o l i z e d f r e e f a t t y a c i d s s l o w l y i n i n i

t i a l s t a g e s o f i n c u b a t i o n c o m p a r e d t o

a g e d m y c e l i u m ( T a b l e 2 ) .T h e c a p a b i l i t y o f P. roqueforti m y c e l i

u m t o p r o d u c e m e t h y l k e t o n e s f r o m f r e e

f a t t y a c i d s i s o f g r e a t p r a c t i c a l s i g n i f i

c a n c e . P r e s e n t s t u d y i n d i c a t e s t h a t t h e m e t h y l k e t o n e s p r o d u c e d b y P. roqueforti m y c e l i u m i n s u b m e r g e d f e r m e n t a t i o n a r e s i m i l a r t o o n e s f o u n d i n B l u e c h e e s e ( A n d e r s o n a n d D a y , 1 9 6 6 ; D a r t e y a n d K i n s e l l a , 1 9 7 1 ) . B a s e d o n t h i s f i n d i n g a t w o - s t a g e c o n t i n u o u s m e t h o d f o r t h e

p r o d u c t i o n o f B l u e c h e e s e f l a v o r b y s u b m e r g e d f e r m e n t a t i o n o f P. roqueforti w a s d e v e l o p e d ( D w i v e d i a n d K i n s e l l a , 1 9 7 4 ) . T h e v a r i a b i l i t y i n t h e r e l a t i v e p r o p o r t i o n c f i n d i v i d u a l m e t h y l k e t o n e s b y c h a n g i n g t h e s u b s t r a t e c o n c e n t r a t i o n ( T a b l e 3 ) m a y b e h e l p f u l i n m a i n t a i n i n g a d e s i r a b l e f l a v o r b a l a n c e i n t h e f e r m e n t e d B l u e c h e e s e f l a v o r p r o d u c t . M i n o r d i f f e r e n c e s i n t h e o b s e r v a t i o n s o f L a w r e n c e a n d H a w k e ( 1 9 6 8 ) a n d t h i s s t u d y m a y b e d u e t o v a r i a b l e s t a t e s o f m y c e l i u m a n d f a t t y

a c i d s u b s t r a t e s . L a w r e n c e a n d H a w k e

( 1 9 6 8 ) u s e d w a s h e d m y c e l i u m a n d p u r e f a t t y a c i d s , w h e r e a s t h i s s t u d y w a s a i m e d

Table 4—The effect of sodium chloride and initial pH of medium used on the production of carbonyl by P. roqueforti mycelium

s.no.

Initial pH of medium

Salted/unsalted

Fatty acid cone /imole/mg mycelium

Total carbonyls, pmole/mg mycelium

Incubation period

6 12 18 24 48

1 4.0 unsalted 20 0.33 0.87 2.14 2.53 2.942 5.8 unsalted 20 0.41 1.40 1.86 2.06 2.333 7.4 unsalted 20 0.24 1.67 2.26 2.33 2.664 7.4 unsalted 40 0.27 0.65 2.06 2.53 3.535 4.0 salted11 20 0.27 0.29 0.41 0.73 2.946 5.8 salted 20 0.24 0.39 1.07 1.80 2.667 7.4 salted 20 0.18 0.30 0.73 1.52 2.948 7.4 salted 40 0.24 0.26 0.30 0.40 1.73

a Concentration = 2.0%

CARBONYL PRODUCTION FROM MILK F A T - 87

Table 5—The effect of salt, free fatty acid level and initial pH of growth medium on methyl ketone production at pH 6.8

Methyl ketone concentration /Limole/mg mycelium Initial pH of growth medium

Time of 4 5.8 7.4 7.4incubation Methyl I FFA 20 jumole/mg mold FFA 20 Mmole/mg mold FFA 20 ^mole/mg mold FFA 40 /imole/mg mold

(hr) ketone Salted Unsalted Salted Unsalted Salted Unsalted Salted Unsalted

6 Cs »C7C9 ,Cj i Tr Tr Tr Tr Tr Tr Tr Tr

12 Cs Tr Tr Tr Tr Tr Tr Tr Trc 7 Tr 0.24 Tr 0.39 Tr 0.63 Tr 0.24C, Tr 0.31 Tr 0.67 Tr 1.07 Tr 0.15c , , Tr Tr Tr Tr Tr Tr Tr Tr

18 Cs Tr 0.14 0.04 Tr Tr 0.10 Tr 0.14Cs Tr 0.63 0.34 0.51 0.22 0.52 Tr 0.87C, Tr 0.96 0.33 0.87 0.24 0.92 Tr 0.64C,, Tr Tr Tr Tr Tr Tr Tr Tr

24 Cs 0.04 0.25 0.16 0.12 0.12 0.18 Tr 0.34c? 0.19 0.62 0.44 0.61 0.35 0.54 Tr 0.87C, 0.24 1.10 0.82 1.02 0.72 1.15 Tr 1.01C ,, Tr 0.33 Tr 0.24 Tr 0.18 Tr Tr

48 Cs 0.50 0.38 0.34 0.18 0.31 0.28 0.31 0.47Cs 0.61 0.52 0.58 0.48 0.53 0.40 0.70 1.18C, 1.18 1.08 1.11 0.98 1.26 0.94 0.49 1.63C ,, 0.30 0.40 0.28 0.22 0.33 0.32 Tr Tr

a t a p r a c t i c a l a p p r o a c h t o p r o d u c i n g c a r b o n y l c o m p o u n d s f o u n d i n B l u e c h e e s e

f l a v o r b y u s i n g l i p o l y z e d c r e a m a n d P. roqueforti m y c e l i a l c u l t u r e a s s u c h w i t h o u t w a s h i n g .

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2 8 9 .

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D e p t , o f F o o d S c ie n c e & N u t r i t i o n , T h e O h io S t a t e U n iv e r s i t y , C o lu m b u s , O H 4 3 2 1 0

ACTIVITY AND STABILITY OF /3-GALACTOSIDASE IMMOBILIZED ON POROUS GLASS

INTRODUCTION

THE POTENTIAL application of immobilized |3-galactosidase to the hydrolysis of lactose in milk and whey has been evident to numerous investigators.

Sharp et al. (1969) were the first to report the attachment of /3-galactosidase (GAS) to a support, being successful in attaching the enzyme to a porous plastic sheet. Bunting and Laidler (1972), as well as Dahlqvist et al. (1973), entrapped /3- galactosidase in polyacrylamide. Olson and Stanley (1973) attached the enzyme to phenol-formaldehyde resins with glutaraldehyde. Woychik and Wondolowski(1972) immobilized the /3-galactosidase on porous glass.

Most investigators have found the immobilized enzyme to be stable in storage and in operation against lactose solutions. Sharp et al. (1969) found /3-galactosidase activity in enzyme bound DEAE cellulose sheets after three years of storage. Olson and Stanley (1973) reported operating columns of the enzyme for periods up to 6 weeks on lactose with no apparent activity loss. However, when using reconstituted nonfat dry milk they found a 10% loss in activity after 17 hr of column operation. Dahlqvist et al. (1973) indicated that some factor(s) present in skimmilk interfere somewhat with the enzyme catalyzed reaction, but that cysteine and serum albumin were activators of the /3-galactosidase immobilized on PAG.

Woychik and Wondolowski (1972) presented the most complete report to date on the competitive inhibition of /3-galactosidase bound to glass by the product galactose.

All of the above reports have been published after the initiation of these studies, with the exception of the reported attachment by Sharp et al. (1969). In addition a number of additional investigations are known to be in progress. This study was undertaken to prepare insoluble enzyme derivatives of (3-galactosidase coupled to porous glass and to define problems encountered in their application to the hydrolysis of lactose in milk related systems.

MATERIALS & METHODS

(5-GALACTOS1DASE (EC 3 .2 .1 .23) was d o n a ted by Miles L ab ora to ries, E lk h a rt, Ind . and D airy land F o o d L abora to ries, W aukesha, Wise.

T he enzym e was b o und through azo-linkages to an ary lam ine derivative o f Z r 0 2 p o ro u s glass. T he glass derivative (po re size 5 5 0 A , 4 0 / 8 0 m esh) was p rovided by C orning Glass Works, C orn ing N.Y.

A nalytical grade reagents and double-distilled w ate r w ere used th ro u g h o u t this w ork. O ther m aterials used w ere: (3-D-glucose, D- galactose, o -n itrophenyl-|3 -D -galac topyranoside (O N PG ), C albiochem ; g lucosta t, g a lac tosta t, W orth ing ton B iochem ical C orp .; lactose, M atheson C olem an and Bell; o -n itro p h en o l, E astm an K odak Co.; and all o th er chem icals, J .T . Baker C hem ical Co.

All lactose so lu tions w ere expressed as percen t lactose (w /v) and con ta in ed 0.05M lactic acid. T he desired pH was ob ta ined by the additio n o f KOH.

C heddar cheese w hey w as ob ta in ed from T he O hio S ta te U niversity Dairy P lant. Whey sam ples w ere h ea ted to 100°C, acidified to pH4.5 w ith lactic acid and allow ed to cool. Coagula ted p ro te in s w ere th en rem oved by filtra tio n th rough W hatm an N o. 1 filte r paper w ith added C elite. Whey was then acid ified to pH 3.5 and

th e lactose co n cen tra tio n was s ta ndard ized to 5 % using a po larim e tric m e th o d (AOAC 1 6 .0 4 9 -1 6 .0 5 0 ) . A nalysis o f the w hey show ed th a t it con ta ined 0.42% galactose and galactosam ine and less th an 0.005% glucose.

B acterial co u n ts w ere d e te rm in ed by the standard p o u r p la te m eth o d . Y east and m old co un ts w ere d e term ined for all co lu m n assays co n d u c ted fo r longer th an 24 hr. Using a su itable d ilu tion o f the feed and p ro d u c t, p la tes w ere in cu b a ted 48 h r using M-P H m edium (B altim ore Biological L ab .), and 72 hr using P o ta to D extrose Agar (D ifco L ab.).

D iazo tization and azo couplingUsing U .S. S tan d ard Sieves, the p o ro u s glass

derivative was sieved to elim inate partic les o th e r th an 4 0 /8 0 m esh. P rior to d iazo tiza tio n the glass derivative was d ried overnight in a desiccator. T he d iazo tiza tio n and coupling p ro cedures as described in B i o m a t e r ia l S u p p o r t s (C orning, 1973) were fo llow ed w ith m od ifications in reac tan t co n cen tra tio n s and evacua tion p rocedures. 5g o f ary lam ine glass w ere added to a trypsin izing flask con ta in ing 150 m i o f 0 .7N

COLUMN REACTOR COOLER DEBUBBLER

BOUNDENZYME

FEED PUMD PRE-HEATERTANK

F ig. 1 —A p p a r a tu s f o r lo n g - t e r m s t a b i l i t y a n d r a te e x p e r im e n t s .

88—JOU RNA L OF FOOD SCIE NC E- Vo lum e 3 9 (1974)

f i -GALACTOS!DASE IMMOBILIZA T IO N -8 9

HC1 and 0.35g o f N aN O a . T he flask, partia lly subm erged in an ice b a th , w as connected to a ro ta ry evapora to r, ro ta te d , and a fte r 5 m in a vacuum was draw n. A fte r 30 m in th e d iazo tized glass was filter-w ashed w ith cold 3% sulfam ic acid and cold distilled w ater, lg o f (3-galactcsi- dase was dissolved in 150 m l o f pH 5.5 citra te- phospha te buffer con tain ing 0 .0 1M 2-m ercapto- e th an o l and the so lu tion was added to the glass derivative con ta ined in a refrigera ted beaker. T he m ix tu re was stirred from overhead fo r 2 hr. The b ound su p p o rt was then filter-w ashed w ith fresh buffer. 100 m l o f 0.1% g lu tara ldehyde was added to the b o u n d su p p o rt con ta ined in a beaker and stirred from overhead fo r 15 min.

T he b o u n d su p p o rt was n ex t filter-w ashed in o rder w ith pH 3.5 glycine-HCl b u ffe r, 100 ml o f 2M KC1 and pH 3.5 glycine-H Cl buffer. The m oist im m obilized enzym e (IM E) was divided in to sub lo ts , w eighted and sto red in buffer a t 4°C . O ne su b lo t was desiccator dried to o b ta in a d ry /w e t conversion.

T he binding reaction was fo llow ed by analyzing fo r lactase activ ity in a liquo ts w ithdraw n at given tim e intervals. S tirring was sto p p ed , the glass w as allow ed to se ttle , a Vi-ml sam ple o f su p e rn a tan t was w ithd raw n and the stirred reactio n w as then allow ed to con tin u e . Assays fo r lactase activ ity w ere also co n d u c ted on the initial b inding so lu tion , final binding so lu tion and all wash so lu tions.

Prior to residual lactase assay g lu tara ldehyde and KC1 wash so lu tions w ere d ialyzed fo r 24 h r in tw o changes o f pH 3.5 buffer.

A ctiv ity assaysThe chrom ogen o-nitrophenyl-/3-D-galacto-

side (ONPG) was used for determ in ing lactase activ ity during the im m obiliza tion p rocedure by a m o d ifica tion o f the m e th o d used by Wen- d o rff and A m undson (1 9 7 1 ). A 0.5 m l a liquo t o f su itably d ilu ted prew arm ed enzym e so lu tion was blow n in to 2.5 m l o f 0.3% ONPG in 0.05M G ly-H C l-buffer (pH 3.5) equ ilib ra ted to 40°C in test tubes con ta ined in a w ater b a th . A t 15 m in, 2.0 m l o f 1M K 2C 0 3 was ad ded to stop the reaction . T he absorbance , co rrec ted fo r a substra te b lank, w as d eterm ined a t 415 nm . A u n it o f activ ity was defined as the hydro lysis o f 1 pM o f ONPG per m in a t the given assay conditions. A standard curve w as estab lished by dissolving various co n cen tra tio n s o f o -n itropheno l in the assay solu tion .Sugar de te rm in a tio n

Lactose hydro lysis was d e term ined by m easuring glucose p ro d u c tio n . G lu co sta t was used to m easure glucose, and w hen applicable galacto- s ta t was used to m easure th e initia l galactose co n cen tra tio n in w hey. D ep ro te in iza tion o f w hey sam ples for glucose or galactose d e te rm inations was done by the usual Som ogyi tech nique as described by W orth ing ton B iochem ical C orp. (1971).R eactor

T he co n tin u o u s assay app ara tu s show n in Figure 1 was designed to o pera te at tem perature con tro lled flow ra tes up to 25 m l/m in . The feed so lu tion was k e p t a t 0°C to co n tro l grow th o f m icroorganism s and p reven t decom position . A p erista ltic pu m p prov ided a steady flow o f substra te to the p reh ea te r , w here the so lu tion was h eated to 50°C and passed through a jacketed colum n con tain ing a fixed bed o f glass beads to elim inate dissolved gas. T he substra te was then cooled to 40°C and passed th rough th e colum n reacto r. T he reac to r , in w hich the enzym atically active su p p o rt was used , con sisted o f a 25 c m x 0.9 cm i.d . jack e ted glass

Table 1—Units of residual enzyme activity in various solutions resulting from the immobilization process

Solution Activity units

Rate aliquots 86Residual in binding 1395Glutaraldehyde + wash 10KCI + wash 450Buffer wash + 18

1959

co lum n. T he im m obilized enzym e (IM E) was supp o rted in the above colum n by a 200 mesh stainless steel screen. T he IME co lum n jack e t tem p era tu re was co n tro lled a t 40°C ± ,02°C an d co lum n tem p era tu re was m o n ito red by a therm ocoup le located below the enzym e bed. T he assay ap p ara tu s was m ade o f glass, connecto rs o f te flon , IME su p p o rt o f stainless steel and lines o f tygon tubing.

RESULTS

Preliminary investigationsSubsequent to preliminary work, with

characterization of both soluble enzyme preparations attention was directed to the Dairyland enzyme preparation because of its greater activity. Investigations on various soluble Dairyland enzyme (SE) and IME preparations conducted in a stirred tank reactor (STR) established the following operational parameters and guide lines:(1) Maximum enzyme activity and stabil

ity was near pH 3.5.(2) Temperatures above 40°C would

slowly inactivate the enzyme.(3) Storage stability at 0°C was in excess

of 3 months.(4) Metal ions did not significantly affect

enzymic activity.(5) One experiment indicated that the

IME preparation was completely stable in excess of 4 days during continuous column operation at 40°C.

Table 3—Effect of added galactose on reaction rate

% Added galactose

Productionratea % Inhibition

0.0 185 000.25 83 550.50 51 721.0 30 84

a Production rate = gM glucose produced/min/g IME at 25% conversion.

(6) Experiments indicated that flow rates greater than 1.6—2.0 ml/min would minimize interparticle diffusion, and a steady state column conversion was reached in less than 30 min when a buffered lactose solution served as substrate in our reactor (Fig. 1).

Enzyme bindingTwo separate stock batches of Dairy-

land enzyme (IME No. 1 and IME No. 2) were immobilized for further study.

The rate of enzyme binding was followed for both preparations, but an additional quantitation of enzyme activity lost in the various wash procedures was conducted for IME No. 2. In Figure 2 the percent activity remaining per ml of binding solution vs. time of reaction is given for IME No. 2. The reaction is relatively rapid since 50% of the enzymic activity available for binding was lost from the soluble phase of the binding solution during the first 10 min.

We attempted to quantitate the enzyme bound by determing residual enzyme activity in the various solutions resulting from the binding process. Table 1 gives units of unbound enzyme. Up to 6710 units of soluble enzyme activity could not be accounted for and this quantity of enzyme was assumed to be in the 5.151 g dry weight of IME. Therefore,

Table 2—Comparison of conversion of 5% lactose solution by SE and IME

LAU/g enzyme Comparative% Conversion SE IMEa hydrolysis ratio

5 6740 2000 3010 3710 1280 3515 2060 1040 5020 1300 915 7025 810 695 8630 630 550 8740 410 370 9050 287 247 8660 217 175 81

a Calculated on the basis of 0.1 og of enzyme/g of IME (LAU) Lactase Activity Unit = production of 1 pm glucose/min

9 0 - J O U R N A L OF FOOD S C IE N C E- Vo lu m e 3 9 (1974)

1 300 units/g dry weight IME were bound. If all of the bound enzyme was active the expected hydrolytic activity of lg dry wt IME would equal that of 0.15g SE. Weight of IME used in all subsequent experiments was on a dry weight basis.

In an attempt to obtain some basis of comparison the relative activity of the soluble enzyme (SE) and the immobilized enzyme (IME) was determined at different conversions of a 5% lactose solution. Stirred tank assay for SE and column assay for IME were carried out at 40°C. The rate was determined for the IME from the slope of the W/F plots for a given fractional conversion, and the rate of the SE was obtained from the slope of a fractional conversion vs. time of reaction times the weight of enzyme per ml plot (X vs. ET) at the given conversion. These rates were considered to be more valid than initial rates because of internal pore diffusion in the IME system at low conversions. In addition, rate comparisons at a given substrate conversion will allow some correction for lactose and galactose concentrations. Data are shown in Table 2.

The Comparative Hydrolysis Ratio, [LAU/g enzyme (IME)/LAU/g enzyme(SE)] x 100, reached a maximum at 40% conversion. Relatively minor differences in the Comparative Hydrolysis Ratio were observed between conversion rates of 25-50%.

StabilityAdditional experiments were con

ducted to determine IME stability during column operation at 40°C in an attempt to duplicate previous stability on prior IME preparations. The feed solution was 5% lactose, pH 3.5, and IME loading 0.327g. The flow rate was controlled at1.9—1.95 ml/min. Fractional conversion of the lactose substrate solution vs. time is shown in Figure 3 as Curve A. An initial activity loss was evident since conversion dropped during the first hours of operation. This experiment was terminated after 62 hr due to mold growth in the IME bed.

The assay apparatus was then autoclaved and reloaded with 0.423g of IME. The IME contained in the column was washed with sterile water and the preheater temperature increased to 60°C. The flow rate was increased to 3.1 ml/ min whereas the lactose concentration and pH remained unchanged. Conversion vs. time is shown as Curve B in Figure 3. An initial activity loss was followed by a slow activity decline until the experiment was terminated at 72 hr due to mold growth within the IME bed. The activity increases noted at 50 and 72 hr for experiment B were attributed primarily to back washing the column 1 hr prior to sample collection. Back washing of the column aided in partially breaking up mold which

F ig . 2 —L o s s o f e n z y m e a c t i v i t y f r o m s o lu b le

p h a s e o f b i n d in g s o lu t io n .

TIME (hr)

F ig . 3 —F r a c t io n a l c o n v e r s io n vs. t i m e o f c o l

u m n o p e r a t i o n a t 4 0 ° C u s in g a 5 % la c to s e f e e d s o l u t i o n p H 3 . 5 w i t h o u t a d d i t i o n o f m ic r o b ia l

in h ib i to r s .

had engulfed portions of the IME. After 50 hr the mold mass had grown to the point where back washing was of little value. The slow activity decline noted in both experiments was related to mold growth within the column. Mold would engulf the IME and thereby effectively prevent sugar transport to and from the enzyme surface.

After 20 hr of operation 5 — 15 colonies of yeast per ml were detected in the effluent of both experiments, whereas none was detected in the feed solutions. Mold was only detected in eluent, 1-2 colonies/ml, between 20 and 40 hr of column operation. It is evident that mold determination in the enzyme column was of questionable value, since after 50 hr of

F ig . 4 - E f f e c t o f i n i t ia l l a c to s e c o n c e n t r a t i o n o n f r a c t io n a l c o n v e r s io n ( X ) vs. W /F .

W / F g I M E / ( M O L E F E E D ) / ( m i n )

F ig . 5 —C o m p a r i s o n o f IM E p r e p a r a t io n s u t i l i z

in g a 5% la c to s e s o l u t i o n p H 3 . 5 a t 4 0 ° C.

column operation approximately 10% of the IME was encased by mold.

Since the addition of a mold inhibitor to this IME system would entail further evaluation and kinetic comparisons of its effect on enzyme activity, all subsequent experiments per sublot of IME were limited to less than 18 hr in duration and mold accumulation was retarded by periodic back washing of the IME column.

Substrate concentrationExperiments were conducted next us

ing IME No. 1 to study the effect of initial lactose concentration in the feed solution on fractional conversion at various flow rates. Results obtained using 2.5, 5.0 and 10.0% lactose feed solutions were ex-

ß -GALACTOSIDASE IMMOBILIZATION - 9 1

0.75 r

W/F g IME/(M0LE FEED)/(min)

F ig . 6 —E f f e c t o f i n i t ia l g a la c to s e c o n c e n t r a t io n o n f r a c t io n a l c o n v e r s io n I X ) vs. W /F .

pressed best with plots of fractional conversion plotted against the weight of the IME/molar flow rate of substrate (X vs. W/F). Results obtained are shown in Figure 4 as one curve. The individual concentration-conversion curves did not superim pose ind icating that fractional conversion was affected to a slight, but significant degree, by initial lactose concentration in the feed solution. Therefore, in all further experimentation lactose concentration was 5% or adjusted to 5%.1ME comparison

In Figure 5 fractional conversion (X) vs. W/F (g IME/mole feed/min) is shown for IME preparations No. 1 and No. 2, which were used in these studies. Since the curves superimpose both preparations contained approximately the same hydrolytic activity, the data obtained were representative of both preparations.Product inhibition

Galactose inhibition was studied by the addition of 0.25, 0.50 or 1.0% D-galactose to a 5% lactose feed solution. The same 1.725g of IME was used for the various concentrations and the data in Figure 6 was generated. Experimentally determined column production rates with added galactose at 25% substrate conversion are given in Table 3.

After this series of experiments, galactose was eliminated from the feed solution to determine if any irreversible inactivation occurred. The corresponding lactose conversion was in agreement with the initial run conducted with no galac

F ig . 7 —E f f e c t o f v a r io u s s u b s t r a te s o n fr a c

t i o n a l c o n v e r s io n ( X ) vs. t i m e o f c o l u m n o p e r a

t i o n a t 4CT C a n d p H 3 .5 .

tose in the feed solution at one chosen flow rate. The addition of .50% B-D-glucose to the feed solution did not measurably affect enzymic activity.Activity on whey

Trials were conducted to determine the conversion of lactose contained :n whey by the IME. The column reactor, loaded with 0.428g of IME, was first as-

Table 4—Column conversion of whey

Column operation Lactose converted(hr) (%)

% 38' k 29

1 2410 21.520 19.840 1572 13

Table 5—Effect of whey on lactase activity

Production % Column rateSubstrate ratea inhibition

Lactose initial 261 00Whey 179 31Lactose reassay 161 38

a Production rate = pM glucose produced/ min/g IME at 15% conversion

F ig . 8 —E f f e c t o f v a r io u s s u b s t r a t e s o n f r a c

t io n a l c o n v e r s io n I X ) vs. W /F f o r c o l u m n o p e r a

t i o n a t 40P C a n d p H 3 .5 . A H s a m p le s t o d e t e r

m i n e f r a c t io n a l c o n v e r s io n w e r e c o l l e c t e d a t s ta b l e c o n d i t io n s .

sayed at pH 3.5 and 40°C with a 5% lactose solution. This assay indicated that the column would convert 30% of the feed solution at 2.0 ml/min. The feed solution was then changed to pH 3.5 whey and the flow rate was maintained at2.0 ml/min. The column conversion was then monitored at various time intervals until the experiment was terminated after 72 hr. The column conversions are given in Table 4. This experiment was terminated after 72 hr due to continual column plugging caused by protein precipitation and mold accumulation. The IME was then thoroughly backwashed and assayed with a 5% lactose solution. An eluent sample collected after 1 hr indicated 16% substrate conversion. The IME was then thoroughly backwashed with 2M KC1 and after 1 hr analysis of the eluent revealed that lactose conversion remained unchanged.

The data in Table 4 would indicate an initial enzyme activation since column conversion increased from 30% on the initial lactose solution assay to 38% initial conversion on whey. Conversion then decreased to 13% after 72 hr of operation. This activity loss was attributed to exposure of the IME to whey and the presence of mold growth. In an attempt to determine the nature and extent of activity loss in whey free of mold growth, a trial was conducted as follows: Various lactose substrate solutions were passed through a column containing 0.393g IME for varying periods of time at a base flow rate of2.7 ml/min. Substrates in order of use and initial base flow rate times are as fol

9 2 - J O U R N A L OF FOOD SCIE NC E- Vo lum e 3 9 (1974)

lows: 5% lactose solution, 4 hr; whey 5% lactose, 5 hr; 5% lactose solution, 1 Vi hr; 5% lactose plus 0.50% galactose solution, 1 hr. During the course of each substrate assay two new flow rates were established to obtain data for W/F plots. The base flow rate was then reestablished for substrates 1 and 2. During substrate changeover the feed system was flushed with water.

Results of the above assays conducted at the base flow rate are plotted as fractional conversion vs. time of column operation in Figure 7.

IME activity decreased significantly during the initial 3 hr of exposure to whey, but activity tended to stabilize upon further exposure. Upon reassay with lactose solution, a stable, but lower, conversion was noted than initially.

By increasing the flow rates for the various substrates used in the course of this experiment we were able to construct a series of fractional conversion vs. W/F plots (Fig. 8) which were then used to construct Table 5.

DISCUSSION

DATA OBTAINED from column studies on the IME were plotted as fractional conversion (X) vs. W/F (g IME/molar flow rate). This method of data representation has been used exiensively by chemical engineers as a basis for reactor scale up. The plots of fractional conversion vs. W/F were helpful in comparing IME preparations, substrate solutions, and studying possible enzyme inactivation. They also proved beneficial during loading of IME columns, since substrate conversion could be directly related to flow rate and weight of IME used. The advent of immobilized enzymes for industrial application will eventually lead to data presentation as fractional conversion vs. W/F, since this method is of more utility.

In an attempt to explain the effectiveness of the IME at various substrate conversions, the relative activity of the SE and IME were compared at different conversions of a 5% lactose solution. The term comparative hydrolysis ratio was coined and defined as [LAU/g enzyme (IME)/LAU/g enzyme (SE)] x 100, to relate the findings of this study. The comparative hydrolysis ratio reached a maximum of 90% at 40% substrate conversions and relatively minor differences were noted between 25 — 50% substrate conversion. At 5% substrate conversion below which all initial enzyme rate measurements are made the comparative hydrolysis ratio was only 30%, indicating a possible source for error when IME kinetics are compared with SE kinetics at low substrate conversions.

The decreased flow rates necessary to obtain increased conversions of a 5% lactose solution indicates the magnitude required to drive lactose conversion to com

pletion. At 20% conversion lg of this IME would convert 7.6 ml/min; at 40% conversion, 2.2 ml/min; at 60% conversion, 0.8 ml/min; and at 80% conversion only 0.25 ml/min were converted. This decrease in reaction rate is related to lower substrate saturation of the enzyme at higher conversions but is due in large measure to the competitive inhibition of galactose one of the reaction products. Product inhibition becomes significant if the Km of lactose and the Kj of galactose obtained from the data of Woychik and Wonolowski (1972) is compared. They give an apparent KM of 0.018 molar and a back calculation of their data indicates a K[ of approximately 0.0008 molar, indicating a twentyfold apparent affinity for galactose by the active site(s) of the enzyme.

To obtain some practical and applicable data on galactose inhibition, we added galactose at various concentrations to the column feed solution. A 0.25% concentration of galactose in the feed solution produced a 55% inhibition of column lactose hydrolysis at 25% lactose conversion. The data presented can be used to determine rate inhibition at 0-1.0% feed galactose concentration and conversions up to 50% depending on initial galactose concentration. These data are significant since whey does contain various amounts of galactose. Cheddar cheese whey used in these studies contained 0.42% galactose and galactosamine.

Initial feed concentrations were found to affect W/F plots. The effect was slight and the results obtained may be due to experimental error. However, by fitting Michaelis-Menton kinetics with competitive product inhibition (Mahler and Cordes, 1971) into the integrated plug flow equation (Levenspiel, 1972), conversion was found to depend on initial substrate concentration. The application of the above equation would require a correction for diffusion.

Experiments conducted to determine IME stability during long term column operation at 40°C in an attempt to predict the possible operational life of the enzyme revealed that operational limits were microbiologically controlled. Loss of enzymic activity by the IME was related to mold growth within the enzyme bed, since mold engulfed the IME and thereby effectively prevented sugar transport to and from the enzyme surface. Attempts to operate under sanitary conditions were unsuccessful and all experiments were terminated after approximately 2—4 days due to column plugging.

The inhibitory effect of milk constituents has been reported previously for polyacrylamide bound lactase acting on milk by Dahlqvist et al. (1973) and phenol-formaldehyde resin bound lactase acting on reconstituted nonfat dried milk by Olson and Stanley (1973).

These present investigations have shown that the exposure of IME to whey will result in the partial, irreversible inhibition of j3-galactosidase activity. Under the conditions reported in one of our systems, this loss was approximately 38% and occurred during the first 3 hr of exposure. The degree of further loss could not be determined due to microbiological activity.

The reason for the irreversible loss in activity is not readily apparent. An attractive assumption would be that whey proteins were binding to the IME or on the surface of the glass beads. However, treatment with 2M KC1, which would be expected to remove adsorbed protein, did not restore the activity of the IME used in these studies. A possible explanation for this type of behavior would be that different conformational or steric restraints are imposed on the enzyme resulting from the immobilization process. These restraints being related to the relative site(s) of the enzyme involved in covalent attachment to the support and covalently linked to itself and neighboring molecules via the glutaraldehyde crosslinking. The extent of these steric restraints would then determine enzyme susceptibility to inactivation during exposure to whey.

These investigations have also shown an activity increase of immobilized lactase acting on Cheddar whey, since in one experiment approximately 32% hydrolysis inhibition occurred during exposure to w'hey as compared to 38% inhibition upon reassay with lactose solution. This activity increase becomes more significant if the competitive inhibition of the initial galactose present in whey used for these studies is considered. The addition of 0.5% galactose to the lactose reassay solution indicated that the galactose inhibition mechanism was still operative; corresponding decreased conversion was about what would have been expected by tile galactose present in whey.

An activation of soluble lactase acting on whey and skim milk was attributed to forewarming treatments and possible protein-protein interactions by Wendorff et al. (1970).

This investigation demonstrates the potential application of immobilized lactase to the hydrolysis of lactose present in whey. Many of the problems encountered in this study can be corrected by the proper choice of reaction conditions, catalyst support, reactor design, and reactor sanitation or catalyst sanitizing. Further investigation to define the partial irreversible inhibitor present in whey v/ould be desirable, but not essential, tc commercial application.

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k i n e t i c s o f / 3 -g a la c to s id a s e a t t a c h e d t o p o r

o u s c e l lu lo s e s h e e t s . B i o t e c h n o l . B io e n g . 1 1 :

3 6 3 .

W e n d o r f f , W . L . , A m u n d s o n , C . H . a n d O l s o n ,

N . F . 1 9 7 0 . T h e e f f e c t o f h e a t t r e a t m e n t o f

m i l k u p o n t h e h y d r o l y z a b i l i t y o f la c t o s e b y

t h e e n z y m e la c t a s e . J . M i l k F o o d T e c h n o l .

3 : 3 7 7 .

W e n d o r f f , W . L . a n d A m u n d s o n , C . H . 1 9 7 1 .

C h a r a c t e r i z a t i o n o f / 3 -g a la c to s id a s e f r o m S a c

c h a r o m y c e s f r a g i le s . J . M i l k F o o d T e c h n o l .

3 4 : 3 0 0 .

W o r t h i n g t o n B i o c h e m i c a l C o r p . 1 9 7 1 . “ G l u c o -

s t a t — f o r t h e E n z y m a t i c D e t e r m i n a t i o n o f

G l u c o s e . ” F r e e h o l d , N . J .

W o y c h i c k , J . A . a n d W o n d o l o w s k i , M . V . 1 9 7 2 .

C o v a l e n t b i n d i n g o f f u n g a l / 3 -g a la c to s id a s e t o

g la s s . B i o c h e m . B i o p h y s . A c t a 2 8 9 : 3 4 7 .

M s r e c e iv e d 6 / 2 8 / 7 3 ; r e v is e d 9 / 2 7 / 7 3 ; a c c e p t e d

9 / 3 0 / 7 3 .

T h i s w o r k w a s s u p p o r t e d b y N S F G r a n t G I - 3 2 1 4 1 .

T h e a u t h o r s a c k n o w l e d g e D r . A l d r i c h S y v e r -

s o n a n d M a r t i n O k o s , D e p t , o f C h e m i c a l E n g i

n e e r in g , T h e O h i o S t a t e U n i v e r s i t y , f o r t e c h n i

c a l a s s is t a n c e t h r o u g h o u t t h e c o u r s e o f t h i s

s t u d y .

C H U N G -S H I H T A N G

D e p t, o f A g r ic u l tu r a l B i o c h e m is t r y , U n iv e r s i t y o f H a w a ii, H o n o lu lu , H I 9 6 8 2 2

BENZYL ISOTHIOCYANATE AS A NATURALLY OCCURRING PAPAIN INHIBITOR

INTRODUCTION

P A PA IN (E C 3 .4 .4 .1 0 ) is one o f the m o s t w ide ly u se d p r o t e o ly t i c e n z y m e s b o th in fo o d in d u s t ry a n d in resea rch l a b o r a to r ies . I t is p re p a re d f ro m th e d r ied la tex o f t h e green p a p a y a f ru i t (C a r ic a p a p a y a L.). C o m m e rc ia l p ap a in varies in q u a l i ty ; rang ing f r o m c ru d e , d r ied la tex w i th o u t a n y f u r t h e r p u r i f ic a t io n to p r o d u c t s d e sc r ibed as “ high p u r i t y ” by the m a n u f a c tu rers . All these d i f f e re n t grades o f p a p a in e x a m in e d in th e p re sen t s tu d y c o n ta in b e n zy lg lu co s in o la te , a n a tu ra l ly o c cu r r in g th io g lu co s id e o f p a p a y a (G m e lin and Kjaer, 1 9 7 0 ; T ang , 1 9 7 1 ) . U p o n th e e n z y m a t ic h y d ro ly s is w i th th iog lucos idase (E C 3.2.3.1), b e n z y l iso t h io c y a n a te (B IT C ) is p r o m p t l y p r o d u c e d acc o rd in g to t h e re a c t io n (E t t l in g e r a n d Kjaer, 1 9 6 8 ) sh o w n in F igure 1.

Prev iously , T an g ( 1 9 7 1 ) r e p o r t e d the d e te c t io n o f free BITC in th e w a x layer as well as th e v a p o r e m a n a te d f ro m in ta c t green p a p ay a s , suggest ing th is c o m p o u n d is a n o r m a l m e ta b o l i t e in th e p a p ay a fru i t . A l th o u g h in th e p re sen t s t u d y BITC was n o t d e te c t e d in c o m m e rc ia l p ap a in sam ples , i t is co n ce ivab le t h a t e n z y m a t i c p r o d u c t io n o f B ITC co u ld be tr iggered by the c o n ta m in a t i o n o f th io g lu co s id ase c o n ta in in g m a te r ia l o r m ic ro o rg an ism s (Kjaer, 1 9 6 0 ) d u r in g th e u t i l i z a t io n o f p a p a in in t h e f o o d in d u s t ry . C e r ta in i s o th io c y a n a te s are well k n o w n re ag e n ts fo r t h e ch em ica l m o d if i c a t io n o f p ro te in m o le cules (K ön igsbe rg , 1967) . Allyl i s o th io c y a n a te m a y fo rm a n o n d is so c iab le c o m plex w i th p a p a in so t h a t th e e n z y m e co u ld n o t reac t w i th i ts a c t iv a to rs such as cy s te in e an d c y an id e (D e s re u x and F ischer , 1947) . In a d d i t i o n , i so th io c y a

n a te s a n d th e i r der iva tives have been s t u d ied as g o i tro g e n ic c o m p o u n d s ( V a n E t t e n e t al., 1 9 6 9 ) . B ITC possesses a p u n g e n t f lavor a n d is liable t o b ac te r ia l d e c o m p o s i t ion to p r o d u c e H 2 S (T an g e t al., 1972) . All these c h a rac te r is t ic s o f i s o th io c y a n a te s suggest th e n e e d fo r a b e t t e r u n d e r s ta n d in g o f th e n a tu r e o f b e n z y lg lu c o s in o la te in p ap a in p ro d u c ts . T h e p re sen t r e p o r t describes th e q u a n t i t a t iv e d e te r m in a t io n o f b e n zy lg lu c o s in o la te in selec ted c o m m e rc ia l p ap a in sam ples a n d the in h ib i to ry e f fe c t o f BITC on p a p a in ac t iv i ty .

EXPERIMENTAL

S A M P L E S o f c o m m e r c i a l p a p a i n w e r e o b t a i n e d f r o m s e v e r a l U . S . c o m p a n i e s w h i c h s u p p l y e n z y m e p r o d u c t s f o r i n d u s t r i a l a n d l a b o r a t o r y u s e s . T w i c e c r y s t a l l i z e d p a p a i n , c r u d e p a p a i n ( T y p e 1 ) , a n d N - c a r b o b e n z o x y - g l y c i n e - p - n i t r o - p h e n y l e s t e r ( C B Z - g l y - p - N P ) w e r e p u r c h a s e d

f r o m S i g m a C h e m i c a l C o . ( S t . L o u i s , M o . ) . B e n z y l i s o t h i o c y a n a t e w a s o b t a i n e d f r o m K a n d K , I n c . ( P l a i n v i e w , N . Y . ) . T h i o g l u c o s i d a s e w a s p r e p a r e d f r o m t h e s a r c o t e s t a o f f r e s h , m a t u r e p a p a y a s e e d s ( T a n g , 1 9 7 3 ) ; 5 g o f s e e d s w e r e m a n u a l l y r u p t u r e d i n 1 0 m l o f d i s t i l l e d w a t e r a n d t h e l i g h t b r o w n e x t r a c t w a s f i l t e r e d a n d u s e d d i r e c t l y a s a c r u d e p r e p a r a t i o n o f t h i o g l u c o s i d a s e .

Determination of benzylglucosinolate in commercial papain

0 . 5 - g s a m p l e s o f p o w d e r e d p a p a i n w e r e d i s

s o l v e d i n 1 5 m l o f d i s t i l l e d w a t e r , 3 5 m l o f a c e t o n e w e r e a d d e d a n d t h e m i x t u r e w a s i n

c u b a t e d i n a 6 0 ° C w a t e r b a t h f o r 1 0 m i n . T h e d e n a t u r e d p r o t e i n s w e r e r e m o v e d b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t w a s e v a p o r a t e d t o n e a r d r y n e s s i n a r o t a r y e v a p o r a t o r w i t h t e m

p e r a t u r e s b e l o w 6 5 ° C . T h e r e s i d u e w a s t h e n t a k e n i n t o 2 5 m l o f d i s t i l l e d w a t e r a n d 3 m l o f t h e f r e s h l y p r e p a r e d c r u d e t h i o g l u c o s i d a s e s o l u

t i o n w e r e a d d e d . A f t e r i n c u b a t i o n a t r o o m t e m p e r a t u r e f o r 1 h r t o a l l o w e n z y m a t i c h y d r o l y s i s o f t h e b e n z y l g l u c o s i n o l a t e , B I T C w a s e x t r a c t e d

w i t h 4 0 m l o f C H C 1 3 . T h e C H C 1 3 l a y e r w a s s e p a r a t e d a n d d r i e d o v e r a n h y d r o u s M g S 0 4 . B I T C c o n t e n t i n t h e C H C 1 3 f r a c t i o n w a s d e t e r m i n e d b y g a s c h r o m a t o g r a p h y a s p r e v i o u s l y r e p o r t e d ( T a n g , 1 9 7 1 ) . T h e a m o u n t o f b e n z y l g l u c o s i n o l a t e i n p a p a i n w a s c a l c u l a t e d a s i t s p o t a s s i u m s a l t f r o m t h a t o f B I T C b a s e d o n t h e i r r e l a t i v e m o l e c u l a r w e i g h t s .

Activation of crystalline papainB o t h t h e a c t i v a t i o n a n d t h e a s s a y o f p a p a i n

w e r e b a s e d o n t h e m e t h o d o f K i r c h a n d I g e l s t r ò m ( 1 9 6 6 ) . T h e a c t i v a t i o n m i x t u r e c o n t a i n e d 0 . 0 2 M p h o s p h a t e b u f f e r , p H 6 . 8 , 1 . 0 X I 0 " 3 M E D T A a n d 3 . 5 X 1 0 ' 4 M c y s t e i n e . T h e f i n a l p a p a i n c o n c e n t r a t i o n w a s u s u a l l y 5 . 2 X 1 0 ‘6 M . T h i s w a s d e t e r m i n e d b y t h e a b s o r b a n c e a t 2 8 0 n m , u s i n g 5 .1 x 1 0 4 M ~' a s t h e e x t i n c t i o n c o e f f i c i e n t . T h e a c t i v a t i o n w a s c a r r i e d o u t

a t r o o m t e m p e r a t u r e f o r 1 h r .

Assay of papain activityP a p a i n a c t i v i t y w a s e v a l u a t e d b y t h e i n

c r e a s e i n t h e a b s o r b a n c e a t 4 0 0 n m w i t h C B Z - g l y - p - N P a s t h e s u b s t r a t e . I n o u r s t a n d a r d

a s s a y m i x t u r e , 1 m l o f 0 . 0 6 M p h o s p h a t e b u f f e r .

F ig. 2 —E f f e c t o f th io g lu c o s id a s e o n c r u d e p a p a

in . C u rv e A , c r u d e p a p a in in t h e a b s e n c e o f th io g lu c o s id a s e ; C u r v e B , w i th b o i l e d t h i o g l u

c o s id a s e (1 0 0 ° C , 1 0 m i n ) ; C u r v e C , w i t h t h i o

g lu c o s id a s e . P a p a in a c t i v i t y w a s e v a l u a te d b y

th e in c r e a s e in t h e a b s o r b a n c e a t 4 0 0 n m as d e s c r ib e d in t h e t e x t .

Q -CH,-C-S-Glu + H20 t h l ° g lu co s ld ase ^~^>-CH 2 N=OS + Glucose + HSO^

N

01

S O j

Benzylglucosinolate BITC

F ig. 1 —B e n z y l g lu c o s in o la t e , u p o n e n z y m a t i c h y d r o ly s i s w i th th io g lu c o s id a s e (E C 3 . 2 . 3 . 1 ) p r o d u c e s B I T C (E t t l i n g e r a n d K ja e r , 1 9 6 8 ) .

9 4 -J O U R N A L O F F O O D S C IE N C E -V o lu m e 3 9 ( 1 9 7 4 )

B I T C A S A P A P A I N I N H I B I T O R -95

Table 1—A m o u n t o f benzylg lucosino la te in com m ercia l papain samples

Sample

mg B enzylg lucosino la te /g papaina-b

1 P u r i f i e d , s t a n d a r d i z e d p a p a i n ,

w h i t e p o w d e r

0.6

2 P u r i f i e d , s t a n d a r d i z e d p a p a i n ,

l i g h t c r e a m c o l o r e d p o w d e r

2.3

3 O f f - w h i t e c o l o r e d p o w d e r 6.54 C r e a m c o l o r e d p o w d e r 10.15 C r u d e p a p a i n , g r a y g r a n u l e , d r i e d

l a t e x f r o m A f r i c a

18.9

a C a l c u l a t e d f r o m t h e a m o u n t o f b e n z y l i s o t h i o c y a n a t e ; m o l w t o f p o t a s s i u m b e n z y l g ! u c o s i n o l a t e : m o i w t o f b e n z y l i s o t h i o c y a n a t e = 3 : 1 .

b A v e r a g e o f t w o e x p e r i m e n t s

p H 6 . 8 , 0 . 1 m l o f t h e a c t i v a t e d p a p a i n , 0 . 1 m l

C H 3 C N a n d 1 . 7 m l H 2 0 w e r e a d d e d t o a c u v e t t e . T h e r e a c t i o n w a s s t a r t e d b y t h e a d d i t i o n

o f 0 . 1 m l , 3 x 1 0 ' 3 M C B Z - g l y - p - N P d i s s o l v e d i n C H 3 C N . T h e a b s o r b a n c e w a s m e a s u r e d w i t h a B e c k m a n D B s p e c t r o p h o t o m e t e r a t 5 - o r 1 0 - s e c

i n t e r v a l s .F o r t h e s t u d y o f i n h i b i t i o n , B I T C w a s f i r s t

d i s s o l v e d i n C H 3 C N . T h e a s s a y m i x t u r e c o n t a i n e d 0 .1 m l a c t i v a t e d p a p a i n , 1 . 0 m l 0 . 0 6 M p h o s p h a t e b u f f e r , p H 6 . 8 , 0 . 1 m l B I T C s o l u t i o n a t v a r i o u s c o n c e n t r a t i o n s a n d 1 . 7 m l H 2 O . T h e r e a c t i o n w a s s t a r t e d b y t h e a d d i t i o n o f t h e s u b s t r a t e .

Effect of thioglucosidase on crude papain

l g o f t h e c r u d e p a p a i n ( T y p e 1 ) w a s s u s p e n d e d i n 1 m l o f 0 . 0 2 M p h o s p h a t e b u f f e r , p H6 . 8 . T h e i n s o l u b l e p o r t i o n w a s r e m o v e d b y l o w s p e e d c e n t r i f u g a t i o n a n d 0 . 1 m l o f t h e r e s u l t i n g s u p e r n a t a n t w a s i n c u b a t e d w i t h 0 . 1 m l o f e i t h e r H j O , t h i o g l u c o s i d a s e o r b o i l e d t h i o g l u c o s i d a s e s o l u t i o n . A f t e r i n c u b a t i o n f o r 4 5 m i n a t r o o m t e m p e r a t u r e , 1 m l o f H 2 O w a s a d d e d t o e a c h s a m p l e a n d f r o m w h i c h , 0 . 1 m l w a s u s e d t o m e a s u r e p a p a i n a c t i v i t y a c c o r d i n g t o t h e s t a n d a r d a s s a y m e t h o d .

Effect of BITC on unactivated papain

2 0 jul o f c r y s t a l l i n e p a p a i n ( 0 . 5 m g ) d i s s o l v e d i n 2 m l o f 0 . 0 2 M p h o s p h a t e b u f f e r , p H6 . 8 w a s t r e a t e d w i t h 0 . 6 6 p m o l e o f B I T C i n 0 . 1

Fig. 3 —S u b s t r a t e p r o t e c t i o n o f a c t i v a t e d p a p a in a g a in s t B I T C in h ib i t i o n . T h e r e a c t io n m i x t u r e c o n ta in e d 0 .0 2 M p h o s p h a t e b u f f e r , p H 6. 8, 1 .7 x 10 '1 M p a p a in , 7 . 7 7 x 1 0 's M c y s t e i n e , 6 .6 7 % a c e to n i tr i l e , w i th o r w i t h o u t B IT C . C u rv e A ,

c o n tr o l ; C u rv e B , 1 0 p i o f 3 . 3 X 7O’ 3 M o f B I T C s o lu t io n w a s a d d e d 6 0 s e c a f t e r t h e a d d i t i o n o f s u b s t r a te ( f in a l v o lu m e 3 m l ) ; C u r v e C , 1 0 p i o f t h e s a m e B I T C s o lu t io n w a s a d d e d t o t h e a s sa y s o lu t io n f o l l o w e d im m e d i a t e l y b y t h e a d d i t i o n

o f s u b s tr a te .

m l C H 3 C N f o r 1 5 m i n . A r e a c t i o n m i x t u r e w i t h o u t B I T C w a s u s e d a s a c o n t r o l . T h e s e t w o p a p a i n s o l u t i o n s w e r e d i a l y z e d s e p a r a t e l y a g a i n s t 1 l i t e r o f 0 . 0 2 M p h o s p h a t e b u f f e r p H6 . 8 c o n t a i n i n g 1 m m o l e o f E D T A f o r 2 0 m i n w i t h t h e f a s t d i a l y s i s m e t h o d . T h e p a p a i n s o l u t i o n s w e r e t h e n a c t i v a t e d b y t h e a d d i t i o n o f c r y s t e i n e t o a f i n a l c o n c e n t r a t i o n o f 1 . 1 7 x 1 0 " 5 M . T h e a c t i v a t i o n w a s c a r r i e d o u t a t r o o m t e m p e r a t u r e f o r 1 h r . A f t e r w a r d s p a p a i n a c t i v i

t i e s w e r e c o m p a r e d b e t w e e n t h e c o n t r o l a n d t h e B I T C - t r e a t e d s a m p l e s .

Fig. 4 — B I T C in h ib i t i o n o f a c t i v a te d , tw ic e r e

c r y s ta l l i z e d p a p a in a n d t h e r e s to r a t io n o f p a p a

in a c t i v i t y b y c y s te in e . A s s a y c o n d i t i o n s a re s im ila r t o t h a t in F ig. 3 . C u rv e A , c o n tr o l ;

C u rv e B , w i th 7.7 X 70 s M B I T C , 0 . 7 m l o f 3 . 5 x 10~2M c y s t e i n e w e r e a d d e d a t 100 s e c a f t e r t h e a d d i t i o n o f s u b s tr a te .


A R E C E N T R E P O R T f ro m o u r l a b o ra to ry in d ic a te d t h a t the b e n zy lg lu co s in o late c o n te n t s o f la te x in f resh g reen pa p a y a ran g ed f ro m 7 . 3 - 1 1 . 6 % on th e d ry w e igh t basis (T an g , 1973) . In th e p re sen t s tu d y , w e e x a m in e d severa l c ru d e a n d p ur i f ied c o m m e rc ia l p a p a in sam p le s a n d f o u n d t h a t t h e y all c o n ta in e d b e n zy lg lu c o s in o la te , a l t h o u g h th e q u a n t i t y varied over a w ide range (T ab le 1). B e n zy lg lu co s ino la te was a lso d e te c t e d in o n e b ra n d o f m e a t t e n d e r iz e r w h ic h c o n ta in e d p apain . BITC was n o t d e te c t e d i f th io g lu co s id ase was n o t a d d e d to these sam ples , i n d ic a t ing the absen ce o f free B ITC as well as the e n z y m e t h a t leads to B ITC p r o d u c t ion . T he c o n s i s t e n t p resence o f b e n z y l g luco s in o la te in p ap a in sam p le s suggests th a t th e p r e se n t m e t h o d s o f re f in in g papa in are u su a l ly in ca p a b le o f c o m p le te rem o v a l o f th is w a te r so lu b le c o n ta m in a n t .

U p o n the a d d i t io n o f th io g lu co s id ase to a c ru d e p a p a in so lu t io n , t h e es terase ac t iv i ty o f h y d r o ly z in g CBZ-gly-p-NP was in h ib i ted . This e f fe c t was n o t o b se rv ed if th io g lu co s id ase was h e a t - d e n a tu r e d p r io r to i ts a d d i t i o n (Fig . 2), o r i f c rys ta l l ine p ap a in was u se d in s tea d . T hese o b se rv a t ions c o u ld b es t be e x p la in e d b y th e e n z y m a t ic p r o d u c t i o n o f B ITC in c ru d e pa pain s o lu t io n . B a h a d u r a n d A t r e y a ( 1 9 6 0 ) r e p o r t e d t h a t ju ic e f r o m p a p a y a f ru i t was in h ib i to r y t o t h e p r o t e o ly t i c a c t iv i ty o f a c ru d e p a p a in p re p a r a t io n . T h e i r o b se rv a t io n is n o t su rp r is ing in v iew o f t h e re p o r t e d p re sen c e o f sm all a m o u n t s o f B IT C a n d th io g lu c o s id ase in r ip e p a p a y a (T ang , 1971) . T h e in h ib i to r y e f fe c t o f BITC in p a p a y a l a te x c o u ld also pa r tia l ly a c c o u n t f o r the loss o f p a p a in ac t iv i ty d u r in g its ha rves t an d s to rag e (H inke l , 1951) , a l th o u g h f u r t h e r s t u d y is n e ed e d to ver ify th is poss ib i l i ty .

T h e B ITC in h ib i t io n o f p apain co u ld be p r e v e n te d b y th e p resence o f su b s t ra te , t h a t is, i f CBZ-gly-p-NP was a d d ed to t h e e n z y m e p r io r t o th e a d d i t io n o f BITC, p a p a in a c t iv i ty was o n ly slightly


Fig. 5 —A L in e w e a v e r - B u r k p l o t o f t h e h y d r o ly s i s o f C B Z -g ly -p -N P b y

p a p a in w i th o r w i t h o u t B IT C . [ E ] = 1 .1 5 X 1 0 6 M ; [ B IT C ] - 4 X

1 0 '5 M ; [ S ] ra n g es f r o m 3 X 1 0 s to 1 X 7 0 ' 1 /W. T h e K i c a lc u la te d f r o m

th is p l o t is a p p r o x i m a t e l y 3 . 9 X 7O ' 6 M .

p apain -SH +

H

< ^ ^ > - C H 2-N=C=S->papain-S-C-N-CH

S

F ig. 6—P r o p o s e d m e c h a n is m fo r t h e r e a c t io n o f a c t i v a t e d p a p a in w i th

B IT C .

a f fe c te d as in d ic a te d by curve A a n d B in F igure 3. H ow ev e r , d ras t ic r e d u c t io n o f ac t iv ity re su l te d (curve C) u p o n reversing this se q u e n c e o f ad d i t io n s . BITC in h ib i t io n c o u ld also be nu l l i f ied by th e a d d i t io n o f an excessive a m o u n t o f cys te ine (Fig. 4 ) . This r e s to r a t io n o f ac t iv i ty , h o w ever, was n o t o bse rved w h e n a low c o n c e n t r a t i o n (e.g., I . l 7 x 10"5 M) o f cy ste ine was used . A Ki o f 3 .9 x 1 0 '6 M was e s t im a te d f r o m a L inew eaver-B urk p lo t o f re ac t io n ra te s a t va r ious su b s t r a te c o n c e n t r a t io n s in th e p resence o r absen ce of BITC (Fig . 5).

T h e o b se rv ed su b s t r a te p r o t e c t io n o f p ap a in aga ins t BITC in h ib i t io n a n d the revival o f B IT C - in h ib i ted p ap a in ac t iv i ty by a d d in g an excessive a m o u n t o f cy ste ine sugges ted a c o m p e t i t iv e ty p e o f in h ib i t io n . H ow eve r , t h e L inew eaver-B urk p lo t sh o w e d t h a t th e r e c ip ro ca l p lo ts w i th

o r w i th o u t BITC m e t a t th e sam e p o in t on th e X axis . I t is possible t h a t the papain -B ITC c o m p le x , o n c e f o r m e d , has a very sm all d isso c ia t io n c o n s t a n t in c o m parison to t h a t o f th e p a p a in -su b s tra te c o m p le x . Th is ten d s to decrease th e a c tua l a m o u n t o f f ree e n z y m e available fo r r e a c t io n , w i t h o u t a f fe c t in g th e b ind ing b e tw e e n p a p a in a n d its su b s t r a te . T h e re fo re , a n o n c o m p e t i t i v e ty p e o f p lo t is o b ta in ed (W estly , 1969) .

T h e genera lly a c c e p te d m o d e l fo r the inac t ive p ap a in is t h a t i t fu n c t io n a l -S H g ro u p is in a b o n d e d fo rm (K le in and Kirsch , 1969) . O nce a c t iv a ted , this su lp h y d ry l g ro u p is l ib e ra te d a n d available fo r r e a c t io n . B ecause BITC is k n o w n to be su b je c t t o n u c le o p h i l i c a t t a c k , the m ec h an ism o f th is i n h ib i t io n is p ro b a b ly as sh o w n in F igure 6.This p r o p o s e d m e c h a n i sm is s u p p o r t e d by

the o b se rv a t io n t h a t B IT C - tr e a te d , u n a c t iv a ted p a p a in , a f t e r t h e r e m o v a l o f BITC by d ialysis fo l lo w e d b y c y s te in e a c t iv a t io n , a ch ieved an ac t iv i ty c o m p a r a ble t o t h a t o f th e u n t r e a t e d sa m p le . It suggests t h a t B IT C o n ly r e a c te d w i th th e ac t iv a ted p a p a in in w h ic h the fu n c t io n a l - SH g roups were available. Th is re su l t , t o g e th e r w i th th e u n u su a l ly h igh re ac t iv i ty o f the fu n c t io n a l -S H g ro u p o f p a p a in ( S lu y te r m a n , 1 9 6 7 ) , favors t h e a r g u m e n t t h a t th e in h ib i t io n is p r im a r i ly d u e to the fo r m a t io n o f a d i th io c a r b a m y l l inkage b e tw een p ap a in a n d BITC.

REFERENCES

Bahadur, K. and Atreya, B.D. 1960. Study of the effect of ripe papaya extract on the proteolytic activity of papain. Enzymologia 21: 325.

Desreux, V. and Fischer, P. 1947. The mechanism of the inactivation of papain by certain war gases. Actualités Biochim. 10: 127.

Ettlinger, M.G. and Kjaer, A. 1968. Sulfur compounds in plants. In “Recent Advances in Phytochemistry,” Ed. Mabry, T.J., Vol 1, p. 59. Appleton-Century-Crofts, New York.

Gmelin, R. and Kjaer. A. 1970. Glucosinolates in the Caricaceae. Phytochem. 9: 591.

Hinkel, E.T. Jr. 1951. The effect of the temperature of drying papaya latex on the initial activity and stability of papain. Ann. N.Y. Acad. Sei. 54: 245.

Kirsch, J.F. and Igelström, M. 1966. The kinetics of the papain-catalyzed hydrolysis of esters of carbobenzoxyglycine. Evidence for an acyl-enzyme intermediate. Biochemistry 5: 783.

Kjaer, A. 1960. Naturally derived isothiocyanates (mustard oils) and their parent gluco- sides. Fortschr. Chem. Org. Naturst. 18: 122.

Klein, I.B. and Kirsch, J.R. 1969. The mechanism of the activation of papain. Bioehem. Biophys. Res. Commun. 34: 57 5.

Königsberg, W. 1967. Subtractive Edman degradation. In “Methods in Enzymology,” Ed. Hirs, C.H.W., Vol 11, p. 461. Academic Press, New York.

Sluyterman, L.A.AE. 1967. Reversible inactivation of papain by cyanate. Bioehem. Biophys. Acta 139: 439.

Tang, C.S. 1971. Benzyl isothiocyanate of papaya fruit. Phytochem. 10: 117.

Tang, C.S. 1973. Localization of benzylglucosinolate and thioglucosidase in Carica papaya fruit. Phytochem. 12: 769.

Tang, C.S., Bhothipaksa, K. and Frank, H.A.1972. Bacterial degradation of benzyl isothiocyanate. Appl. Microbiol. 23: 1145.

VanEtten, C.H., Daxenbichler, M.E. and Wolff,I.A. 1969. Natural glucosinolates (thiogluco- sides) in foods and feeds. J. Agr. Food Chem. 17: 483.

Westly, J. 1969. Inhibition, activation, inactivation, pH effects. In “Enzyme Catalysis,” p. 42. Harper and Row, Publishers, New York.

Ms received 6/18/73; revised 8/18/73; accepted8/20/73.__________________ ___

Portions of the results were presented at the 164th National Meeting of the American Chemical Society in New York, 1972.

Journal Series No. 1633 of the Hawaii Agricultural Experiment Station.

The author gratefully acknowledges the advice and aid given by Dr. Wen-Jing Tang.

J . C. C H A N G , M A R Y W. R E N O L L a n d P. M . T. H A N S E N

D e p t , o f F o o d S c ie n c e & N u t r i t i o n , T h e O h io S t a t e U n iv e r s i ty , C o lu m b u s , O H 4 3 2 1 0

ZONAL ELECTROPHORESIS OF FOOD STABILIZERS IN MALONATE BUFFER

INTRODUCTION

T H E C O M M O N S T A B I L I Z E R add it ives used b y th e f o o d in d u s t ry a re p o ly sac char ide g u m s o b ta in e d m a in ly f ro m seaw eed e x t ra c ts , t r ee e x u d a te s , seed g u m s and cel lu lose der iva tives (K lose a n d G lick sm an , 1968) . T hese g u m s are ra re ly used as single en t i t ie s in f o o d sy s tem s; r a th e r m a n u f a c tu r e r s o f c o m m e rc ia l stab il izers su p p ly , u n d e r b ra n d nam es, se lec ted m ix tu re s fo r spec if ic pu rposes . Several an a ly t ica l m e th o d s fo r t h e id e n t i f ica t io n o f h y d ro c o l lo id s have b e e n c o m piled b y G l ick sm an ( 1 9 6 9 ) . H ow ever , c o m p le te m e th o d s fo r t h e d e te r m in a t io n o f ind iv idua l g u m s in f o o d m ix tu r e s are still n o t avai lable a n d th e d e te r m in a t io n is c o m p l ic a te d by th e lo w c o n c e n t r a t io n levels genera l ly used.

E le c t ro p h o re s is is o n e o f th e a p p ro a ch e s w h ic h m a y be u se d fo r th e id en t i f ic a t io n o f h y d ro c o l lo id m ix tu re s . T he e le c t r o p h o r e t i c c h a ra c te r i s t ic s o f h y d ro p h i l ic c o l lo ids has b e e n e x a m in e d by m ov ing b o u n d a r y e le c t ro p h o re s is (H ida lgo an d H an sen , 1 9 6 8 a ) , and th e resu l ts suggested th a t th is e le c t r o p h o r e t i c t e c h n iq u e m ay be u se fu l in s tu d ies on in te ra c t io n s o f f o o d stab il ize rs w i th o th e r c o m p o n e n t s . H ow ever , t h e t e c h n iq u e w o u ld be o f l i t t le va lue fo r r o u t in e an a l ysis o f s tab i l ize rs b e cau se o f th e l im i ta t io n s o n t im e a n d th e n u m b e r o f sam ples w h ich can be d e te r m in e d s im u lta n eo u s ly .

Briggs et al. ( 1 9 5 6 ) e m p lo y e d e le c t ro phores is o n glass f i l te r p a p e r fo r se p a ra t io n o f c a r b o h y d r a te s in b o r a te bu ffer . N eu t ra l a n d acid ic p o ly sa cc h a r id e s were d e te c te d b y a lka l ine p o ta s s iu m p e r m a n g an a te a n d v a r io u s m e th y l a n d ace ty l derivatives o f sugars b y 1 -n a p h th o l in sulfuric acid. E l e c t r o p h o r e t i c analysis o f a n u m b e r o f gell ing an d th ic k e n in g agen ts o n cel lu lose a c e ta te s t r ip s by using b o ra te b u f fe r h a s b e en r e p o r t e d by P a d m o y o and Miseriz ( 1 9 6 7 ) . T h e y suggested th a t t h e s e p a ra ted g u m s co u ld be id en t i f ie d by to lu id in e b lue , p e r io d ic ac id -Sch if f rea gent (PA S), o r a t a n n in -P A S s ta in ing t e c h n ique. Hsu et al. ( 1 9 7 2 ) used z o n e e lec t ro p h o re s is o f m u c o p o ly s a c c h a r id e s on cellulose a c e ta te in th re e b u f fe r s : (a) 0 .1 N HCl, (b ) 0 .2M b u ty la m in e an d (c) 0 .0 5 M ca lc iu m a ce ta te in 40 % d ie th y le n e glycol. E le c t ro p h o re s is o n cel lu lose a ce

t a te o f p o ly sa cc h a r id e s t h a t had been p rev ious ly t r e a te d w i th P ro c io n d y e -s tu f fs was e m p lo y e d by D u d m a n a n d Bishop( 1 9 6 8 ) and b y A n d e r so n et al. ( 1 9 7 1 ) . A c o m b in a t io n o f agar gel e le c t ro p h o re s is an d p a p e r e le c t ro p h o re s is was u sed by D ie tr ich a n d D ie tr ich ( 1 9 7 2 ) fo r th e i d e n t i f ic a t io n o f m u c o p o ly sa c c h a r id e s a f te r t h e a c t io n o f specif ic en zy m es .

M ean ingfu l z o n e e le c t ro p h o re s i s o f s tab il ize r add it ives in f o o d req u ire s th e a p p l i c a t io n o f n o n d e s t r u c t iv e m e th o d s fo r t h e in it ia l i so la t io n o f th e collo ids. We have p rev ious ly r e p o r t e d (H an sen a n d Chang, 1 9 6 8 ) t h a t CMC m ay be re co v e red q u a n t i t a t iv e ly f ro m p a s teu r ize d m ilk by a m e th o d involving a c o m b in a t io n o f t ry p s in d ig es t io n a n d e x t r a c t io n w i th t r ic h lo ro a c e t i c acid fo l lo w ed b y p re c ip i ta t io n w i th se lec ted o rgan ic solvents . G ra h am ( 1 9 6 8 , 1 9 7 2 ) p u b l ish ed m e th o d s for th e s e p a ra t io n o f CMC an d ca rra g ee n an f ro m da iry fo o d s e m p lo y in g p ap a in d ig es t io n an d c o m p le x in g o f t h e h y d r o co llo ids w i th c e ty l p y r id in iu m ch lo r ide .

T h e p re se n t s tu d y was u n d e r t a k e n to d e te r m in e th e c o m p o s i t i o n o f m ix tu r e s o f c o m m o n fo o d stab il ize rs a f te r th e i r rec overy f ro m m ilk p ro d u c ts . T h e inves tigat io n has dea l t w i th th e se lec t io n o f a p p ro p r ia te b u f fe r sy s te m s a n d su i tab le m e th o d s fo r th e s e p a ra t io n a n d id e n t i f ic a t io n o f a n io n ic a n d n e u tra l s tab il izers on cel lu lose a c e ta te strips.

MATERIALS & METHODS

S a m p l e p r e p a r a t i o n

T h e f o l l o w i n g m a t e r i a l s w e r e u s e d : s o d i u m s a l t s o f l a m b d a - a n d k a p p a - c a r r a g e e n a n ( M a r i n e C o l l o i d s , I n c . , S p r i n g f i e l d , N . J . ) ; s o d i u m a l g i n a t e ( K e l c o C o . , S a n D i e g o , C a l i f . ) ; s o d i u m c a r - b o x y m e t h y l c e l l u l o s e ( H e r c u l e s P o w d e r C o . , W i l m i n g t o n , D e l . ) ; p e c t i n ( N a t i o n a l P e c t i n , C h i c a g o , 111.); a n d c o m m e r c i a l g r a d e s o f g u m a r a b i c , g u a r g u m a n d l o c u s t b e a n g u m . T h e m a t e r i a l s w e r e d i s p e r s e d in d i s t i l l e d w a t e r o r m i l k a t 6 0 ° C b y s p r i n k l i n g t h e p o w d e r o n t o t h e s u r f a c e o f t h e l i q u i d w i t h r a p i d a g i t a t i o n . T h e s a m p l e s w e r e t h e n h o m o g e n i z e d s e v e r a l t i m e s w i t h a h a n d - o p e r a t e d l a b o r a t o r y h o m o g e n i z e r t o f a c i l i t a t e c o m p l e t e d i s p e r s i o n .

I s o l a t i o n p r o c e d u r e

T h e m e t h o d d e s c r i b e d f o r C M C ( H a n s e n a n d C h a n g , 1 9 6 8 ) w a s u s e d t o r e c o v e r m i x t u r e s o f s t a b i l i z e r s f r o m m i l k . T h e m e t h o d i n v o v e s

t r e a t i n g a 1 0 - m l s a m p l e w i t h 0 . 2 % t r y p s i n ( D i f c o c e r t i f i e d e n z y m e 1 : 3 0 0 o r 1 : 2 5 0 ) a t 4 0 ° C / 3 h r m a i n t a i n i n g p H a t 8 . 5 w i t h I N N a O H . 1 0 m l o f 0 . 4 % d i o c t y l s u l f o s u c c i n a t e ( A m e r i c a n C y a n a m i d e C o . ) is t h e n a d d e d t o t h e

h y d r o l y s a t e f o l l o w e d b y 2 0 m l o f t r i c h l o r o a c e t i c a c i d ( 2 5 % ) . T h e m i x t u r e is c e n t r i f u g e d a t

4 0 0 0 - 5 0 0 0 X G f o r 3 0 m i n a n d t h e h y d r o c o l l o i d i s p r e c i p i t a t e d f r o m t h e s u p e r n a t a n t b y t h e

a d d i t i o n o f 1 v o l o f a b s o l u t e e t h a n o l a n d 2 . 5 v o l o f e t h y l a c e t a t e ( i f p h a s e s e p a r a t i o n o c c u r s , m o r e e t h a n o l is a d d e d ) . T h e m i x t u r e is a l l o w e d t o s t a n d f o r 3 0 m i n w i t h i n t e r m i t t e n t s h a k i n g a f t e r w h i c h t i m e t h e f l o c c u l a t e d h y d r o c o l l o i d is c o l l e c t e d b y c e n t r i f u g a t i o n . T h e p r e c i p i t a t e d

h y d r o c o l l o i d is d i s p e r s e d i n d i s t i l l e d w a t e r a n d t h e p H a d j u s t e d t o b e t w e e n 7 - 8 w i t h s o d i u m h y d r o x i d e ( i n d i c a t o r p a p e r ) . T h e i s o l a t i o n o f h y d r o c o l l o i d f r o m c o n c e n t r a t e d m i l k s i s d e s c r i b e d i n t h e t e x t .

I n s t r u m e n t a n d g e n e r a l t e c h n i q u e

E l e c t r o p h o r e s i s w a s c o n d u c t e d w i t h t h e

P h o r o S l i d e E l e c t r o p h o r e s i s S y s t e m ( M i l l i p o r e C o r p . , B e d f o r d , M a s s . ) a t r o o m t e m p e r a t u r e . T h e c e l l w a s f i l l e d w i t h b u f f e r a n d a p p r o x i m a t e l y 0 . 3 p i o f s a m p l e w a s a p p l i e d t o t h e s t r i p a c c o r d i n g t o t h e r e c o m m e n d e d o p e r a t i n g i n

s t r u c t i o n s .

N e u t r a l p o l y s a c c h a r i d e s

B o r a t e b u f f e r ( p H 1 0 , i o n i c s t r e n g t h 0 . 1 3 ) w a s p r e p a r e d a c c o r d i n g t o t h e p r o c e d u r e o f P a d m o y o a n d M i s e r i z ( 1 9 6 7 ) . F o l l o w i n g t h e

e l e c t r o p h o r e s i s f o r 2 5 m i n a t lO O v t h e s t r i p w a s s t a i n e d b y t h e P A S r e a c t i o n ( H o t c h k i s s , 1 9 4 8 ) .

A n i o n i c p o l y s a c c h a r i d e s

S o d i u m m a l o n a t e b u f f e r ( p H 2 . 9 , 0 . 0 7 5 M ) w a s p r e p a r e d b y d i s s o l v i n g 7 . 8 g o f m a l o n i c a c i d ( B a k e r , A . R . ) i n 8 0 0 m l d i s t i l l e d w a t e r . T h e p H w a s a d j u s t e d t o 2 . 9 w i t h I N N a O H a n d t h e v o l u m e w a s a d j u s t e d t o 1 l i t e r . T h e s p e c i f i c r e s i s t a n c e o f t h e b u f f e r a t 0 ° C w a s 4 5 0 o h m s / c m . F o l l o w i n g e l e c t r o p h o r e s i s f o r 1 0 m i n a t lO O v ,

t h e s t r i p w a s s t a i n e d w i t h 0 . 2 % t o l u i d i n e b l u e a c c o r d i n g t o t h e m e t h o d o f P a d m o y o a n d M i s e r i z ( 1 9 6 7 ) .


S T A B IL IZ E D fo o d p r o d u c t s m ay c o n ta in b o th n e u t r a l and ion ic p o ly sacch ar id es . P re l im in ary s tu d ies revea led t h a t such m ix tu re s o f p o ly sa cc h a r id e s co u ld be s e p a ra ted by e le c t ro p h o re s i s o n cellulose a c e ta te s t r ip s a n d th e z o n es v isualized by a p p r o p r i a te s ta ins . H ow ever , it was fo u n d th a t no single t e c h n iq u e cou ld a d e q u a te ly sa t is fy all poss ib le c o m b in a t io n s o f stabi-

V o lu m e 39 < 1974)—J O U R N A L O F F O O D S C IE N C E -9 7


lizers. F o r th is re a so n , d i f f e re n t m e th o d s w ere u se d fo r n e u t r a l a n d a n io n ic p o ly sacchar ides . In i t ia lly , a t t e n t i o n was given to t h e ana lyses o f t h e n e u tra l types .

A na lys is o f n e u t r a l s tab il izersT h e basic r e q u i r e m e n t fo r t h e ap p l ic a

t io n o f e le c t r o p h o r e t i c t e c h n iq u e s is th a t th e c o m p o n e n t s t o be se p a ra te d possess e lectr ica l charges. A t t e m p t s w e re m a d e to se p a ra te n e u t r a l s tab i l ize rs u s ing b o r a te b u f f e r b y th e m e t h o d o f P a d m o y o and Miseriz ( 1 9 6 7 ) . In such a sy s te m , t h e c o n s t i tu e n t s ach ieve e le c t r o p h o r e t i c m o b i l i ty

as t h e resu l t o f i n te r a c t io n w i th b o r a te ions ( N c r th c o t e , 1954) .

O u r resu lts , u s ing t h e P A S s ta in in g te c h n iq u e , i n d ic a te d t h a t c e r t a in n e u t r a l s tab il izers w e re read i ly d e te c t e d , w h e rea s in genera l , t h e a n io n ic s tab i l ize rs w e re n o t as in te n se ly s ta in e d b y th is m e t h o d , w i th

F ig. 1 —E l e c t r o p h o r e t i c p a t t e r n s o f n e u t r a l s ta b i l i z e r s in b o r a te b u f f e r a n d s ta in e d b y P A S r e a g e n t: ( A ) M ix tu r e o f g u a r g u m a n d l o c u s t b e a n g u m ; ( C) M ix tu r e o f g u a r g u m a n d lo c u s t b e a n g u m ; (D ) L o c u s t b e a n g u m ; (E ) M ix tu r e o f g u a r g u m , l o c u s t b e a n g u m , a n d g u m a r a b ic . ( V o l ta g e : 1 0 0 v ; T im e :

2 5 m in )

iALGINATE----- -¿IMP.

Fig. 2 —E l e c t r o p h o r e t i c s e p a r a t io n o f s ta b i l i z e r s a t tw o d i f f e r e n t p H v a lu e s ( a n a ly s e s in d u p l i c a t e ) : (A ) N a - m a lo n a te b u f f e r , p H 2 .9 ; (B ) B o r a te b u f f e r , p H 10. (S ta in in g : T o lu id in e b lu e ; V o l ta g e : 1 0 0 v ; T im e : 1 0 m in )

ZONAL ELECTROPHORESIS OF FOOD STABILIZERS-99

Fig. 3 —E l e c t r o p h o r e t i c p a t t e r n s o f f o u r a n io n ic s ta b i l i z e r s in N a - m a lo n a te b u f f e r a n d s ta in e d b y t o lu id in e b lu e : la ) C M C ;

lb ) M ix tu r e r e c o v e r e d f r o m s k i m m i l k ; (c ) A lg in a te ; Id ) M ix tu r e r e c o v e r e d f r o m s k i m m i l k ; (e ) K a p p a -c a r r a g e e n a n ; I f ) M ix tu r e r e c o v e r e d f r o m s k i m m i l k ; Ig ) L a m b d a -c a r r a g e e n a n ; Ih ) M ix tu r e r e c o v e r e d f r o m s k i m m i l k ; ( i) M ix tu r e in w a te r d is p e r s io n ; fj) M ix tu r e r e c o v e r e d f r o m s k i m m i lk . I V o l ta g e : 1 0 0 v ; T im e : 10 m in )

Fig. 4 —E l e c t r o p h o r e t i c p a t t e r n s o f m i x t u r e s c o n ta in in g g u a r g u m a n d lo c u s t b e a n g u m in b o r a te b u f f e r a n d s ta in e d b y P A S r e a g e n t: IA ) M ix tu r e r e c o v e r e d f r o m s k i m m i l k ; IB ) M ix tu r e in w a te r d i s p e r s io n ; I V o l ta g e : 1 0 0 v ; T im e : 2 5 m in )

th e e x c e p t io n o f gu m arabic . T h e e le c t ro p h o re t ic p a t t e rn s (Fig . 1) sh o w ed t h a t the degree o f h o m o g e n e i ty was d i f f e re n t fo r guar g u m a n d lo cu s t b e a n g u m . T h e guar g u m p a t t e r n sh o w e d a s t ro n g s ta t io n a ry z o n e w h ic h was ab se n t f r o m lo cu s t b e a n gum . T h e m ig ra t in g zo n es fo r th e tw o stabil izers w ere su f f ic ie n t ly d i f f e re n t to p e rm i t d i f f e re n t i a t io n . T h e m ix tu r e o f th e t w o s tab il ize rs w i th g u m arab ic gave rise t o a s t ro n g ly d i f fu sed z o n e in d ica t in g th a t e le c t r o p h o r e t i c id e n t i f ic a t io n o f in d iv idual s tab i l ize rs in c o m p le x m ix tu re s by th is m e t h o d w o u ld n o t be possible. T h e m o b i l i t ie s in r e la t io n to lo cu s t b e an g um are p r e se n te d in Tab le 1.

Selection of buffer for anionic stabilizers

T h e e le c t r o p h o r e t i c se p a ra t io n o f a m ix tu re o f t h e f o u r s tab il izers , l am b d a - ca r rag een an , k a p p a -c a r ra g ee n an , a lg ina te an d CMC at tw o d i f f e re n t p H va lues and s ta ined w i th to lu id in e b lue is sh o w n in F igure 2. S e p a ra t io n o f th is m ix tu r e was n o t o b ta in e d in b o r a te b u f fe r , p H 10 be cause t h e m o b i l i t ie s o f th e fo u r s tabilizers were n ea r ly th e sa m e a n d th e zo n es were ove r lapp ing . H ow eve r , a c o m p le te separa t io n was poss ib le in m a lo n a te b u f fe r o f

1 0 0 - JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

p H 2.9. T he re a so n fo r th e im p ro v e d s e p a ra t io n a t th is pH m ay be fo u n d in the lo w e r d egree o f d is so c ia t io n o f th e w e a k ly acid ic c a rb o x y l ic g ro u p s in a lg ina te and CMC in c o n t r a s t to th e m o r e c o m p le te ly d isso c ia ted su lfa te g ro u p s o f c ar rageenan .

T h e se lec ted m a lo n a te b u f fe r was app lied to m ea su re th e m o b i l i t ie s o f various a n io n ic s tab il izers in re la t io n to a se lec ted lam b d a -c a r rag e en a n . T h e resu lts are sh o w n in Tab le 2 and reveal t h a t so m e stab il izers are h o m o g e n e o u s , w h e reas o th e r s are h e te ro g e n e o u s . D e f in i te sep a ra t io n was n o t ach ieved o f all o f th e s ta b i lizers s ince so m e o f these possessed id e n tical m ob il i t ie s . In t h e case o f CMC, there was ev idence t h a t h igh v iscosi ty sam ples (H P ) m ig ra ted w i th a lo w e r m o b i l i ty t h a n sam ples w i th re d u c e d v iscosi ty (MP andLP). As e x p e c te d , th e re was also a n o t ic e able e f fec t d i rec t ly a t t r i b u ta b le to var ia t io n s in th e degree o f su b s t i tu t io n . A n a p p r o x im a te id e n t i f ic a t io n o f t h e fo l lo w ing ty p e s was poss ib le based u p o n th e i r m o b il i t ie s re la tive to th e se lec ted lam b d a - car rageenan .

CMC a n d g um k a ray a ( m in o r c o m p o n e n t s )

Pec t inCMC, g u m arabic ,

g u m k a ray a AlginC arrageenan

R X = 0 R \ = 0.11

RX = 0 . 2 2 - 0 . 2 5 R X = 0 .50 RX = 0-72 1.11

Analysis o f s tab i l ize r m ix tu re sin sk im m ilk

T w o m ix tu re s , o n e c o n ta in in g fo u r a n io n ic s tab i l ize rs a n d th e o t h e r c o n ta i n ing tw o n e u tra l s tab il izers , w ere ad d ed (0 .1% fo r each s tab il ize r ) t o sk im m ilk at 60 C /3 0 m in a n d t h e n recove red by the iso la t io n p ro c ed u re . T h e c o m p o s i t io n o f th e m ix tu r e s was a n a ly ze d qu a l i ta t iv e ly by zo n a l e le c t ro p h o re s i s te c h n iq u e s as d e scr ibed above. T h e e le c t r o p h o r e t i c pat-

Table 1 —Relative mobilities of selected stabilizers i buffer(100v/10min)

S tab ilize rC oncen tra tion

(% )

Q u a n tityapplied

(u l)

D istancemoved(m m ) ^ L o c u s t 3

L o c u s t b e a n g u m 0 . 3 0 . 9 h 1 . 0 0

G u a r g u m 0 . 3 0 . 9 0 0 . 0 0

1 3 1 . 1 8

G u m a r a b i c b 2 . 0 0 . 9 2 0 - 2 4 1 . 8 2 - 2 . 1 8

a R L o c u s t v a l u e = m i g r a t i o n o f z o n e ________, m i g r a t i o n o f l o c u s t g j mD D i f f u s e z o n e

Table 2 —Relative m o b ilitie s o f various an ion ic stab ilizers in m a lonate b u ffe r (100v /1 0 m in)

C oncen tra tionS tab ilize r (%)

Q u a n tityapplied

(Ml)

Distancemoved(m m ) R x a

H i - m e t h o x y l p e c t i n 0 . 3 0 . 9 2 0 . 1 1

N a t u r a l p e c t i n 0 . 3 0 . 9 2 0 . 1 1

C M C b ( D . S . , 0 . 9 , H P ) 0 .1 0 . 3 4 0 . 2 2

C M C ( D . S . , 0 . 9 , M P ) 0 .1 0 . 3 4 . 5 0 . 2 5

C M C ( D . S . , 0 . 7 , H P ) 0 .1 0 . 3 4 0 . 2 2

C M C ( D . S . , 0 . 7 , M P ) 0 .1 0 . 3 4 . 5 0 . 2 5

C M C ( D . S . , 0 . 7 , L P ) 0 .1 0 . 3 4 . 5 0 . 2 5

C M C ( D . S . , 0 . 4 , H P ) 0 . 3 0 . 3 0 0 . 0 0

C M C ( D . S . , 0 . 4 , M P ) 0 . 3 0 . 3 2 0 . 1 1

G u m a r a b i c 2 . 0 0 . 9 4 . 5 0 . 2 5

G u m k a r a y a 0 . 3 0 . 9 0 0 . 0 0

4 . 5 0 . 2 5

A l g i n , H P 0 .1 0 . 3 9 0 . 5 0

A l g i n , L P 0 .1 0 . 3 9 0 . 5 0

L a m b d a - c a r r a g e e n a n 0 .1 0 . 3 1 8 1 . 0 0

K a p p a - c a r r a g e e n a n 0 .1 0 . 3 1 3 0 . 7 2

1 8 1 . 0 0

C a r r a g e e n a n S e a k e m 2 0 .1 0 . 9 1 4 0 . 7 8

2 0 1 . 1 1

S e a k e m 1 4 0 .1 0 . 9 1 4 0 . 7 8

2 0 1 .1 1

S e a k e m D 0 .1 0 . 9 1 5 0 . 8 3

2 0 1 . 1 1

a _ m ig ra tio n o f a g iven zone - m o b i l i t y o f A.-carrageenan

k D . S . = d e g r e e o f s u b s t i t u t i o n ; H P , M P , L P = h i g h , m e d i u m a n d l o w v i s c o s i t y

Fig. 5 - E l e c t r o p h o r e t i c p a t t e r n s o f s ta b i l i z e r m i x t u r e s r e c o v e r e d f r o m c h o c o l a t e m i l k : ( A ) K n o w n m i x t u r e in w a te r d is p e r s io n ; (B ) M i x t u r e r e c o v e r e d from c h o c o l a t e m i l k ; ( C ) M a te r ia l r e c o v e r e d f r o m a c h o c o l a t e m i l k t o w h ic h n o s ta b i l i z e r w a s a d d e d ; (D ) M ix tu r e r e c o v e r e d f r o m c h o c o l a t e m i l k ; (E ) M a te r ia l r e c o v e r e d f r o m a c o m m e r c i a l c h o c o l a t e m i l k ; (F ) M ix tu r e r e c o v e r e d f r o m c h o c o l a t e m i l k . (S ta in in g : T o lu id in e b lu e ; V o l ta g e : lO O v; T im e : 1 0 m in )

ZONAL ELECTROPHORESIS OF FOOD STABILIZERS- 1 0 1

4 - J , j-~i

Crude k AF ig. 6— E l e c t r o p h o r e t ic p a t t e r n s o f c a r r a g e e n a n (0 .4 % ) in 0 . 0 7 5 M c a lc iu m m a io n a te b u f f e r , p H 2 .9 ,

w i th 15% a b s o lu te e th a n o l a d d e d : C r u d e , S e a k e m 2 ; t t-C a r ra g e e n a n ; \ -C a r r a g e e n a n . ( V o l ta g e : 1 0 0 v ; T im e : 1 5 m in )

H “

A B C DFig. 7 —E l e c t r o p h o r e t i c p a t t e r n s o f h y d r o c o l lo id s r e c o v e r e d f r o m e v a p o r a te d m i l k a n d i n f a n t m i l k : (A ) C o n tr o l o f S e a k e m 2 ; (B ) H y d r o c o l lo id f r o m e v a p o r a te d m i l k ; (C ) H y d r o c o l lo id f r o m in f a n t

m i lk ; (D ) C o n tr o l o f S e a k e m 2 . (C a lc iu m m a lo n a te b u f f e r , p H 2 . 9 w i th 15% a b s o lu t e e th a n o l a d d e d . 1 0 0 v /1 5 m in , s ta in in g w i th t o tu id in e b lu e )

t e rn s are i l lu s t ra ted in F igure s 3 a n d 4. Indiv idual s tab il izers d ispersed in d istil led w a te r w e re used as c o n tro ls . T he p a t t e rn s in F igure 3 in d ic a te t h a t all o f t h e an ion ic stab il izers w ere re co v e red f ro m th e m ilk a n d th a t the e le c t r o p h o r e t i c m o b i l i té s o f th e c o n s t i tu e n t s tab il izers w ere s im ila r to th o se o f th e respec t ive c o n tro ls . T h e simila r i ty o f th e p a t t e rn s and th e iden t ica l m ob il i t ie s suggest t h a t th e re la tive c o n c e n t r a t io n s a n d ind iv idua l c h arac te r i s t ic s o f t h e s tab i l ize rs w e re n o t s ign if ican tly a f fec ted b y th e iso la t io n t r e a tm e n t . H o w ever a fa in t s t a t i o n a r y z o n e was usua lly o b ta in e d in t h e recove red m ix tu re s f ro m milk, w h ich m ay be d u e to na tive m ilk po lysacchar ides .

N e u t ra l s tab il izers w ere also read i ly recovered an d id en t i f ied f ro m m ilk m ix tu re s (Fig. 4 ) . H ow ever , in th is case, the

zo n es f ro m th e recove red s tab il izers were s o m e w h a t w eak in c o m p a r is o n w i th the co n tro ls . N everthe less , th e p re sen ce o f b o t h guar a n d lo cu s t b e a n g u m was easily estab l ished .

Analysis o f s tab i l ize r m ix tu re s in c h o c o la te m ilk

T h e e le c t r o p h o r e t i c p a t t e rn s o f a m ix tu re c o n ta in in g f o u r an io n ic stab il izers reco v e red f ro m c h o c o la t e m ilk a re sh o w n in F igure 5. T h e re su l ts revea led th a t a d d i t io n a l zo n es a p p e a re d in t h e p a t t e rn s resu l t ing f ro m a m ate r ia l p re sen t in c h o c o la te m ilk to w h ic h n o s tab il ize r was a d d e d (Fig. 5). Th is c o n f i r m s th e resu lts o f o u r p rev ious r e p o r t s (H a n se n and Chang , 1 9 6 8 ) w h ic h sh o w ed an in te r f e r ing e f fec t o f c h o c o la te c o n s t i t u e n t s on th e q u a n t i t a t i v e d e te r m in a t io n o f CM C in

c h o c o la te milk. O n e o f th e in te r fe r in g su b s ta n c e s w as a p p a r e n t ly p e c t in w i th a z o n e close t o t h e origin. A n o th e r c o m p o u n d w as ev id e n t w i th a m o b i l i ty w h ich was i n te r m e d ia t e t o a lg ina te a n d CMC. This c o m p o u n d m a y be na t iv e to c h o c o la te o r it co u ld have b e en a d d e d b y th e m a n u f a c tu r e r . T h e p a t t e r n o f t h e k n o w n m ix tu r e was also c o m p a r e d w i th t h a t o f t h e m ate r ia l r e c o v e red f ro m a c o m m e rc ia l c h o c o la te m ilk (Fig. 5e, f). T h e p a t t e rn s in d ic a te d t h a t th e c o m m e rc ia l sam ple c o n ta in e d a m ix tu r e o f ca r ra g ee n an , p re d o m in a n t ly o f t h e k a p p a - ty p e . T h e zo n es w i th lo w e r m o b i l i t ie s w ere p r e su m a b ly f ro m c o n s t i t u e n t s o f c h o c o la te . The s t ro n g s t a t i o n a r y z o n e in th e recove red m ix tu r e m a y b e a n i n te r a c t io n p r o d u c t resu l t ing f ro m c h o c o la t e c o m p o n e n t s and m ilk c o n s t i t u e n t s such as a t a n n i n / p r o t e in c o m p le x .

A nalysis o f c a r rag een an in s te r il ized m ilk p r o d u c t s

D iff icu l t ie s w ere e n c o u n te r e d in using th e m a lo n a te b u f f e r fo r t h e r e so lu t io n o f a n u m b e r o f ca r ra g ee n an sam ples , w h ich r e p e a te d ly y ie ld e d s t r e a k y p a t t e rn s . H o w ever, th ese d i f f icu l t ie s c o u ld be p a r t ly o v e rc o m e b y in t r o d u c in g ca lc iu m and e th a n o l i n to th e b u f f e r (Fig. 6 ) a n d it was o f in te re s t to n o t ic e t h a t in th is b u f fe r sy s te m , th e re la tive m o b i l i ty o f k appa- ca r ra g ee n an was g re a te r t h a n fo r l am b d a - c a r rag een an . Th is e f fe c t m a y poss ib ly be th e resu l t o f a t e n a c io u s b in d in g o f calciu m to s o m e o f t h e s u lp h a te g ro u p s in lam b d a -c a r rag e en a n caus ing an e f fec t ive r e d u c t io n in t h e n e t ion ic charge . T he p a t t e r n fo r th e lam b d a -c a r r a g e e n a n was d if fu se w hile k a p p a -c a r r a g e e n a n p r o d u c e d o n e s t a t i o n a r y a n d t w o m ig ra t in g zones .

This b u f f e r sy s te m was a p p lied to th e e le c t r o p h o r e t i c e x a m in a t io n o f h y d r o c o l lo ids reco v e red f ro m e v a p o ra te d m ilk and f r o m an in f a n t m ilk p r o d u c t . B ecause o f th e low c o n c e n t r a t i o n o f c a r r a g ee n an in these p ro d u c ts , it b e c a m e n ecessa ry to m o d ify t h e re c o v e ry p r o c e d u r e t o in crease y ield and m in im iz e th e adverse e f fec ts o f t h e acid ic c o n d i t i o n s o n th e p o ly sacch ar id es . T h e sam p le size was in creased to 30g to w h ic h a n eq u a l v o lu m e o f w a te r w as a d d e d p r io r t o t ry p s in d iges t ion . T h e d ig es t io n p e r io d was in creased to 24 h r w i th pH m a in ta in e d at8 .5. T h e h y d r o ly s a te was t h e n c o o le d in ice w a te r an d all su b s e q u e n t s teps , in c lu d ing e x t r a c t io n w i th t r ic h lo ro a c e t i c acid (1 2 .5 % final c o n c e n t r a t i o n ) and so lven t p re c ip i t a t io n w ere c o m p le te d a t 5 C o r b e lo w . T h e h y d ro c o l lo id was p re c ip i t a te d f ro m th e t r ic h o lo ro a c e t i c acid f i l t r a te by a d d in g 2 vol o f a b so lu te e th a n o l . T he small a m o u n t o f p re c ip i t a te was co llec ted b y c e n t r i f u g a t io n an d dissolved in p h o s p h a te b u f f e r (p H 7 .5 ) and th e n t rea te d w i th a c a t io n ex ch a n g e resin (A m b e r l i te C G -1 2 0 ) o n a w a rm sp o t p la te p r io r to e le c t ro p h o re s is .

1 0 2 -JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

The patterns shown in Figure 7 reveal that the isolated hydrocolloids were considerably changed in comparison with the unprocessed carrageenan. Although these differences may reflect artifacts induced by the isolation/recovery procedure, the possibility of processing-induced alterations to carrageenan must not be discounted. In view of the relative ease by which carrageenan and other stabilizers were isolated without apparent effect on their electrophoretic properties from milk products having received only a moderate heat treatment, the latter possibility has been a major concern and has been the subject for further studies the results of which are presented in another paper (Hansen and Renoll, 1974).

REFERENCESAnderson, D.M.W., Hendrie, A., Millar, J.R.A.

and Munro, A.C. 1971. Electrophoresis of dyed polysaccharides on “PhoroSlides.” Analyst 96: 870.

Briggs, D.R., Garner, E.F. and Smith, F. 1956. Separation of carbohydrates by electrophoresis on glass filter paper. Nature 178: 154.

Dietrich, C.P. and Dietrich, S.M.C. 1972. Simple micro method for identification of heparin and other acidic mucopolysaccharides from mammalian tissues. Anal. Bio- chem. 46: 209.

Dudman, W.C. and Bishop, C.T. 1968. Electrophoresis of dyed polysaccharides on cellulose acetate. Can. J. Chem. 46: 3079.

Glicksman, M. 1969. “Gum Technology in the Food Industry,” Ch 14. Academic Press, New York.

Graham, H.D. 1968. Quantitative determination of carrageenan in milk and milk products using papain and cetyl pyridinium chloride. J. Food Sci. 33: 390.

Graham, H.D. 1972. Determination of carboxymethylcellulose from milk. J. Dairy Sci. 55: 42.

Hansen, P.M.T. and Chang, J.C. 1968. Quantitative recovery of carboxymethylcellulose from milk. J. Agr. Food Chem 16: 77.

Hansen, P.M.T. and Renoll, M.W. 1974. Heat induced changes in the zonal electrophoretic patterns of carrageenan stabilizers. J. Food Sci. 39: 000.

Hidalgo, J. and Hansen, P.M.T. 1968a. Moving boundary electrophoresis of food stabilizers. J. Food Sci. 33: 7.

Hidalgo, J. and Hansen, P.M.T. 1968b. Interactions between food stabilizers and /3-lactoglobulin. J. Dairy Sci. 51: 945.

Hotchkiss, R.D. 1948. A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 16: 131.

Hsu, D., Hoffman, P. and Mashburn, T.A. Jr.1972. Micro colorimetric method of dye staining and its application to cellulose polyacetate strip electrophoresis for the determination of acid glycosaminoglycans. Anal. Biochem. 46: 156.

Klose, R.E. and Glicksman, M. 1968. “Handbook of Food Additives,” p. 315. The Chemical Rubber Co., Cleveland.

Northcote, D.H. 1954. Electrophoresis of some neutral polysaccharides. Biochem. J. 58: 353.

Padmoyo, M. and Miseriz, A. 1967. Identification of gelling and thickening agents permitted in Switzerland by electrophoresis and staining on cellulose acetate strips (in German). Mitt. Geb. Lebensmittelunters Hyg. 58: 31.


Article 7-73, Dept, of Food Science & Nutrition. Investigation was supported by U.S. Public Health Service Research Grant FD-00117. Office of Research & Training Grants, FDA.

P O U L M. T. H A N S E N a n d M A R Y W. R E N O L L

D e p t, o f F o o d S cience & N u t r it io n , The O h io S ta te U n iv e rs ity , C o lu m b u s , O H 4 3 2 1 0

HEAT-INDUCED CHANGES IN THE ZONE ELECTROPHORETIC PATTERNS OF CARRAGEENAN STABILIZERS

INTRODUCTION

T H E P E R M I T T E D U S E o f c a r r a g e e n a n i n

e v a p o r a t e d m i l k ( F e d e r a l F o o d , D r u g a n d C o s m e t i c A c t , 1 9 6 7 ) i s a r e c o g n i t i o n o f

t h e e f f e c t i v e n e s s o f t h i s s t a b i l i z e r i n r e t a r d i n g c r e a m i n g . C a r r a g e e n a n h a s a l s o b e c o m e t h e s t a b i l i z e r o f c h o i c e i n m a n u

f a c t u r e d i n f a n t m i l k p r o d u c t s w h e r e c r e a m i n g i s r e g a r d e d a s a p a r t i c u l a r l y s e r i o u s d e f e c t b e c a u s e i t s i g n a l s t h e i n c i p i e n t f o r m a t i o n o f i n s o l u b l e f a t t y p a r t i c l e s , t h e p r e s e n c e o f w h i c h m a y h a m p e r i n f a n t f e e d i n g b y o b s t r u c t i n g t h e f e e d i n g n i p p l e s .

S i n c e c a r r a g e e n a n f u n c t i o n a l i t y a p p a r e n t l y d e p e n d s u p o n u n i q u e s t r u c t u r a l f e a t u r e s ( R e e s , 1 9 6 9 ; L i n a n d H a n s e n , 1 9 7 0 ; C h a k r a b o r t y a n d R a n d o l p h , 1 9 7 2 ) ,

T a b le 1—C o m p o s i t i o n o f m ilk sa l t s o l u t i o n 1 (pH 6 .7)

C a t io n s A n io n s(m M /l i te r ) (m M /l i te r )

Na 18.3 P 11.6K 39.4 Cl 32.4Ca 9.0 so„ 1.0Mg 3.2 co7 2.2

Citr. 9.6

a J en n e s s an d K o o p s (1 9 6 2 )

i t f o l l o w s t h a t a n y i n c i d e n t a l d a m a g e t o t h e c a r r a g e e n a n s t r u c t u r e d u e t o p r o c e s s

i n g m a y a l s o l e a d t o s t a b i l i t y p r o b l e m s o f t h e p r o d u c t i n w h i c h i t i s u s e d . F o r t h i s

r e a s o n , m e t h o d s a r e n e e d e d t o d e t e r m i n e a c c u r a t e l y t h e e x t e n t t o w h i c h f o o d s t a b i l i z e r s , p r e s e n t i n c o m p l e x f o o d s y s t e m s , w i t h s t a n d e x t r e m e s o f p r o c e s s i n g t r e a t

m e n t s .

I n a p r e v i o u s r e p o r t ( C h a n g e t a l . ,1 9 7 4 ) , i t w a s e s t a b l i s h e d t h a t a n u m b e r

o f f o o d s t a b i l i z e r s , i n c l u d i n g c a r r a g e e n a n , m a y b e r e c o v e r e d f r o m a t r y p t i c d i g e s t o f

m i l k b y s o l v e n t p r e c i p i t a t i o n o f t h e h y d r o c o l l o i d s f r o m a t r i c h l o r o a c e t i c a c i d e x t r a c t . Z o n a l e l e c t r o p h o r e t i c p a t t e r n s r e

v e a l e d t h a t c a r r a g e e n a n r e c o v e r e d f r o m m i l k h a v i n g r e c e i v e d o n l y l o w h e a t t r e a t m e n t w a s n o t s i g n i f i c a n t l y a l t e r e d . H o w

e v e r , t h e p a t t e r n s o b t a i n e d f o r c a r r a g e e n a n r e c o v e r e d f r o m s t e r i l i z e d , c o n c e n t r a t e d m i l k s w e r e n o l o n g e r s i m i l a r t o t h e i r

*

4 1-

M Û *

4 '- r t

4 0 C

2 0 C

m

0 . 5 % 0 . 4 % 0 . 3 % 0 . 2 % 0 . 1 %

C o n cen tra tio n appliedFig. 1—E ffe c t o f sam p le c o n c e n tra t io n a n d a m b ie n t te m p e ra tu re o n th e e le c tro p h o re t ic p a tte rn o f S eakem 2 carrageenan. E le c tro p h o re s is a t 4 0 ° C was p e r fo rm e d in an in c u b a to r w ith ce lls a n d b u f fe r p re c o n d it io n e d a t 4 0 ° C.

Volume 3 9 (1 9 7 4 ) - J O U R N A L OF FOOD SCIENCE- 1 0 3

1 0 4 - JO U RN AL OF FOOD S CI EN C E-V o lu me 3 9 (1974)

c o n t r o l . T h e r e f o r e , t h e p u r p o s e o f t h i s

r e s e a r c h h a s b e e n t o e x a m i n e t h e p o s s i b i l i t y t h a t h e a t - i n d u c e d a l t e r a t i o n s t o

c a r r a g e e n a n s m a y b e r e f l e c t e d i n t h e i r z o n e e l e c t r o p h o r e t i c p a t t e r n s .

EXPERIMENTALA M IL K -S A L T SO L U T IO N ( J & K B u ffer) sim ulating m ilk u ltrafiltra te was prepared according to Jen n ess and K oops (1 9 6 2 ) and had

the com position show n in T able 1. T h e pH o f the freshly prepared solution was 6 .7 . Low er pH values were achieved by the dropwise addition o f IN HC1. Carrageenan samples were obtained from Marine Colloids, In c ., Springfield,N .J. and included sodium salts o f A- and «-carrageenan obtained by potassium fraction ation o f Chondrus crispus e x tra c t, sodium salt o f E. spinosum e x tra c t (i-carrageenan) and Seakem 2 carrageenan w hich is a calcium salt o f un fractionated Chondrus ex tract.

The carrageenan sam ples w ere dispersed in the J & K bu ffer at 0 .4 or 0 .5 % co n cen tra tio n at 6 0 °C /3 0 min and then were cooled to room tem perature prior to pH ad ju stm en ts. C o n cen trations low er than 0 .4% were achieved by dilution with bu ffer. T he carrageenan solutions then were placed in 5 or 10 ml am poules which were heat scaled. T he filled am poules were heated for the specified tim e in an oil bath m aintained at 122 ± 0 .5 °C and then im m ed iately cooled. Prior to e lectrop horesis, the sam ples

pH 6 . 7 pH 5 . 9 pH 5 . 4

. . , j p - - . m&m*

Vs

........... ] _ ;___ [ IH C C H C H

H = heated at I22°C/3Q min, C

(Electrophores is in Ca-malonate/EtOH2. 9 , 100 V/15 min)

pH 4 . 6

H C

u n heated

buffer, pH

Fig. 2 —H e a t e ffe c ts o n th e e le c tro p h o re t ic p a tte rn s o f'S e a k e m 2 carrageenan.

pH 6 .5 pH 3 .7 pH 5 . 1 p H 4 . 5

----------- -¿mod; i

: i

+im i .... |

:: ' 1.......:...... - •• _ .V.:;,.. i . .......i

C H H C H C C H

H - heated !22°C/30 min, C = un heated

(Electrophoresis in Ca-malonate/EtOH buffer, pH2.9, 100 V/15 min)

P '9 ’ 3 —H e a t e ffe c ts o n th e e le c tro p h o re t ic p a tte rn s o f K -carrageenan (C. c ris p u s }.

ELECTROPHORETIC PATT ERN S OF C A R R A G E E N A N STABILIZERS - 105

were pretreated w ith about 25 m g/m l o f a cation exchange resin (A m berlite C G -1 2 0 ) in the sodium form .

Sam ples o f degraded «-carrageenan were obtained from Marine C olloids, In c ., R ocklan d , Me. T he series had been prepared by degrading a sample o f carrageenan ( 9 0 - 9 5 % kappa) at pH 4 under selected cond ition s follow ed by neutralization and ion exchange to the sodium form . T he viscosities are those reported by Marine Colloids for a 1.5% solution at 7 5°C .

E lectrophoresis was cond ucted w ith the PhoroSlide E lectrophoresis System (M illipore C orp ., B edford , M ass.). T he bu ffer was prepared by dissolving 7 .8g o f m alonic acid (C .R .) in 8 0 0 m l o f w ater and adding solid calcium hyd roxide until pH 2 .9 was reached. T he volume was then adjusted to 1 0 0 0 m l, and 150 ml o f absolute ethan ol was added. Follow ing electrophoresis for 15 min at lOOv, the strips were stained fo r 2 m in w ith 0 .2% toluid ine blue and then rinsed w ith 0 .0 2 5 % potassium acid phthal- ate.

D en sitom eter tracings o f e lectrop h oretic patterns were recorded by reflectan ce from the stained and dried PhoroSlides on a double-beam recording m icro d ensitom eter ( Jo y ce C hrom oscan) using a linear cam . N o attem p t was made to relate the peak area to con cen tration .

RESULTS

A s r e p o r t e d p r e v i o u s l y ( C h a n g e t a l . ,1 9 7 4 ) , z o n a l e l e c t r o p h o r e s i s i n c a l c i u m m a l o n a t e / e t h a n o l b u f f e r p r o d u c e s c h a r a c

t e r i s t i c p a t t e r n s f o r c a r r a g e e n a n . T h e p a t t e r n f o r S e a k e m 2 c a r r a g e e n a n i s s h o w n i n F i g u r e 1 . C a r r a g e e n a n o f t h i s t y p e i s r e p o r t e d t o b e a n u n f r a c t i o n a t e d e x t r a c t

f r o m C. crispus h a v i n g r e c e i v e d o n l y a

m i l d a l k a l i t r e a t m e n t a n d f i n d i n g c o n s i d e r a b l e a p p l i c a t i o n f o r s t a b i l i z i n g f l u i d

m i l k p r o d u c t s . T h e e l e c t r o p h o r e t i c p a t

t e r n i s r e p r e s e n t e d b y a s t a t i o n a r y b a n d a n d t w o m i g r a t i n g z o n e s w h i c h s h o w e v i

d e n c e o f p o s s i b l e i n t e r a c t i o n . T h e l e a d i n g z o n e h a s b e e n i d e n t i f i e d p r e v i o u s l y a s K - c a r r a g e e n a n w h i l e t h e s l o w e r a n d m o r e i n t e n s e b a n d w a s m o v i n g w i t h t h e m o b i l

i t y o f A - c a r r a g e e n a n . T h e s e m o b i l i t y r e l a t i o n s h i p s a r e c u r i o u s b e c a u s e t h e y a r e r e v e r s e d i n N a - m a l o n a t e b u f f e r . S i n c e e l e c t r o p h o r e t i c s e p a r a t i o n o f c o l l o i d s o n c e l

l u l o s e a c e t a t e i s p r i m a r i l y a f u n c t i o n o f

c h a r g e d i f f e r e n c e s , i t w o u l d b e e x p e c t e d t h a t A - c a r r a g e e n a n w i t h t h e h i g h e s t s u l f a t e c o n t e n t w o u l d m o v e f a s t e r t h a n k -

c a r r a g e e n a n . T h i s w a s n o t t h e c a s e ; t h e r e f o r e , i t a p p e a r s t h a t t h e s e l e c t e d b u f f e r

s y s t e m p r o d u c e s e f f e c t s w h i c h a r e o f f s e t t i n g t h e d i f f e r e n c e s i n s u l f a t e c o n t e n t .

A p o s s i b l e i n t e r a c t i o n b e t w e e n t h e

t w o m i g r a t i n g z o n e s w a s n o t i c e d b e c a u s e p u r i f i e d A - c a r r a g e e n a n u s u a l l y s h o w e d a

m o r e b l o t c h y a n d d i f f u s e z o n e w h e n r u n s i n g l y t h a n i n m i x t u r e s w i t h K - c a r r a g e e n -

a n . F u r t h e r m o r e , e l e c t r o p h o r e s i s a t 4 0 ° C

( F i g . 1 , t o p ) r e s u l t e d i n a s h a r p e r s e p a r a t i o n o f t h e s e t w o b a n d s .

T h e s t a t i o n a r y b a n d i n F i g u r e 1 i s a

c h a r a c t e r i s t i c o f t h e p o t a s s i u m a n d c a l c i u m s e n s i t i v e c a r r a g e e n a n s . T h e f o r m a

t i o n o f t h i s b a n d i s e v i d e n t l y t h e r e s u l t o f t h e a g g r e g a t i n g i n f l u e n c e o f c a l c i u m i o n s

a n d i t d i d n o t o c c u r w h e n N a - o r L i -

m a l o n a t e b u f f e r w a s u s e d . T h e i n c r e a s e d i n t e n s i t y o f t h e b a n d w i t h i n c r e a s i n g c a r

r a g e e n a n l o a d , w h i c h i s p a r t i c u l a r l y a p

p a r e n t a t 2 0 ° C ( F i g . 1 , b o t t o m ) , s u g g e s t s t h a t t h e a g g r e g a t i o n m a y b e c o n c e n t r a t i o n a s w e l l a s t e m p e r a t u r e d e p e n d e n t .

H e a t e f f e c t s , a t v a r i o u s p H l e v e l s , o n t h e e l e c t r o p h o r e t i c p a t t e r n s o f d i f f e r e n t c a r r a g e e n a n s w e r e e x a m i n e d b y i n c o r p o

r a t i n g 0 . 4 % o f t h e s t a b i l i z e r s i n a s y n t h e t i c m i l k - s a l t s y s t e m , a d j u s t i n g t h e p H o f t h e s y s t e m f r o m 6 . 7 t o 4 . 6 a n d e x p o s i n g t h e s o l u t i o n s t o 1 2 2 ° C f o r 3 0 m i n . T h e

c h o i c e o f t h e m i l k - s a l t s o l u t i o n w a s m a d e f o r t h e p u r p o s e o f r e p r o d u c i n g t h e i o n i c e n v i r o n m e n t o f m i l k w i t h o u t i n t r o d u c i n g i n t e r f e r e n c e i n t h e s u b s e q u e n t e l e c t r o p h o r e s i s b y t h e o t h e r m i l k c o n s t i t u e n t s .

M i l k s a l t s a r e i m p o r t a n t t o t h e f u n c t i o n o f c a r r a g e e n a n b e c a u s e t h e h i g h c o n c e n t r a t i o n o f c a l c i u m a n d p o t a s s i u m ( T a b l e 1 ) i s s u f f i c i e n t t o i n d u c e g e l a t i o n a n d m a y , t h e r e f o r e , i n f l u e n c e t h e r e s i s t a n c e

o f c a r r a g e e n a n t o h e a t . T h e o n l y p r e t r e a t

m e n t w h i c h w a s r e q u i r e d f o r e l e c t r o p h o r e s i s o f t h e c a r r a g e e n a n w a s i o n e x c h a n g e

t o r e m o v e t h e e f f e c t o f t h e g e l l i n g i o n s . W i t h o u t i o n e x c h a n g e , i t w a s d i f f i c u l t t o

d e p o s i t g e l l e d s a m p l e s u n i f o r m l y o n t h e c e l l u l o s e a c e t a t e m e m b r a n e s w i t h t h e c a l i

b r a t e d a p p l i c a t o r s .

T h e e f f e c t o f h e a t o n t h e p a t t e r n s o f

S e a k e m 2 c a r r a g e e n a n i s s h o w n i n F i g u r e2 . H e a t t r e a t m e n t c a u s e d t h e s t a t i o n a r y

z o n e t o v a n i s h a t a l l p H l e v e l s . I n g e n e r a l , h e a t i n g c a u s e d a m o r e d i s t i n c t s e p a r a t i o n o f t h e t w o m i g r a t i n g z o n e s , a n e f f e c t w h i c h w a s i n e v i d e n c e e v e n f o r t h e s a m

p l e s o f p H 6 . 7 . A s p H w a s l o w e r e d t o w a r d s 4 . 5 , t h e s e t w o b a n d s b e c a m e w e a k e r a n d t h e i r m o b i l i t i e s i n c r e a s e d s o m e w h a t .

A n e a r l y i d e n t i c a l s e q u e n c e o f e v e n t s w a s r e c o r d e d f o r K - c a r r a g e e n a n p r e p a r e d a s t h e p o t a s s i u m g e l l i n g f r a c t i o n o f C.

pH 6 .7 pH 5 .5 pH 4 .5

- 1

—

>*/•

' ¡Spy i -•v ; / , . •• !r ! > ¿>1

‘--‘ J

M ij

. »¡SSglgi:

4- .

H C C H H C

H = heated 122°C/30 min, C = unheated

(Electrophoresis in Ca-malonate/EtOH buffer, pH2.9, 100V/15 min)

Fig. 4 - H e a t e ffe c ts o n th e e le c tro p h o re t ic p a tte rn s o f \-ca rra geen an (C. c rispus).

■\06 -JOURNAL OF FOOD S CI EN C E- Vo lu m e 3 9 (1974)

pH 6 . 6 pH 5 . 6 pH 5 . 0 pH 4 . 5

C H H C C H H C

H ■ heated at 122°C/30 min , C = un heated

(Electrophoresis in Ca-malonate/EtOH buffer, pH2.9, 100 V/15 min)

Fig. 5 —H e a t e ffe c ts o n th e e le c tro p h o re t ic p a tte rn s o f i-carrageenan (E. s p in o s u m ).

crispus e x t r a c t ( F i g . 3 ) . H o w e v e r , t h e u n

h e a t e d c o n t r o l p a t t e r n s ( c ) r e v e a l e d t h e p r e s e n c e o f m o r e t h a n o n e c o m p o n e n t , o n e o f w h i c h m i g r a t e d w i t h t h e m o b i l i t y

o f X - c a r r a g e e n a n . S u c h h e t e r o g e n e i t y m a y p o s s i b l y h a v e b e e n c a u s e d b y t h e c a r r y o v e r o f a s i g n i f i c a n t a m o u n t o f t h e n o n

g e l l i n g X - c o m p o n e n t s u g g e s t i n g t h a t p o t a s s i u m f r a c t i o n a t i o n m a y b e f a r f r o m i d e a l f o r p u r i f i c a t i o n o f c a r r a g e e n a n . H e a t i n g o f K - c a r r a g e e n a n d e s t r o y e d t h e

s t a t i o n a r y z o n e a n d c a u s e d i n c r e a s e d m o b i l i t i e s o f t h e m i g r a t i n g z o n e s . T h e r e w a s e v i d e n c e t h a t t h e f r a c t i o n i n t h e X-

z o n e w a s d e s t r o y e d p r e f e r e n t i a l l y l i k e i t

w a s i n S e a k e m 2 .H e a t a n d p H e f f e c t s o n X - c a r r a g e e n a n

( F i g . 4 ) m a y b e m o r e d i f f i c u l t t o a s s e s s b e c a u s e p u r i f i e d X - c a r r a g e e n a n d o e s n o t p r o d u c e a s h a r p z o n e o n c e l l u l o s e a c e t a t e

i n a n y o f t h e b u f f e r s y s t e m s w e h a v e e x a m i n e d . T h e a p p a r e n t e f f e c t o f h e a t w a s t o p r o d u c e a s h a r p e r z o n e w i t h i n c r e a s e d m o b i l i t y . H o w e v e r , i t i s a t l e a s t p o s s i b l e t h a t t h e r e m a i n i n g z o n e i n t h e h e a t e d s o l u t i o n a t p H 4 . 5 m a y n o t r e p r e s e n t X - c a r r a g e e n a n b u t m a y b e a m o r e

h e a t r e s i s t a n t c o n t a m i n a n t .C a r r a g e e n a n e x t r a c t s f r o m E. spino

sum ( t - c a r r a g e e n a n ) p r o d u c e d a v e r y i n t e n s e s t a t i o n a r y e l e c t r o p h o r e t i c z o n e ( F i g . 5 ) w i t h o n l y f a i n t m a r k i n g s f o r m i g r a t i n g z o n e s . A g a i n t h e e f f e c t o f h e a t a n d l o w p H w a s t o d e s t r o y t h e s t a t i o n a r y z o n e a n d t o p r o d u c e n e w m i g r a t i n g z o n e s

i n t h e p a t t e r n . T h i s w a s m o s t e v i d e n t a t p H 4 . 5 .

T h e e f f e c t o f v a r y i n g t h e h e a t i n g t i m e i n t h e p H r a n g e 6 . 5 —6 . 7 i s s h o w n b y t h e d e n s i t o m e t e r t r a c i n g s i n F i g u r e 6

( S e a k e m 2 c a r r a g e e n a n ) a n d i n F i g u r e 7 ( K - c a r r a g e e n a n ) . I n b o t h c a s e s , t h e r e w a s

a g r a d u a l l o s s o f t h e s t a t i o n a r y z o n e a s w e l l a s a g r a d u a l s e p a r a t i o n o f t h e m i g r a t i n g z o n e s . T h e m a i n d i f f e r e n c e t o b e

n o t i c e d b e t w e e n t h e t w o t y p e s w a s n o t i n t h e e l e c t r o p h o r e t i c m o b i l i t i e s b u t i n t h e

m a g n i t u d e s o f t h e t w o h e a t - d i s s o c i a t e d p e a k s . T h e a p p e a r a n c e o f a s p l i t s t a t i o n

a r y z o n e , i n s o m e c a s e s ( e . g . , F i g . 7 , 1 5 m i n ) , w a s a n a r t i f a c t c a u s e d b y t h e u n

e v e n d e p o s i t i o n o f s a m p l e f r o m t h e

d o u b l e w i r e a p p l i c a t o r .A c o m p a r i s o n o f t h e v a r i o u s e l e c t r o

p h o r e t i c p a t t e r n s s h o w a g e n e r a l l o s s o f s t a i n i n g i n t e n s i t y f o r t h e s t r o n g l y h e a t e d

c a r r a g e e n a n . T h i s i s p a r t i c u l a r l y e v i d e n t i n t h e d e n s i t o m e t e r t r a c i n g s o f h e a t e d

K - c a r r a g e e n a n ( F i g . 7 ) . I t m a y b e n o t i c e d t h a t t h e d i s a p p e a r a n c e o f t h e i n t e n s e s t a

t i o n a r y z o n e d i d n o t r e s u l t i n a n i n c r e a s e i n t h e m i g r a t i n g p e a k s . T h i s l o s s i n d y e b i n d i n g c a p a c i t y t h r o u g h h e a t i n g i s c u r r e n t l y u n d e r i n v e s t i g a t i o n .

T h e h e a t - i n d u c e d c h a n g e s w e r e s h a r p l y a c c e l e r a t e d a s t h e p H o f t h e s y s t e m w a s l o w e r e d . T h e d e n s i t o m e t e r t r a c i n g s i n F i g u r e 8 o f d e g r a d e d K - c a r r a g e e n a n s h o w t h a t c o n t r o l l e d a c i d h y d r o l y s i s l e a d t o n e a r l y i d e n t i c a l c h a n g e s i n t h e c a r r a g e e n a n p a t t e r n s t o t h o s e o b s e r v e d f o r t h e s t r o n g l y h e a t e d c a r r a g e e n a n a t p H 6 . 5 ( F i g . 7 ) . T h e o n e e x c e p t i o n n o t e d w a s t h e a b s e n c e o f a n i n c r e a s e i n t h e m o b i l i t y o f t h e m i g r a t i n g z o n e s i n F i g u r e 8 .

DISCUSSION

T H E R E S U L T S o f t h i s s t u d y h a v e s h o w n t h a t w h o l e c a r r a g e e n a n a n d i t s f r a c t i o n s

a r e s u b j e c t t o h e a t - i n d u c e d a l t e r a t i o n s u n d e r s e v e r e h e a t t r e a t m e n t , p a r t i c u l a r l y a t l o w p H v a l u e s . S u c h e f f e c t s a r e n o t n e c e s s a r i l y l i m i t e d t o c a r r a g e e n a n b e c a u s e

f e w c o l l o i d s w o u l d r e m a i n i n e r t t o s u c h - d r a s t i c t r e a t m e n t s . K i r a t s o u s e t a l . ( 1 9 7 3 ) h a v e s h o w n t h a t b o t h c o l o r a n d v i s c o s i t y

o f a r a n g e o f f o o d s t a b i l i z e r s a r e a f f e c t e d b y h i g h - t e m p e r a t u r e p r o c e s s i n g .

T h e c h a n g e s i n c a r r a g e e n a n a r e r e f l e c t e d i n t h e i r z o n a l e l e c t r o p h o r e t i c p a t t e r n s

b y ( a ) a l o s s i n t h e i n t e n s i t y o f a c a l c i u m - a g g r e g a t e d s t a t i o n a r y z o n e w h i c h i s c h a r

a c t e r i s t i c f o r b o t h K - a n d t - c a r r a g e e n a n

a n d ( b ) a n i n c r e a s e i n t h e m o b i l i t y o f t w o m i g r a t i n g z o n e s , c h a r a c t e r i s t i c f o r k - a n d

X - c a r r a g e e n a n .

O u r p r e v i o u s f i n d i n g ( C h a n g e t a i . ,

1 9 7 4 ) t h a t c a r r a g e e n a n s t a b i l i z e r s r e c o v e r e d f r o m p r o c e s s e d m i l k d o n o t

m a t c h t h e i r r e s p e c t i v e c o n t r o l s c a n p e r h a p s b e e x p l a i n e d a s a c o n s e q u e n c e o f t h e s e h e a t e f f e c t s . I f s o , i t w o u l d m a k e

e l e c t r o p h o r e t i c i d e n t i f i c a t i o n o f h y d r o c o l

l o i d s i n t h e s e p r o d u c t s d i f f i c u l t . H o w e v e r , t h e o b s e r v e d c h a n g e s w e r e p r o n o u n c e d o n l y a t e x t r e m e s o f h e a t t r e a t m e n t a n d

w e f o u n d n o s i g n i f i c a n t a l t e r a t i o n s t o c a r r a g e e n a n p a t t e r n s u n d e r m o d e r a t e h e a t i n g ( 1 2 2 ° C / 5 m i n ) a t p H 6 . 7 .

T h e n a t u r e o f t h e c h a n g e s w h i c h t a k e p l a c e h a v e n o t b e e n d e t e r m i n e d b u t s i n c e t h e y a r e a c c e l e t a t e d a t l o w p H , i t i s p o s s i b l e t h a t h y d r o l y t i c r e a c t i o n s m a y b e i n v o l v e d . I t s h o u l d b e e m p h a s i z e d t h a t z o n e

s e p a r a t i o n o n c e l l u l o s e a c e t a t e i s a f u n c t i o n o f c h a r g e r a t h e r t h a n p o l y m e r s i z e a n d t h e r e f o r e t h i s m e t h o d w o u l d n o t b e

a p p r o p r i a t e f o r a s t u d y o f p o l y m e r b r e a k d o w n .

ELECTROPHORETIC PATT ERN S OF C A R R A G E E N A N STAB!L /Z E f f S - 1 0 7

Fig. 6 —E ffe c t o f h e a tin g t im e o n e le c tro p h o

re tic p a tte rn s o f Seakem 2 carrageenan he a te d a t p H 6 .7 I R e fle c ta n ce d e n s ito m e te r scan).

F ig. 7 —E ffe c t o f h e a tin g t im e o n e le c tro p h o

re tic p a tte rn s o f K -carrageenan a t p H 6 .5 (R e

fle c ta n ce d e n s ito m e te r scan).

Fig. 8—Electrophoretic patterns of acid degraded K-carrageenan (Reflectance densitometer scan).

F r o m a t e c h n i c a l p o i n t o f v i e w , i t i s o f i n t e r e s t t o d e t e r m i n e i f t h e o b s e r v e d h e a t

i n d u c e d c h a n g e s t o c a r r a g e e n a n s t r u c t u r e s m a y a l s o l e a d t o a l t e r e d f u n c t i o n a l p r o p e r t i e s . A n i n d i c a t i o n t h a t t h i s m a y b e t h e

c a s e w a s o b t a i n e d i n a s t u d y o f t h e s t a b i l i z a t i o n o f r e n n e t t r e a t e d m i l k p r o t e i n s

b y c a r r a g e e n a n ( O ’ L o u g h l i n a n d H a n s e n ,1 9 7 3 ) b u t n o r e p o r t i s a v a i l a b l e r e l a t i n g

t o c o m m e r c i a l f o o d p r o d u c t s . B e s i d e s

t h e s e c o n s i d e r a t i o n s , t h e r e c e n t c o n c e r n s e x p r e s s e d a b o u t t h e s a f e t y o f d e g r a d e d c a r r a g e e n a n i n f o o d ( I F T E x p e r t P a n e l ,1 9 7 3 ) c l e a r l y s u g g e s t t h a t r e s e a r c h is

n e e d e d t o d e t e r m i n e t h e f a t e o f c a r r a g e e n a n i n p r o c e s s e d f o o d s .

REFERENCES

C h a k r a b o r t y , B .K . a n d R a n d o l p h , H . E . 1 9 7 2 . S t a b i l i z a t i o n o f c a l c i u m s e n s i t i v e p l a n t p r o t e i n s b y K - c a r r a g e e n a n s . J . F o o d S c i . 3 7 : 7 1 9 .

C h a n g , J . C . , R e n o l l , M .W . a n d H a n s e n , P . M . T .1 9 7 4 . Z o n e e l e c t r o p h o r e s i s o f f o o d s t a b i l i z e r s i n m a i o n a t e b u f f e r . J . F o o d S c i . 3 9 : 0 0 0 .

F e d e r a l F o o d , D r u g a n d C o s m e t i c A c t . 1 9 6 7 . D e f i n i t i o n s a n d S t a n d a r d s , P a r t 1 8 , p . 3 a n d 5: M i lk a n d C r e a m . A m e n d m e n t s i n F e d e r a l R e g i s t e r , F e b r u a r y 1 , 1 9 6 7 .

I F T E x p e r t P a n e l o n F o o d S a f e t y & N u t r i t i o n .1 9 7 3 . C a r r a g e e n a n . J . F o o d S c i . 3 8 : 3 6 7 .

J e n n e s s , R . a n d K o o p s , J . 1 9 6 2 . P r e p a r a t i o n a n d p r o p e r t i e s o f a s a l t s o l u t i o n w h i c h s i m u l a t e s m i l k u l t r a f i l t r a t e . N e t h . M i lk & D a i r y J . , 1 6 : 1 5 3 .

K i r a t s o u s , A .S . , F r a n c i s , F . J . a n d Z a h r a d n i k , J .W . 1 9 6 2 . E f f e c t o f h i g h - t e m p e r a t u r e s h o r t - t i m e p r o c e s s i n g o n t h e v i s c o s i t y a n d c o l o r o f

f ive t h i c k e n i n g a g e n t s . F o o d T e c h n o l . 1 6 ( 7 ) : 1 1 1 .

L i n , C . F . a n d H a n s e n , P . M . T . 1 9 7 0 . S t a b i l i z a t i o n o f c a s e i n m i c e l l e s b y c a r r a g e e n a n . M a c r o m o l e c u l e s 3 : 2 6 9 .

O ’L o u g h l i n , D . K . a n d H a n s e n , P . M . T . 1 9 7 3 . S t a b i l i z a t i o n o f r e n n e t t r e a t e d m i l k p r o t e i n s b y c a r r a g e e n a n . J . D a i r y S c i . 5 6 : 6 2 9 .

R e e s , D . A . 1 9 6 9 . S t r u c t u r e , c o n f o r m a t i o n a n d m e c h a n i s m in t h e f o r m a t i o n o f p o l y s a c c h a r i d e ge ls a n d n e t w o r k s . A d v . C a r b o h y d . C h e m . 2 4 : 2 6 7 .

M s r e c e i v e d 6 / 2 8 / 7 3 ; r e v i s e d 1 0 / 4 / 7 3 ; a c c e p t e d 1 0 / 8 / 7 3 . _____________________________________

A r t i c l e 9 - 7 3 , D e p t , o f F o o d S c i e n c e & N u t r i t i o n . I n v e s t i g a t i o n w a s s u p p o r t e d b y U .S . P u b l i c H e a l t h S e r v i c e R e s e a r c h G r a n t F D - 0 0 1 1 7 , O f f i c e o f R e s e a r c h a n d T r a i n i n g G r a n t s , F D A a n d b y a g r a n t f r o m R o s s L a b o r a t o r i e s , C o l u m b u s , O h i o . A p p r e c i a t i o n is e x p r e s s e d t o D . S t a n c i o f f , M a r i n e C o l l o i d s , I n c . f o r s u p p l y i n g a s e r i e s o f d e g r a d e d k -c a r r a g e e n an s .

P O -M IN U N a n d J. G. L E E D E R

D ept, o f F o o d Science, R u tg e rs U n iv e rs ity

The S ta te U n iv e rs ity o f N e w Jersey, N e w B ru n s w ic k , N J 0 8 9 0 3

MECHANISM OF EMULSIFIER ACTION IN AN ICE CREAM SYSTEM

INTRODUCTION

I N T H E P A S T , w h e n t h e f a t e m u l s i o n i n s o f t - s e r v e i c e c r e a m m i x e s b e g a n t o d e s t a b i l i z e o r “ c h u r n , ” t h e c o m m o n i n d u s t r y p r a c t i c e w a s t o a d d m o r e e m u l s i f i e r a n d ,

p r e s u m a b l y , r e d i s p e r s e t h e f a t b a c k i n t o t h e m i x . F o r m a n y y e a r s , i t h a s b e e n a s s u m e d t h a t e m u l s i f i e r s , w h e n a d d e d t o a n i c e c r e a m m i x , i m p a r t t h e i r b e n e f i c i a l p r o p e r t i e s s o l e l y b y r e d u c i n g t h e i n t e r f a c i a l t e n s i o n b e t w e e n t h e f a t p h a s e a n d t h e a q u e o u s p h a s e o f t h e e m u l s i o n . T h i s p r a c t i c e w a s b e l i e v e d t o p e r m i t a f i n e r d i s p e r s i o n o f t h e f a t i n t h e m i x , p r o d u c e a f i n e r a i r c e l l s t r u c t u r e a n d i m p r o v e w h i p p i n g a b i l i t y . L a t e r , i n v e s t i g a t i o n s b y K l o s e r a n d K e e n e y ( 1 9 5 9 ) s h o w e d t h a t e m u l s i f i e r s m a y a c t u a l l y d e e m u l s i f y t h e f a t e m u l s i o n a n d t h i s d e e m u l s i f i c a t i o n w a s s u g g e s t e d a s b e i n g r e s p o n s i b l e f o r d r y n e s s i n i c e c r e a m . R e c e n t i n v e s t i g a t o r s h a v e d e m o n s t r a t e d t h a t t h e n o n f a t m i l k

s o l i d s a l o n e , a t t h e l e v e l n o r m a l l y u s e d i n i c e c r e a m , h a v e s u f f i c i e n t e m u l s i f y i n g

c a p a c i t y t o e m u l s i f y t h e f a t p r e s e n t i n a s t a n d a r d i c e c r e a m m i x . T h e a d d e d c h e m i c a l e m u l s i f i e r , r e g a r d l e s s o f i t s H L B o r

c o n c e n t r a t i o n , c o n t r i b u t e s l i t t l e t o t h e e m u l s i f i c a t i o n o f t h e m i x ; i n s t e a d , i t a c t u a l l y p r o m o t e s d e e m u l s i f i c a t i o n w h e n t h e m i x i s f r o z e n i n a n i c e c r e a m f r e e z e r

( G o v i n a n d L e e d e r , 1 9 7 1 ) . A m o r e s y s t e m a t i c s t u d y b y t h e m h a s a l s o s h o w n t h a t t h e d e g r e e o f f a t d e e m u l s i f i c a t i o n

c a u s e d b y a n e m u l s i f i e r , a t a n y c o n c e n t r a t i o n , i s r e l a t e d t o i t s H L B . T h e m o r e h y d r o p h i l i c t h e e m u l s i f i e r , t h e g r e a t e r t h e

d e e m u l s i f i c a t i o n .

I t h a s b e e n g e n e r a l l y b e l i e v e d t h a t e m u l s i f i e r s a r e a d s o r b e d a n d c o n c e n t r a t e d a t t h e o i l a n d a q u e o u s i n t e r f a c e , t h u s r e d u c i n g t h e i n t e r f a c i a l t e n s i o n a n d m a k i n g a s t a b l e e m u l s i o n . H o w e v e r , a m a r k e d c o a g u l a t i o n o f p r o t e i n ( c a s e i n ) a t t h e o i l - w a t e r i n t e r f a c e h a s b e e n r e p o r t e d ( W a l k e r , 1 9 6 0 ) . L a t e r e v i d e n c e b y D u r h a m e t a l . ( 1 9 6 2 ) s h o w e d t h a t a s t r o n g c o m p l e x is f o r m e d b e t w e e n f a t t y a c i d ( p r e s e n t i n c o m m e r c i a l m o n o g l y c e r i d e ) a n d a m i d o g r o u p s i n c a s e i n . K e e n e y ( 1 9 6 2 ) r e p o r t e d t h a t t h e f o a m p o r t i o n o f m e l t e d i c e c r e a m w a s h i g h e r i n f a t a n d l o w e r i n e m u l s i f i e r c o n t e n t , w h i l e t h e s e r u m p o r t i o n w a s h i g h e r i n e m u l s i f i e r a n d l o w e r i n f a t c o n t e n t . B a s e d o n t h e s e o b s e r v a t i o n s , G o v i n ( 1 9 6 9 ) s u g g e s t e d t h a t t h e r e m i g h t b e a n i n t e r a c t i o n b e t w e e n e m u l s i f i e r a n d p r o t e i n .

T h e e f f e c t o f e m u l s i f i e r s a n d c o r n s y r u p s o l i d s o n f a t d e s t a b i l i z a t i o n i n s o f t - s e r v e i c e c r e a m m i x h a s a l s o b e e n r e

p o r t e d b y K l o t z e k e t a l . ( 1 9 6 3 ) . H o w e v e r , a l a t e r i n v e s t i g a t i o n b y M a h d i a n d B r a d l e y ( 1 9 6 9 ) s h o w e d t h a t t h e a m y l o s e

f r a c t i o n o f c o r n s w e e t e n e r s u s e d i n f r o z e n d e s s e r t s i s i n v o l v e d i n a c o m p l e x w i t h a s

a n d p c a s e i n .S i r . c e t h e r e i s l i t t l e i n f o r m a t i o n p u b

l i s h e d o n t h e m e c h a n i s m s c o n c e r n i n g

e m u l s i f i e r a c t i o n s i n i c e c r e a m , t h i s i n v e s t i g a t i o n w a s p l a n n e d w i t h t h e f o l l o w i n g

o b j e c t i v e s :

( 1 ) T o c o n f i r m t h e r e l a t i o n s h i p b e t w e e n t h e H L B o f a d d e d e m u l s i f i e r a n d i t s

e f f e c t o n f a t d e e m u l s i f i c a t i o n .( 2 ) T o i n v e s t i g a t e t h e i n t e r a c t i o n b e

t w e e n e m u l s i f i e r s a n d m i l k p r o t e i n s , a n d t h e r e l a t i o n s h i p o f t h e i n t e r

a c t i o n t o t h e f a t e m u l s i o n s t a b i l i t y i n i c e c r e a m .

( 3 ) T o e s t a b l i s h a m e c h a n i s m o f e m u l s i f i e r a c t i o n i n a n i c e c r e a m s y s t e m .

EXPERIMENTAL

Preparing the ice cream mixT h roughou t the study, all the ice cream

mixes were prepared using the following formula:

Milk fat 12%Nonfat milk solids 11%Sucrose 15%Emulsifier 0.1%Water 61.9%

Emulsifiers o f different HLB’s were pre-pared as follows:

HLB value Composit ion4 88% Span 80 & 12% Span 85B 65% Span 80 & 35% T ween 80

12 28% Span 80 & 72% T ween 8016 60% Tw een 20 & 40% Tween 80

F or each trial, 1 kg o f mix was made. The required am o u n ts o f nonfa t milk solids and sucrose were blended and stirred thoroughly into one-third o f the total required am o u n t o f water . The emulsif ier and ano ther one-third of the water were then added and mixed. Finally, the weighed am oun t o f fat (sweet bu t te r ) and the rest o f the water, which had been warmed to 5 0 ° C, were added to the m ixture . T he mixtures were pasteurized at 71°C for 30 min and homogenized at the same tem pera tu re in a Man- ton-Gaulin two-stage single piston homogenizer. A homogenizing pressure o f 2000 and 500 psi was used on the first- and second-stage valves, respectively. The homogenized mixes were

cooled and stored a t 4 .4°C for 24 hr before freezing. A small am o u n t o f unf rozen m ix from each trial was saved for the emulsion stability rest. T he mixes were frozen in a m odel 5P Electro Freeze soft-serve batch ice cream freezer at - 7 . 2 ° C for 9 min.

Emulsion stability test o ffrozen and unfrozen ice cream mixes

The emulsion stability test em ployed was the light transmission m ethod o f Keeney and Josephson (1958) as modified by Govin and Leeder (1971).

Isolation and prepara tion o f milk proteins

The casein and serum prote ins from u n frozen ice cream mixes or m elted f rozen ice cream mixes were isolated by the m ethod de- s c ib e d by Haddad (1970).

T o prepare a casein sample for elec trophoresis, an am o u n t o f the freeze-dried protein sufficient to obta in a final p ro te in concen tra tion o f 15% was weighed and dispersed with 7M urea solution. The m ix tu re was w armed to 5C°C and held 15 min and then centrifuged with a h a r d centrifuge to facilita te the separation o f the milk fat which was then discarded by means o f a water aspirator . T h e remaining solution was then ready for e lec trophore tic analysis.

To prepare a sample o f serum pro te ins for electrophoresis, the dry serum pro te ins were dissolved in distilled water to give a 10% solution.

Electrophoretic analysis o f the interaction between milk pro teins and emulsif iers

To investigate the possibility o f an interaction, two types of zonal e lectrophoresis were utilized. One was cellulose ace ta te e lec tro phoresis, the o ther was polyacrylam ide gel electrophoresis.

In cellulose acetate electrophoresis , the elect rophore t ic pa t terns were developed using 5 /al of protein solution in a buffer solution o f pH 3.60 and ionic strength of 0.05 under 200v for 2 hr.

In polyacrylamide gel e lectrophoresis, the geis for serum pro te in analysis were prepared according to the m e th o d o f Davis and Ornstein(1959), whereas the gels for casein analysis were prepared by the m ethod described by Grindrod and Nickerson (1968). A current of2.5 n iAmp per column was used.

Model system studiesMilk proteins were isolated from raw milk as

follows: Skimmilk was prepared f rom raw milk by centrifuging it at 4 ,0 0 0 x G for 15 min. The skimmilk was then tempered to 50°C and acidified to pH 4 .60 with IN HC1. T he m ix tu re was allowed to react for 15 min and was then filtered. The precipitate (casein) was washed several times with warm distil led water (50°C)

W 8 - J O U R N A L OF FOOD S CI EN C E-V o lu me 3 9 (1974)

MECHANISM OF EMULSIFIER IN ICE CREAM - 1 0 9

to remove the residual whey and then dried. 4% casein solution was prepared by dispersing the casein into 7M urea solution.

The whey ob ta ined f rom the p repara t ion o f the casein fraction was filtered twice through Whatman No. 1 filter paper , then dialyzed against distilled water at refr igeration tem perature (4 .4°C) for 3 days with the water being changed twice a day. T he dilute serum prote in solution in the cellulose dialyzing tubing was then pervaporated a t refr igeration tem pera tu re until a concen tra t ion o f a bou t 1.5% o f serum proteins was obtained.

Various emulsif iers used in this study are listed in Table 1. 4% emulsif ier solution was made so tha t its final concen tra t ion w ould be0.4% when 0.1 ml o f it was mixed w ith 0.9 ml of milk p ro te in solution. The m ix tures were then kep t at pas teurization tem pera tu re (7 1 3C) for 30 min with cons tan t stirring.

The detec t ion o f an in terac tion between milk pro teins and emulsif iers was again per form ed by the two e lec trophore tic techniques. The materials, appara tus and procedures were the same as previously described.


The effect of HLB of emulsifier at constant concentration on fat deemulsification of frozen and unfrozen ice cream mixes

I n t h i s e x p e r i m e n t , a s e r i e s o f m i x e s c o n t a i n i n g n o e m u l s i f i e r ( a s c o n t r o l ) a n d

e m u l s i f i e r s w i t h F I L B ’s o f 4 , 8 , 1 2 a n d 1 6 w e r e p r e p a r e d , h o m o g e n i z e d a n d p a s t e u r i z e d . A p o r t i o n o f e a c h m i x s a m p l e w a s

u s e d f o r t h e d e t e r m i n a t i o n o f f a t d e - e m u l s i f i c a t i o n . T h e r e m a i n i n g m i x w a s

f r o z e n i n a n i c e c r e a m f r e e z e r . T h e d e g r e e o f f a t d e e m u l s i f i c a t i o n w a s d e t e r m i n e d a f t e r t h e s a m p l e h a d b e e n m e l t e d .

I n F i g u r e 1 , t h e e f f e c t o f i n c r e a s i n g t h e H L B o f e m u l s i f i e r o n f a t d e e m u l s i f i c a t i o n i s p r e s e n t e d . I n c u r v e s A a n d A c , t h e e f f e c t a p p e a r s t o b e s i m i l a r t o t h a t p r e s e n t e d b y G o v i n a n d L e e d e r ( 1 9 7 1 ) , t h a t i s , a t a c o n s t a n t e m u l s i f i e r c o n c e n t r a t i o n , f a t d e e m u l s i f i c a t i o n i n c r e a s e d w i t h

i n c r e a s i n g H L B o f t h e e m u l s i f i e r . I n c o m p a r i n g c u r v e s B a n d B e , t h e r e s u l t s d o n o t s h o w a s i g n i f i c a n t i n c r e a s e i n t h e s t a b i l i t y o f t h e f a t e m u l s i o n o f t h e u n f r o z e n i c e c r e a m m i x w i t h i n c r e a s i n g H L B . H o w

e v e r , i n c o m p a r i n g t h e f a t e m u l s i o n s t a b i l i t y o f i c e c r e a m m i x e s b e f o r e a n d a f t e r

f r e e z i n g ( C u r v e s A a n d B ) , i t i s q u i t e e v i d e n t t h a t f r e e z i n g a n d a g i t a t i o n h a v e a p r o n o u n c e d e f f e c t o n t h e r a t e o f f a t d e

e m u l s i f i c a t i o n .

Study of the interaction of milk proteins and emulsifiers in frozen and unfrozen ice cream mixes

T h e r e s u l t s f r o m b o t h c e l l u l o s e a c e t a t e a n d p o l y a c r y l a m i d e g e l e l e c t r o p h o r e

t i c a n a l y s i s s h o w e d t h a t n o a p p r e c i a b l e c h a n g e o c c u r r e d i n t h e e l e c t r o p h o r e t i c

p a t t e r n s o f c a s e i n a n d s e r u m p r o t e i n s b e t w e e n t h e c o n t r o l s a n d t h e i r r e s p e c t i v e f r o z e n a n d u n f r o z e n m i x e s c o n t a i n i n g

e m u l s i f i e r s . A p p a r e n t l y t h e i n t e r a c t i o n

Table 1—List o f emulsifiers, the ir sources and code numbers

Code # Emulsifier Source

i Monoglycerides (HLB 3.1) Durkee Co.,40% mono; 1 % f.f.a. Cleveland, Ohio

ii Span 80 (HLB 4.3) Atlas Chemical Co., W ilm ington, Del.

iii Tween 65 (HLB 10.5) Atlas Chemical Co.iv Tween 80 (HLB 15.0) Atlas Chemical Co.V Lecithin (vegetable) W.A. Cleary Co.,

New Brunswick, N.J.vi Sodium dodecyl sulfate Matheson Company,

East Rutherford, N.J.vii Sodium stearoyl-2-lactylate Pateo Company,

Kansas C ity, Mo.

b e t w e e n t h e m i l k p r o t e i n s a n d t h e e m u l s i

f i e r s d i d n o t o c c u r o r w a s n o t s i g n i f i c a n t e n o u g h t o b e d e t e c t e d b y e l e c t r o p h o r e s i s . I t i s a l s o p o s s i b l e t h a t t h e e m u l s i f i e r - p r o t e i n c o m p l e x d i s s o c i a t e d d u r i n g t h e c o u r s e o f i s o l a t i o n .

I n m o d e l s y s t e m s t u d i e s w h e r e o n l y m i l k p r o t e i n s a n d e m u l s i f i e r s w e r e u s e d t o e l i m i n a t e t h e c o m p l e x i t y o f a n i c e c r e a m m i x , t h e a b o v e t w o t y p e s o f e l e c t r o p h o r e s i s w e r e a g a i n c o n d u c t e d . T h e e l e c t r o p h o r e t i c p a t t e r n s o f c a s e i n , c o n

t a i n i n g n o e m u l s i f i e r ( a s c o n t r o l ) a n d t h o s e c o n t a i n i n g d i f f e r e n t e m u l s i f i e r s , a r e p r e s e n t e d i n F i g u r e 2 , w h i l e t h o s e o f

s e r u m p r o t e i n s a r e p r e s e n t e d i n F i g u r e 3 .

S i m i l a r e l e c t r o p h o r e t i c p a t t e r n s w e r e o b t a i n e d f r o m c a s e i n ( C j , Q j , C m , C j v )

Fig. 1 —E m u ls io n s ta b ilit ie s o f fro z e n a n d u n

fro z e n ice c re am m ix e s w ith e m u ls if ie rs o f d i f

fe re n t H L B 's . (A ) F ro ze n ice c re am m ix ; (B ) U n fro z e n ice c re am m ix ; (A c ) C o n t ro l w ith no e m u ls if ie r in fro z e n ice c re am m ix ; (B e) C o n tro l w ith n o e m u ls if ie r in u n fro z e n ice c re am m ix .

a n d f r o m s e r u m p r o t e i n s ( S ; , S q , S p ; ,

S jv ) , w h i c h c o n t a i n e d n o n i o n i c e m u l s i f i e r s . S u b s t a n t i a l c h a n g e s w e r e o b s e r v e d

( F i g . 2 a n d 3 ) i n t h e e l e c t r o p h o r e t i c p a t

t e r n s o f c a s e i n ( C v , C v j , C v j j ) a n d s e r u m

p r o t e i n s ( S v , S v j , S v i j ) , w h i c h c o n t a i n e d i o n i c e m u l s i f i e r s . T h i s i n d i c a t e s t h a t n o n i o n i c e m u l s i f i e r s d o n o t i n t e r a c t w i t h

m i l k p r o t e i n s w h e r e a s i o n i c e m u l s i f i e r sd o . T h i s r e s u l t i s i n a g r e e m e n t w i t h t h a t o f C h e e s e m a n ( 1 9 6 8 ) w h o s t u d i e d t h e i n

t e r a c t i o n o f c a s e i n a n d s o d i u m d o d e c y l s u l f a t e . T h i s r e s u l t i s a l s o i n a g r e e m e n t

w i t h T s e n ( u n d a t e d ) w h o d e m o n s t r a t e d b y m e a n s o f g e l c h r o m a t o g r a p h y t h a t c a l c i u m s t e a r o y l - 2 - l a c t y l a t e c a n f o r m a s t a

b l e c o m p l e x w i t h p r o t e i n . I n b o t h c a s e s , s o d i u m d o d e c y l s u l f a t e a n d c a l c i u m s t e a r - o y l - 2 - l a c t y l a t e a r e i o n i c e m u l s i f i e r s w h i c h c a n i n t e r a c t o r f o r m a c o m p l e x w i t h p r o t e i n s .

A proposed mechanism of emulsifier action in an ice cream system

B e f o r e p r o p o s i n g t h e m e c h a n i s m o f

e m u l s i f i e r a c t i o n i n i c e c r e a m , i t w o u l d b e h e l p f u l t o s u m m a r i z e s o m e o f t h e e a r l i e r

r e p o r t s o n f a t d e s t a b i l i z a t i o n i n i c e c r e a m . 7 . h a s b e e n r e p o r t e d t h a t c o a l e s c e n c e o f m i l k f a t g l o b u l e s o c c u r r e d d u r i n g t h e f r e e z i n g p r o c e s s a n d t h e d e g r e e o f f a t d e s t a b i l i z a t i o n i n c r e a s e d d i r e c t l y w i t h

f r e e z i n g t i m e ( K e e n e y , 1 9 5 8 ; F r a z e u r ,

1 9 5 9 , 1 9 6 0 ; A l s a f a r a n d W o o d , 1 9 6 6 ; K l o t z e k a n d L e e d e r , 1 9 6 6 ; B e r g e r a n d W h i t e , 1 9 7 1 ) . S h e r m a n ( 1 9 6 6 ) n o t e d t h a t t h e d e g r e e a n d r a t e o f c o a l e s c e n c e d e p e n d o n t h e e x t e n t t o w h i c h t h e a d s o r b e d e m u l s i f i e r - p r o t e i n l a y e r s a r o u n d t h e f a t g l o b u l e s w e r e r u p t u r e d d u r i n g f r e e z i n g . A l s a f a r a n d W o o d ( 1 9 6 6 ) n o t e d t h a t s o m e e m u l s i f i e r m a y b e a d s o r b e d o n a r e a s o f t h e g l o b u l e s u r f a c e n o t c o m p l e t e l y f i l l e d w i t h a d s o r b e d p r o t e i n p a r t i c l e s f r o m t h e s e r u m , a n d t h a t s o m e o f t h e e m u l s i f i e r m a y r e p l a c e m o s t o f t h e a d s o r b e d p r o t e i n p a r t i c l e s . K l o t z e k a n d L e e d e r ( 1 9 6 6 ) s t a t e d t h a t i n t h e f r e e z e r

t h e e m u l s i f i e r w a s b r o k e n o r d i s l o d g e d

1 1 0 - J O U R N A L OF FOOD SC IE N C E -V o lu m e 3 9 (1974)

c c . c . c . . . C , C C , C -

O I II iv v vi VII

F ig . 2 —P o ly a c ry la m id e ge l e le c tro p h o re s is p a tte rn s o f caseins tre a te d w ith d i f fe r e n t e m u ls if ie rs .

( C0 ) Caseins w ith n o e m u ls if ie r as c o n t ro l; ( C¡1 Caseins w ith m o n o g ly c e r id e s ; ( C /j) Caseins w ith S pan ; (C m ) Caseins w ith Tw een 6 5 ; (C jv) Caseins w ith Tw een 8 0 ; ( Cv) Caseins w ith le c i th in ; (C vj) Caseins w ith s o d iu m d o d e c y i s u lfa te ; (C vj j ) Caseins w ith s o d iu m s te a ro y l-2 - la c ty la te .

f r o m t h e f a t s u r f a c e , c a u s i n g a c o a l e s c e n c e o f t h e f a t g l o b u l e s i n t o c l u s t e r s a n d b u t t e r g r a n u l e s . T h e w o r k o f K e e n e y( 1 9 6 2 ) a l s o s u p p o r t s t h e c o n c l u s i o n t h a t

e m u l s i f i e r s a r e d i s l o d g e d f r o m t h e s u r f a c e

o f t h e f a t g l o b u l e s .

T h e r e s u l t s i n F i g u r e 1 i n d i c a t e t h a t

t h e c o m b i n e d p h y s i c a l a c t i o n s i n t h e f r e e z e r a r e t h e m a i n f a c t o r s o n t h e d e s t a

b i l i z a t i o n o f t h e f a t e m u l s i o n . T h e r e s u l t s f r o m e l e c t r o p h o r e t i c a n a l y s e s s h o w t h a t

t h e r e i s n o c o m p l e x f o r m a t i o n b e t w e e n

m i l k p r o t e i n s a n d e m u l s i f i e r s i n i c e c r e a m a n d t h e a s s u m p t i o n t h a t s u c h a n i n t e r

a c t i o n m a y l e a d t o f a t d e e m u l s i f i c a t i o n is t h u s d o u b t f u l . T h i s s u g g e s t s t h a t a n e m u l s i f i e r - p r o t e i n c o m p l e x i n g i s n o t n e c e s s a r y

f o r t h e a d s o r p t i o n o f m i l k p r o t e i n s o r

a d d e d e m u l s i f i e r s o n t h e f a t g l o b u l e s . A p r o p o s e d m e c h a n i s m i s p r e s e n t e d t o e x

p l a i n t h e a c t i o n s o f e m u l s i f i e r s a n d t h e i r e f f e c t o n f a t d e e m u l s i f i c a t i o n i n a n i c e

c r e a m s y s t e m .

F a t g l o b u l e s i n t h e i r n a t u r a l s t a t e a v e r a g e 4 — 10jU i n d i a m e t e r . E a c h i s c o v e r e d w i t h a “ m e m b r a n e ” c o m p o s e d o f m e m b r a n e p r o t e i n s a n d n a t u r a l e m u l s i f i e r s ( e . g . , p h o s p h o l i p i d s ) . A f t e r p r o p e r h o m o

g e n i z a t i o n , t h e f a t g l o b u l e s a r e r e d u c e d i n s i z e t o a b o u t 1 - 2 / u w i t h a t r e m e n d o u s l y i n c r e a s e d t o t a l s u r f a c e . T h e o r i g i n a l m e m b r a n e m a t e r i a l s a r e n o t s u f f i c i e n t t o c o v e r t h e n e w l y c r e a t e d s u r f a c e a r e a ( T h a r p ,

1 9 6 8 ) . T h u s , t h e a d d e d e m u l s i f i e r s , c a s e i n , a n d s e r u m p r o t e i n s i n t h e s u r r o u n d i n g s e r u m a d s o r b o n t h e s e n e w l y

c r e a t e d s u r f a c e s , f o r m i n g a n e w m e m b r a n e o n t h e s u r f a c e o f t h e s m a l l e r f a t

g l o b u l e s ( J e n n e s s a n d P a t t o n , 1 9 5 9 ; W e b b a n d J o h n s o n , 1 9 6 5 ; S h e r m a n , 1 9 6 6 ; B e r g e r e t a l . , 1 9 7 2 ) . T h e c h a n c e f o r t h e s e

m a t e r i a l s t o a d s o r b o n t h e f a t g l o b u l e s

d e p e n d s o n t h e i r c h e m i c a l n a t u r e s a n d t h e i r r e s p e c t i v e c o n c e n t r a t i o n s i n t h e s e r u m p o r t i o n o f t h e m i x . W h e n a l o w

H L B e m u l s i f i e r i s u s e d , b e c a u s e o f i t s s t r o n g l i p o p h i l i c g r o u p , t h e e m u l s i f i e r m o l e c u l e b e c o m e s f i r m l y a s s o c i a t e d w i t h t h e f a t g l o b u l e . D u r i n g t h e p r o c e s s o f

f r e e z i n g a n d a g i t a t i o n i n t h e f r e e z e r , m o s t o f t h e f a t g l o b u l e s s t i l l r e t a i n t h e i r i d e n t i t i e s , a n d l e s s f a t d e e m u l s i f i c a t i o n o c c u r s . O n t h e o t h e r h a n d , w h e n a h i g h H T B

e m u l s i f i e r i s u s e d , b e c a u s e o f i t s s t r o n g h y d r o p h i l i c g r o u p a t o n e e n d a n d w e a k l i p o p h i l i c g r o u p a t t h e o t h e r , t h e e m u l s i f i e r m o l e c u l e i s t h u s l o o s e l y a d s o r b e d o n t h e f a t g l o b u l e . D u r i n g a g i t a t i o n i n t h e f r e e z e r t h e m e c h a n i c a l f o r c e o f t h e b l a d e s i s s o g r e a t t h a t i t s w e e p s o f f t h e w e a k e r b o u n d e m u l s i f i e r s f r o m t h e f a t g l o b u l e s , a l l o w i n g t h e m t o f o r m c l u s t e r s b e c a u s e o f a n i n c r e a s e i n s u r f a c e t e n s i o n . T h e c l u s t e r e d f a t g l o b u l e s a r e t h e n f u r t h e r c o a l e s c e d a n d f o r m a l a r g e f a t m a s s .

M a h d i a n d B r a d l e y ( 1 9 6 9 ) s u g g e s t e d t h a t a p r o t e i n - c a r b o h y d r a t e c o m p l e x a d s o r b e d o n t h e s u r f a c e o f f a t g l o b u l e s w a s r e m o v e d b y a g i t a t i o n a n d a b r a s i o n i n t h e

f r e e z e r , a l l o w i n g t h e a g g r e g a t i o n o f t h e

g l o b u l e s . T h e r e s u l t s o f t h i s s t u d y s u g g e s t t h a t t h e r e i s n o c o m p l e x f o r m e d b e t w e e n p r o t e i n a n d e m u l s i f i e r a n d t h a t s u c h a c o m p l e x i s n o t n e c e s s a r y f o r f a t d e e m u l s i f i c a t i o n .

A l s a f a r a n d W o o d ( 1 9 6 6 ) o b s e r v e d t h a t t h e m e m b r a n e m a t e r i a l s u r r o u n d i n g t h e f a t g l o b u l e s i n i c e c r e a m c o n t a i n i n g

T w e e n 8 0 e m u l s i f i e r ( H L B 1 5 . 0 ) a p p e a r s t o b e t h i n n e r a n d w e a k e r t h a n t h a t i n t h e s a m p l e s c o n t a i n i n g S p a n 6 0 e m u l s i f i e r ( H L B 4 . 7 0 ) . T h i s o b s e r v a t i o n s u p p o r t s t h e p r e s e n t t h e o r y s i n c e i t i s b e l i e v e d t h a t

m o r e e m u l s i f i e r m o l e c u l e s w o u l d b e r e m o v e d f r o m t h e f a t g l o b u l e s u r f a c e w h e n a h i g h H L B e m u l s i f i e r is u s e d t h a n w h e n a l o w H L B e m u l s i f i e r i s u s e d . H o w e v e r , f u r

t h e r i n v e s t i g a t i o n i s n e c e s s a r y t o s u p p o r t

t h i s t h e o r y o f e m u l s i f i e r a c t i o n i n i c e c r e a m .

CONCLUSIONS

T H E R E S U L T S d o n o t s h o w a s i g n i f i c a n t i n c r e a s e i n t h e s t a b i l i t y o f t h e f a t e m u l s i o n p r i o r t o f r e e z i n g i n t h o s e i c e c r e a m

m i x e s c o n t a i n i n g e m u l s i f i e r s . H o w e v e r , t h e e m u l s i o n c o u l d b e b r o k e n w h e n i t w a s s u b j e c t e d t o f r e e z i n g . T h e f a t e m u l s i o n s t a b i l i t y w a s a f f e c t e d b y t h e d e g r e e o f t h e h y d r o p h i l i c c h a r a c t e r ( o r H L B v a l

u e ) o f t h e e m u l s i f i e r . C o m p a r i n g t h e f a t e m u l s i o n s t a b i l i t y o f i c e c r e a m m i x e s b e f o r e a n d a f t e r f r e e z i n g , t h e r e s u l t s s t r o n g

l y i n d i c a t e d t h a t f r e e z i n g a n d a g i t a t i o n

MECHANISM OF EMULSIFIER IN ICE CREAM - 111

Fig . 3 —P o ly a c ry la m id e g e l e le c tro p h o re s is p a tte rn s o f s e ru m p ro te in s tre a te d w ith d if fe re n t e m u ls ifie rs . IS 0 ) S e ru m p ro te in s w ith n o e m u ls if ie r as c o n t ro l ; (S/) S e ru m p ro te in s w ith m o n o

g ly c e r id e s ; IS n l S e ru m p ro te in s w ith Span 8 0 ; (S jj j) S e ru m p ro te in s w ith Tw een 6 5 ; (S jv) S erum p ro te in s w ith Tw een 8 0 ; IS v) S e ru m p ro te in s w ith le c i th in ; IS V/) S e ru m p ro te in s w ith s o d iu m d o d e c y l s u lfa te ; (S vj j ) S e ru m p ro te in s w ith s o d iu m s te a ro y l-2 - la c ty la te .

h a v e a p r o n o u n c e d e f f e c t o n f a t d e e m u l s i f i c a t i o n i n i c e c r e a m .

T h e e l e c t r o p h o r e t i c s t u d i e s s h o w e d t h a t a d d e d e m u l s i f i e r s d o n o t s i g n i f i

c a n t l y a f f e c t t h e m i l k p r o t e i n s ( b o t h c a s e i n a n d s e r u m p r o t e i n s ) . T h e i n t e r a c t i o n o r c o m p l e x i n g , i f a n y , b e t w e e n e m u l

s i f i e r a n d m i l k p r o t e i n s w a s n o t d e t e c t a b l e . H o w e v e r , i n a m o d e l s y s t e m s t u d y , i t

w a s f o u n d t h a t i o n i c e m u l s i f i e r s a f f e c t e d t h e e l e c t r o p h o r e t i c p r o p e r t i e s o f t h e m i l k p r o t e i n s w h i l e n o n - i o n i c e m u l s i f i e r s d i d n o t . T h e r e f o r e , t h e a s s u m p t i o n t h a t a n i n t e r a c t i o n b e t w e e n e m u l s i f i e r a n d m i l k

p r o t e i n s i s r e s p o n s i b l e f o r f a t d e e m u l s i f i c a t i o n i n i c e c r e a m i s d o u b t f u l .

S i n c e t h i s s t u d y i n d i c a t e d t h e p h y s i c a l a c t i o n s i n t h e i c e c r e a m f r e e z e r p l a y a m a j o r r o l e i n t h e f a t d e e m u l s i f i c a t i o n , a t h e o r y o f t h e m e c h a n i s m o f e m u l s i f i e r a c t i o n i n i c e c r e a m w a s p r o p o s e d . H o w e v e r , f u r t h e r i n v e s t i g a t i o n i s n e c e s s a r y t o s u p p o r t t h i s t h e o r y .

REFERENCESA l s a f a r , T . a n d W o o d , F . W . 1 9 6 6 . M i c r o s c o p i c a l

e x a m i n a t i o n o f f a t d i s p e r s i o n o f i c e c r e a m . P r o c . 1 7 t h I n t . D a i r y C o n g . 5 : 4 0 1 .

B e r g e r , K . G . a n d W h i t e , G .W . 1 9 7 1 . A n e l e c

t r o n m i c r o s c o p i c a l i n v e s t i g a t i o n o f f a t d e s t a b i l i z a t i o n i n i c e c r e a m . J . F o o d T e c h n o l . 6: 2 8 5 .

B e r g e r , K . G . , B u l l i m o r e , B .K . , W h i t e , G .W . a n d W r i g h t , W .B . 1 9 7 2 . T h e s t r u c t u r e o f ice c r e a m —P a r t 1 . D a i r y I n d u s t r i e s , 3 7 ( 8 ) : 4 1 9 .

C h e e s e m a n , G .C . 1 9 6 8 . A p r e l i m i n a r y s t u d y b y ge l f i l t r a t i o n a n d u l t r a c e n t r i f u g a t i o n o f t h e i n t e r a c t i o n o f b o v i n e m i l k c a s e in s w i t h d e t e r g e n t s . J . D a i r y R e s . 3 5 : 4 3 9 .

D a v is , B . J . a n d O r n s t e i n , L . 1 9 5 9 . A n e w h ig h r e s o l u t i o n e l e c t r o p h o r e s i s m e t h o d . D e l iv e r e d a t t h e S o c i e t y f o r t h e S t u d y o f B l o o d , N e w Y o r k A c a d e m y o f M e d i c i n e .

D u r h a m , K . , J o n e s , T . G . , S h a w , D .J . a n d W a l k e r , D . A . 1 9 6 2 . T h e p h y s i c a l p r o p e r t i e s o f i c e c r e a m . F i r s t I n t e r n a t i o n a l C o n g r e s s o f F o o d S c i e n c e & T e c h n o l o g y , L o n d o n . ( M i m e o g r a p h e d . )

F r a z e u r , D . R . 1 9 5 9 , 1 9 6 0 . S o m e f a c t o r s a f f e c t i n g c h u r n i n g o f b u t t e r f a t i n s o f t - s e r v e i ce c r e a m . I c e C r e a m F i e l d . 7 3 ( 3 ) : 1 8 ; 7 3 ( 5 ) : 3 2 ; 7 4 ( 1 ) : 4 8 .

G o v i n , R . 1 9 6 9 . S t u d i e s o f e m u l s i f i e r a c t i o n i n i c e c r e a m u t i l i z i n g t h e H L B c o n c e p t . P h . D . t h e s i s . R u t g e r s U n i v . , N e w B r u n s w i c k , N . J .

G o v i n , R . a n d L e e d e r , J . G . 1 9 7 1 . A c t i o n o f e m u l s i f i e r s i n i ce c r e a m u t i l i z i n g t h e H L B c o n c e p t . J . F o o d S c i . 3 6 ( 5 ) : 7 1 8 .

G r i n d r o d , J . a n d N i c k e r s o n , T . A . 1 9 6 8 . E f f e c t o f v a r i o u s g u m s o n s k i m m i l k a n d p u r i f i e d m i l k p r o t e i n s . J . D a i r y S c i . 5 1 : 8 3 4 .

H a d d a d , G .S . 1 9 7 0 . E f f e c t s o f v a r y i n g h e a t t r e a t m e n t s o n t h e s o l u b i l i t y a n d s t a b i l i z a t i o n e f f e c t i v e n e s s i n i c e c r e a m o f c o m m o n w a t e r - b i n d i n g a g e n t s . P h . D . t h e s i s , U n iv . o f G e o r g i a , A t h e n s , G a .

J e n n e s s , R . a n d P a t t o n , S . 1 9 5 9 . “ P r i n c i p l e s o f D a i r y C h e m i s t r y . ” J o h n W i le y & S o n s , I n c . ,N . Y .

K e e n e y , P . G . 1 9 5 8 . F a t s t a b i l i t y p r o b l e m s . I c e C r e a m F i e l d 7 2 : 1 .

K e e n e y , P . G . 1 9 6 2 . O b s e r v a t i o n o f t h e e m u l s i f i e r c o n t e n t a n d f a t t y a c i d d i s t r i b u t i o n i n t h e l i p i d s a s s o c i a t e d w i t h t h e a i r c e l l s t r u c t u r e i n i c e c r e a m . J . D a i r y S c i . 4 5 ( 5 ) : 6 5 8 .

K e e n e y , P . G . a n d J o s e p h s o n , D . V . 1 9 5 8 . A m e a s u r e o f f a t s t a b i l i t y i n t h e i c e c r e a m a n d i t s r e l a t i o n s h i p t o d r y n e s s . I c e C r e a m T r a d e J . 5 4 ( 5 ) : 3 2 .

K l o s e r , J . J . a n d K e e n e y , P . G . 1 9 5 9 . A s t u d y o f s o m e v a r i a b l e s t h a t a f f e c t f a t s t a b i l i t y a n d d r y n e s s i n i c e c r e a m . I c e C r e a m R e v . 4 2 ( 1 0 ) : 3 6 .

K l o t z e k , L . M . , L e e d e r , J . G . , W el ls , P . R . a n d B a t t , R . M . 1 9 6 3 . “ S p e c k i n g ” o f c h o c o l a t e s o f t - s e r v e i c e c r e a m . I c e C r e a m R e v . 4 6 ( 9 ) :2 8 .

K l o t z e k , L . M . , a n d L e e d e r , J . G . , 1 9 6 6 . F a c t o r s a f f e c t i n g t h e c h u r n i n g o f s o f t - s e r v e i c e c r e a m : h o m o g e n i z a t i o n p r e s s u r e s a n d e m u l s i f i e r t y p e s . I c e C r e a m R e v . 4 9 ( 1 1 ) : 2 0 .

L e o , A . a n d B e t s c h e r , J . J . ( u n d a t e d ) . U s e o f s o d i u m s t e a r o y l - 2 - l a c t y l a t e t o i m p r o v e t h e l i p o - p r o t e i n m e m b r a n e i n n o n d a i r y c o f f e e c r e a m e r s . P a t c o P r o d u c t s C o . , K a n s a s C i t y , M o .

M a h d i , S . R . a n d B r a d l e y , R . L . 1 9 6 9 . F a t d e s t a b i l i z a t i o n i n f r o z e n d e s s e r t s c o n t a i n i n g l o w d e x t r o s e e q u i v a l e n t c o r n s w e e t e n e r s J . D a i r y S c i . 5 2 ( 1 1 ) : 1 7 3 8 .

S h e r m a n , P . 1 9 6 6 . T h e t e x t u r e o f i c e c r e a m . 3. R h e o l o g i c a l p r o p e r t i e s o f m i x a n d m e l t e d i c e c r e a m . J . F o o d S c i . 3 1 : 7 0 7 .

T h a r p , B .W . 1 9 6 8 . A d v a n c e s i n e m u l s i f i c a t i o n o f f r o z e n d e s s e r t s —2 . I c e C r e a m W o r l d , M a y 1 7 : 1 2 .

T s e n , C .C . ( u n d a t e d ) . U n p u b l i s h e d w o r k . M a n h a t t a n , K a n s a s . ( C i t e d f r o m L e o a n d B e t s c h e r . )

W a l k e r , D . A . 1 9 6 0 . T h e a g in g o f l i p o - p r o t e i n f i l m s a t t h e o i l - w a t e r i n t e r f a c e . T h i r d I n t e r n a t i o n a l C o n g r e s s o f S u r f a c e A c t i v i t y , C o l o g n e , V o l 4 ( 4 1 ) : 3 5 1 .

W e b b , B .H . a n d J o h n s o n , A . H . 1 9 6 5 . “ F u n d a m e n t a l s o f D a i r y C h e m i s t r y . ” p . 4 8 1 . T h e A v i P u b l i s h i n g C o . , I n c . , W e s t p o r t , C o n n .

M s r e c e i v e d 7 / 2 / 7 3 ; r e v i s e d 9 / 1 3 / 7 3 ; a c c e p t e d 9 / 1 9 / 7 3 .______________________________________________

P a p e r o f t h e J o u r n a l S e r i e s , N e w J e r s e y A g r i c u l t u r a l E x p e r i m e n t S t a t i o n , R u t g e r s U n i v e r s i t y —T h e S t a t e U n i v e r s i t y o f N e w J e r s e y , N e w B r u n s w i c k , N . J .

T h e a u t h o r s w i s h t o a c k n o w l e d g e t h e a d v ic e a n d h e l p o f D r . D i c k K l e y n a n d D r . E n d e l K a r - m a s i n t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t .

T. P. L A B U Z A a n d H. E. C H O U

D ep t, o f F o o d S cience & N u t r it io n , U n iv e rs ity o f M in n e s o ta , St. P au l, M N 5 5 1 0 1

DECREASE OF LINOLEATE OXIDATION RATE DUE TO WATER AT INTERMEDIATE WATER ACTIVITY

INTRODUCTIONI N A S T U D Y o f l i p i d o x i d a t i o n o f i n t e r m e d i a t e m o i s t u r e s y s t e m s b y H e i d e l b a u g h e t a l . ( 1 9 7 1 ) , H e i d e l b a u g h a n d K a r e l ( 1 9 7 0 ) , L a b u z a e t a l . ( 1 9 7 1 ) , T j h i o e t a l .

( 1 9 6 9 ) , C h o u e t a l . ( 1 9 7 3 ) a n d C h o u a n d L a b u z a ( 1 9 7 4 ) , i t w a s f o u n d t h a t w a t e r c o n t e n t a n d w a t e r a c t i v i t y ( A w ) h a d a p r o f o u n d e f f e c t o n o x i c l a t i o n r a t e . W h e n a s w e l l a b l e s o l i d s u p p o r t w a s u s e d , t h r e e

f a c t o r s w e r e a p p a r e n t : ( 1 ) A t l o w A w a n i n c r e a s e i n m o i s t u r e d e c r e a s e s o x i d a t i o n r a t e ; ( 2 ) I n c r e a s i n g t h e w a t e r a c t i v i t y i n t o t h e i n t e r m e d i a t e m o i s t u r e r a n g e i n c r e a s e d t h e r a t e o f o x i d a t i o n ; a n d ( 3 ) S y s t e m s p r e p a r e d b y d e s o r p t i o n ( D M ) o x i d i z e d f a s t e r t h a n d i d t h o s e p r e p a r e d t o t h e

s a m e w a t e r a c t i v i t y b y h u m i d i f i c a t i o n ( D H ) . T h e s e l a t t e r h a d a l o w e r w a t e r c o n t e n t d u e t o s o r p t i o n h y s t e r e s i s . S i m i l a r r e s u l t s o c c u r r e d f o r a n o n s w e l l a b l e c e l - l u l o s i c s y s t e m a t l o w t r a c e m e t a l c o n t e n t ;

h o w e v e r , a t h i g h a d d e d t r a c e m e t a l s (~ 1 0 0 0 p p m ) t h e r a t e o f o x i d a t i o n d e c r e a s e d a s A w i n c r e a s e d a t h i g h A w .

T h e e f f e c t o f w a t e r o n t h e l o w m e t a l s y s t e m a s w e l l a s t h e s w e l l i n g s y s t e m w a s

e x p l a i n e d o n t h e b a s i s o f m o b i l i t y a n d s o l u b i l i t y o f c a t a l y s t s . A t l o w A w w a t e r i n a c t i v a t e s c a t a l y s t s a n d h y d r o g e n b o n d s p e r o x i d e s s h o w i n g a n a n t i o x i d a n t e f f e c t . A s m o i s t u r e i n c r e a s e s f u r t h e r t h e a q u e o u s p h a s e b e c o m e s l e s s v i s c o u s , t h u s i n c r e a s i n g m o b i l i t y o f m e t a l c a t a l y s t s . S e c o n d l y , t h e h i g h e r t h e m o i s t u r e l e v e l i n t h i s r a n g e t h e g r e a t e r t h e s o l u b i l i t y o f c a t a l y s t s . I n f a c t , a s m o i s t u r e i n c r e a s e s , n e w c a t a l y s t s m a y b e e x p o s e d a s t h e s y s t e m s w e l l s a n d t h e n m a y b e d i s s o l v e c . T h e p u r p o s e o f

t h i s r e s e a r c h w a s t o v e r i f y a n d d e t e r m i n e t h e c a u s e f o r t h e r a t e r e v e r s a l i n t h e c e l l u l o s e s y s t e m a t h i g h a d d e d t r a c e m e t a l s i n t e r m s o f A w a n d m o i s t u r e c o n t e n t . I n

t h i s s y s t e m a s A w i n c r e a s e d f u r t h e r t h e o x i d a t i o n r a t e d e c r e a s e d .

MATERIALS & METHODSTH E P R O C E D U R E S used have been described in detail by Chou et al. ( 1 9 7 3 ) . Basically a system com prised o f 1 0 pt m ethyl lin oleate, 40 pt g lycerol (spectrograde) and 50 pt m icrocrystalline cellulose (A vicel, FM C C orp., Marcus H ook, Pa.) was either m ixed directly with water (DM ) fo r the d esorption system or was hum idified (D H ) to a given A w. T o get cellulose o f varying m etal co n ten t, it was initially m ixed with a so lution o f co b a lt ch loride, then freeze dried to give m etal co n ten ts from 2 0 - 1 0 0 0

ppm on a solids (including glycerol) basis. The co n tro l cellulose contain ed less than 3 ppm trace m etals (solids basis). A fter preparation, oxygen uptake was m easured by the Warburg techn ique and the data were analyzed according to the m ethods o f Labuza (1 9 7 1 ) . The tim e to reach 3% oxid ation (m ole oxygen /m ole lipid) was used as the ind uction tim e.

RESULTST H E R E S U L T S o f o x y g e n u p t a k e i n t h i s s t u d y a r e s h o w n i n F i g u r e 1 . A c o m p l e t e r e v e r s a l o f t h e r a t e o f o x i d a t i o n o c c u r s a s

t h e m e t a l c o n t e n t i n c r e a s e s . A t l o w m e t a l c o n t e n t t h e h i g h e r t h e A w t h e f a s t e r t h e o x i d a t i o n r a t e a n d t h e D M s y s t e m o x i -

Table 1-M o is tu re content, theoretical viscosity and theoretical metal concentration3

0.75 0.84 0.89

System DM DH DM DH DM DH

Moisture content Theoretical

34 28 61 47 78 63

viscosity (cp) 4.16 5.37 2.25 3.00 1.80 2.10

Theoretical metal concentration in aqueous phase

Metal content (ppm/solid basis)

(ppm/H 2 O basis)

DM DH DM DH DM DH

5 14.7 17.8 8.2 10.7 6.3 7.910 29.4 35.7 16.5 21.5 12.6 15.820 58.8 71.4 33.0 43.0 25.2 31.650 147 - 82.5 - 63.0 -

100 294 - 165 - 126 -500 1470 - 825 - 630 -

1000 2940 3570 1650 2150 1260 1580

a DM —desorption systems; DH—adsorption systems

Fig. 1 —O x id a t io n e x te n t a t 3 5 ° C as a fu n c t io n o f s o rp t io n hys te res is fo r va rio u s trace m e ta l co n te n ts . D M re fe rs to d e s o rp t io n sys tem s; D H to a d s o rp t io n system s.

U 2 - J O U R N A L OF FOOD S C /E N C E -V o /u m e 3 9 (1974)

L I N O L E A T E O X I D A T I O N R A T E D E C R E A S E - 113

d i z e s f a s t e r t h a n t h e D H . A s t h e m e t a l

c o n c e n t r a t i o n i s i n c r e a s e d t o 5 0 p p m , a l l

s y s t e m s o x i d i z e a t a b o u t t h e s a m e r a t e .

A t 5 0 0 p p m t h e s y s t e m o x i d a t i o n r a t e p a t t e r n h a s c o m p l e t e l y r e v e r s e d w i t h t h e r a t e o f o x i d a t i o n b e i n g f a s t e r a t t h e l o w e r

w a t e r c o n t e n t a n d l o w e r A w .

T h e s e r e s u l t s c a n b e e x p l a i n e d o n t h e b a s i s o f a d i l u t i o n e f f e c t . A t h i g h m e t a l

c o n c e n t r a t i o n , i n c r e a s i n g t h e A W ) a n d t h e r e f o r e t h e a m o u n t o f w a t e r , s i g n i f i

c a n t l y d e c r e a s e s t h e f r e e m e t a l c o n c e n t r a t i o n i n d i r e c t c o n t a c t w i t h t h e l i p i d p h a s e , e v e n t h o u g h t h e p h a s e v i s c o s i t y i s

r e d u c e d . T h i s d i l u t i o n e f f e c t , w h i c h d e c r e a s e s t h e e f f e c t i v e m e t a l c a t a l y s t c o n

c e n t r a t i o n f o r o x i d a t i o n , p r e d o m i n a t e s i n t h e s y s t e m w h e r e t h e m e t a l s a r e f a i r l y

f r e e . I n t h e w o r k o f C h o u e t a l . ( 1 9 7 3 ) , t h e h i g h m e t a l c o n t e n t a m y l o p e c t i n d i d n o t s h o w t h i s b e c a u s e t h e m e t a l s w e r e t i g h t l y b o u n d a n d t h e r e f o r e i n e f f e c t i v e . T h i s e f f e c t o f d i l u t i o n i s i l l u s t r a t e d i n

T a b l e 1 . F o r e x a m p l e , i n t h e D M s y s t e m ,

a b o u t a 5 0 % r e d u c t i o n i n t r a c e m e t a l c o n t e n t o c c u r s i n g o i n g f r o m A w 0 . 7 5 t o A w

0 . 8 9 f o r a l l s y s t e m s . H o w e v e r , a t h i g h m e t a l c o n t e n t a s s u m i n g t h e s a m e s u r f a c e a r e a o f l i q u i d t o f a t p h a s e i s a v a i l a b l e , t h e

r e d u c t i o n a t h i g h m e t a l c o n t e n t i s m u c h m o r e p r o n o u n c e d d u e t o d i l u t i o n . T h i s

d i l u t i o n f a c t o r m o r e t h a n c o m p e n s a t e s f o r t h e i n c r e a s e d m o b i l i t y d u e t o c h a n g e

i n v i s c o s i t y . A s s h o w n i n T a b l e 1 , t h e v i s c o s i t y c h a n g e i s a b o u t a f a c t o r 2 x l a r g e r t h a n t h e d i l u t i o n e f f e c t , b u t i t d o e s n o t p r e d o m i n a t e a t t h e h i g h m e t a l c o n c e n t r a t i o n .

o

2 0 PPM ■ □M ETAL

IOOOPPM A AM E T A L

1001___________ ,___________ .---------0-7 0-3 0 -9

A.,,

- ig . 2 —O x id a t io n in d u c t io n t im e fo r ce llu lo se

n o d e ! sys tem s as a fu n c t io n o f w a te r a c t iv ity .

i n o r d e r t o f u r t h e r e l u c i d a t e t h e e f f e c t

o f A w a n d w a t e r c o n t e n t o n o x i d a t i o n , t h e r e s u l t s o f t h i s s t u d y a n d t h o s e o f C h o u e t a l . ( 1 9 7 3 ) w e r e e x a m i n e d i n l i g h t

o f t h e s o r p t i o n h y s t e r e s i s e f f e c t . A s s h o w n b y L a b u z a e t a l . ( 1 9 6 6 ) , a t l o w m o i s t u r e c o n t e n t w a t e r e x e r t s a n a n t i o x i d a n t e f f e c t , t h r o u g h h y d r a t i o n o f m e t a l

c a t a l y s t s l o w e r i n g t h e i r a c t i v i t y a n d h y d r o g e n b o n d i n g p e r o x i d e s t h e r e b y r e t a r d i n g c h a i n i n i t i a t i o n . A s w a t e r c o n t e n t

i n c r e a s e s , t h e p h a s e v i s c o s i t y d e c r e a s e s ,

i n c r e a s i n g c a t a l y s t m o b i l i t y ; h o w e v e r , t h e e f f e c t i v e m e t a l c o n c e n t r a t i o n d e c r e a s e s , w o r k i n g i n t h e o p p o s i t e d i r e c t i o n . D e

p e n d i n g o n t h e r e l a t i v e m e t a l c o n c e n t r a t i o n t h e r a t e c a n e i t h e r i n c r e a s e o r d e

c r e a s e .

T h e w a t e r a c t i v i t y o f t h e s y s t e m i s a n

i n d e x o f r e a c t i v i t y s i n c e i t m e a s u r e s t h e a v a i l a b i l i t y o f t h e w a t e r . T h e l o w e r t h e

A w , t h e m o r e s t r o n g l y b o u n d i s t h e w a t e r t o t h e s y s t e m c o m p o n e n t s . T h u s , w h e n

w a t e r i s a n a n t i o x i d a n t , t h e r a t e o f o x i d a t i o n s h o u l d d e c r e a s e a s A vv i n c r e a s e s s i n c e t h e w a t e r b e c o m e s m o r e a v a i l a b l e . O n t h e

o t h e r h a n d , w h e n w a t e r a c t s a s a p r o - o x i

d a n t t h e r a t e o f o x i d a t i o n s h o u l d i n c r e a s e a s A w i n c r e a s e s , a s m o r e w a t e r b e c o m e s a v a i l a b l e .

T h e s e e f f e c t s a r e i l l u s t r a t e d i n F i g u r e s 2 a n d 3 i n w h i c h t h e i n d u c t i o n t i m e w a s p l o t t e d v s . A w o r m o i s t u r e c o n t e n t , f o r t h e c e l l u l o s e s y s t e m . W h a t i s o b v i o u s i s t h a t i n t h e i n t e r m e d i a t e m o i s t u r e r a n g e i t i s t h e m o i s t u r e c o n t e n t t h a t i s t h e i m p o r t a n t f a c t o r i n c o n t r o l l i n g t h e r a t e o f o x i d a t i o n s i n c e s i m i l a r s y s t e m s f a l l o n t h e s a m e c u r v e . A s s e e n i n F i g u r e 3 , t h e r e i s v e r y l i t t l e d i f f e r e n c e i n t h e o x i d a t i o n r a t e

b e t w e e n t h e t w o h y s t e r e s i s b r a n c h e s i f t h e s y s t e m s a r e c o m p a r e d a t s i m i l a r w a t e r c o n t e n t a n d m e t a l c o n t e n t . T h u s , a t h i g h

A w , w h e r e t h e w a t e r i s n o t b o u n d t i g h t l y ,

i t i s t h e m o i s t u r e c o n t e n t w h i c h c o n t r o l s o x i d a t i o n r a t e . I n a s y s t e m w h e r e m e t a l s

a r e f a i r l y f r e e , g r e a t e s t s t a b i l i t y i s o b

t a i n e d a t t h e l o w e s t m o i s t u r e , o r o n t h e o t h e r h a n d , b y g o i n g t o v e r y h i g h m o i s

t u r e c o n t e n t s . T h u s , i n t h e m a n u f a c t u r e o f a n i n t e r m e d i a t e m o i s t u r e f o o d , o n e

c o u l d g o t o a s h i g h a m o i s t u r e a s p o s s i b l e t o a t t a i n t h e d e s i r e d t e x t u r a l c h a r a c t e r i s

t i c s w i t h o u t m u c h c h a n g e i n o x i d a t i o n r a t e , t h e o n l y l i m i t a t i o n b e i n g m i c r o b i a l g r o w t h .

REFERENCESChou, H.E., A cott, K.M. and Labuza, T.P.

1973. Sorption hysteresis and chemical reactivity: Lipid oxidation. J. Food Sci. 38: 316.

Chou, H.E. and Labuza, T.P. 1974. Antioxidant effectiveness in interm ediate m oisture conten t m odel systems. J. Food Sci. In press.

Heidelbaugh, N.D. and Karel, M. 1970. Effect of water binding agents in the catalyzed oxidation of m ethyl linoleate. JAOCS 47: 539.

Heidelbaugh, N.D., Yeh, C.P. and Karel, M. 1971. Effects of m odel system composition on autoxidation of m ethyl linoleate. J. Agr. Food Chem. 19: 140.

Labuza, T.P., Heidelbaugh, N.D., Silver, M. and Karel, M. 1971. Oxidation at interm ediate m oisture contents. JAOCS 48: 86.

Labuza, T.P. 1971. Kinetics of lipid oxidation in foods. Critical Rev. Food Tech. 2: 355.

Labuza, T.P., Maloney, J.F . and Karel, M. 1966. A utoxidation of m ethyl linoleate in a freeze-dried m odel system: Effect of water on the cobalt catalyzed oxidation. J. Food Sci. 31: 885.

Tjhio, K., Labuza, T.P. and Karel, M. 1969. Effects of hum idification on catalysts and antioxidants in m odel systems. JAOCS 46: 577.

Ms received 8/3 /73; revised 9 /27 /73 ; accepted 9 /3 0 /73.

This is paper no. 8396, University of Minnesota Experim ent Station. The research was supported in part by University of Minnesota Agricultural Experim ent S tation, Project No. 18-72 and NASA contract NAS 9-12650, Lyndon Johnson Space Center, Houston, Texas.

Fig . 3 —O x id a t io n in d u c t io n t im e as a fu n c t io n o f m o is tu re c o n te n t fo r

ce llu lo s e m o d e l system s.

J. M O U S S E R I, M. P. S T E IN B E R G , A . I. N E L S O N a n d L . S. W E I

D ep t, o f F o o d Science, U n iv e rs ity o f I l l in o is , U rba na , IL 6 1 8 0 1

BOUND WATER CAPACITY OF CORN STARCH AND ITS DERIVATIVES BY NMR

INTRODUCTIONS C I E N T I S T S a n d e n g i n e e r s s t u d y i n g d e h y d r a t i o n p h e n o m e n a h a v e l o n g r e c o g n i z e d t h a t m o r e e n e r g y i s r e q u i r e d t o r e m o v e t h e f i n a l p o r t i o n o f w a t e r t h a n t h e i n i t i a l m o i s t u r e . T h i s i n d i c a t e s t h a t t h i s f i n a l p o r t i o n i s m o r e t i g h t l y b o u n d t o t h e s o l i d . T h e c o n c e p t o f b o u n d w a t e r ( B W ) i s o f s p e c i a l i m p o r t a n c e i n o t h e r u n i t o p e r a t i o n s a s w e l l a n d t o a l l u n i t p r o c e s s e s i n v o l v e d i n t h e p r e s e r v a t i o n o f f o o d s . F o r i n s t a n c e , i n d e v e l o p m e n t o f i n t e r m e d i a t e m o i s t u r e f o o d s i t i s o f i m p o r t a n c e t o k n o w m e r e a b o u t B W a n d

t h e f a c t o r s w h i c h a f f e c t i t .N u c l e a r M a g n e t i c R e s o n a n c e ( N M R )

h a s b e e n u s e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f t o t a l w a t e r d i s p e r s e d i n s o l i d s

( M i l l e r a n d K a s l o w , 1 9 6 3 ; C o n w a y e t a l . , 1 9 5 7 ) . W i d e - l i n e N M R m e a s u r e s a l l t h e l a b i l e p r o t o n s o r h y d r o g e n a t o m s p r e s e n t .

T h e h y d r o g e n a t o m s o f s o l i d s s u c h a s i c e a r e r e s t r i c t e d i n t h e i r m o t i o n a n d t h e r e

f o r e g i v e a b r o a d r e s o n a n c e l i n e w h i l e

h y d r o g e n a t o m s o f l i q u i d s g i v e s h a r p p e a k s ( D y e r , 1 9 6 5 ) . B e c a u s e o f t h i s , i t i s p o s s i b l e t o a d j u s t t h e N M R i n s t r u m e n t i n

s u c h a w a y t h a t o n l y t h e s i g n a l c o m i n g f r o m a l i q u i d w i l l b e c o n s i d e r e d ( V a r i a n

A s s o c i a t e s , 1 9 6 6 ) .T o l e d o e t a l . ( 1 9 6 8 ) u s e d t h i s i n s t r u

m e n t a l c a p a b i l i t y t o d e v e l o p a m e t h o d

f o r m e a s u r i n g B W i n w h e a t f l o u r . H e f o u n d t h a t d e c r e a s i n g t e m p e r a t u r e t h r o u g h t h e f r e e z i n g p o i n t c a u s e d a m a r k e d r e d u c t i o n i n s i g n a l a t t r i b u t e d t o s o l i d i f i c a t i o n o f f r e e w a t e r . T h e r e m a i n i n g s i g

n a l w a s a t t r i b u t e d t o B W , d e f i n e d a s w a t e r t h a t d o e s n o t f r e e z e . T h e B W s i g n a l g r a d u a l l y d e c r e a s e d a s t e m p e r a t u r e w a s l o w e r e d s o h e b a s e d t h e B W r e a d i n g o n a n a r b i t r a r y t e m p e r a t u r e . T o t h a t e x t e n t , t h e m e t h o d w a s e m p i r i c a l .

R e c e n t l y , a n N M R t e c h n i q u e f o r d i r e c t d e t e r m i n a t i o n o f B W w a s d e v e l o p e d . T h e m e t h o d ( S h a n b h a g e t a l . , 1 9 7 0 ) w a s b a s e d o n t h e f i n d i n g t h a t a t t h e l o w e s t r a d i o - f r e q u e n c y ( r f ) a t t e n u a t i o n ( h i g h e s t p o w e r ) , h y d r o g e n n u c l e i f r o m f r e e w a t e r g a v e a n e g l i g i b l e s i g n a l b u t h y d r o g e n f r o m B W g a v e a s t r o n g s i g n a l . I t w a s a l s o f o u n d t h a t , w i t h i n a g i v e n s e t o f i n s t r u m e n t

p a r a m e t e r s , t h e N M R s i g n a l f r o m B W w a s i n d e p e n d e n t o f t h e s o l i d b i n d i n g t h e w a t e r . T h e r e f o r e , a “ U n i v e r s a l ” N M R c a l i b r a t i o n c o n s t a n t p e r u n i t w e i g h t B W

c o u l d b e d e r i v e d f o r a n y f o o d p r o d u c t .

T h e w e i g h t o f B W p e r u n i t w e i g h t o f d r y

m a t t e r i n c r e a s e d l i n e a r l y w i t h i n c r e a s i n g m o i s t u r e c o n t e n t o n a d r y m a t t e r b a s i s t o

a m a x i m u m , c a l l e d b o u n d w a t e r c a p a c i t y ( B W C ) , a n d t h e r e a f t e r r e m a i n e d c o n s t a n t . A p l o t o f B W p e r u n i t w e i g h t o f t o t a l

s a m p l e a g a i n s t m o i s t u r e c o n t e n t o n a w e t b a s i s g a v e a n a s c e n d i n g s t r a i g h t l i n e f o l l o w e d b y a d e s c e n d i n g l i n e ; t h e i n t e r s e c

t i o n g a v e t h e B W C .T h e o b j e c t i v e o f t h i s s t u d y w a s t o

d e t e r m i n e t h e N M R p r o p e r t i e s o f c o r n

s t a r c h s a m p l e s a t d i f f e r e n t m o i s t u r e l e v e l s a n d t o d e t e r m i n e t h e B W C o f c o r n s t a r c h a n d i t s h y d r o l y t i c p r o d u c t s a n d o f s e v e r a l m o d i f i e d c o r n s t a r c h e s .

MATERIALS & METHODSM a t e r i a l s s t u d i e d

BWC d eterm inations o f the follow ing products were m ade:

( 1 ) Raw Corn S tarch - Argo Brand, CPC In ternational, In c., New Y ork .

(2 ) W axy Maize S ta rc h -C P C In ternational, In c ., New Y ork .

(3 ) Pregelatinized W axy S ta rc h -C P C In ternation al, In c ., New Y ork.

(4 ) P h o sp h ated W axy Sorghum Starch (h y d ro p h ilic )-C P C In ternational, In c., New Y ork .

(5 ) Corn Syrups, low and high D extrose E q u iv a le n t-C P C In tern ation al, In c., New Y ork .

(6 ) D - M a l t o s e - N u t r i t io n a l B iochem ical C orp., Cleveland, Ohio.

(7 ) D-glucose, anhydrous, analytical rea- g en t-M a llin ck ro d t Chem ical W orks, St. Louis, Mo.

P r e p a r a t i o n o f m a t e r i a l s

Sam ples o f each o f the above m aterials were dried in a vacuum oven at 28 in. Hg and 6 0 °C for 3 6 - 4 8 hr. This was su fficien t to remove essentially all the w ater as evidenced by the fact that the samples no longer gave an NM R signal a t r f 2 8 . T h e sam ples were then le ft in desiccators for 24 hr at 22° C befo re any treatm en t was made. The m oisture co n ten t was raised, as needed, by adding the calculated am ount o f w ater, m ixing thoroughly and equilibrating at 2 2 °C for 2 hr.

A bout 15-g sample o f each o f these m oistened starches was added to a tared 4 0 ml K im ax tube. Transfer o f the sample to the tube was made w ithout touching the wall above the level o f the sample to prevent “ noisy” signals. Sam ple weight was determ ined on an analytical balance to an accuracy o f 0.1 mg.

M o i s t u r e d e t e r m i n a t i o n

T h e m oisture co n ten t o f the corn starch,

d extrose and m altose was determ ined by the AOAC (1 9 7 0 ) vacuum oven m eth od , excep t that the holding tim e in the vacuum oven was extended to 3 6 - 4 8 hr. T h e m oisture co n ten t o f the corn syrup was determ ined by the vacuum oven m ethod for syrups described by the C IR F (1 9 6 5 ) with a slight m o d ifica tio n ; a folded filter paper was placed in the drying dish to in crease drying surface area.

N M R a n a l y z e r

T he Varian (Palo A lto , C alif.) m odel PA-7 NMR Process Analyzer was used. T h e follow ing instrum ent param eters were k ep t con stan t throughout the study:

Tim e co n stan t 0 .5 secSw eep tim e 0 .5 minSweep am plitude 2 .0 gaussM odulation am plitude 0 .5 gaussThreshold 0.1 mvW eight setting 1 0 0 0 equal to 1 0 .Og

The Sensitivity dial, the R eadout M ultiplier dial and the Signal M ultiplier dial were adjusted to give m inim um “ n oise” and m axim um Integral R eadout w ithin the paper scale o f 1 0 0 . All values were standardized to a sensitivity o f 2 0 0 , R eadout M ultiplier o f 2 0 and Signal M ultiplier o f 1. T h e standardized Integral R eadout will be referred to as NM R units per g sample.

RESULTS & DISCUSSION C o r n s t a r c h

N M R s i g n a l s f r o m c o m s t a r c h a t s i x m o i s t u r e c o n t e n t s f r o m 1 2 . 4 9 —6 8 . 6 7 %

w e t b a s i s w e r e o b t a i n e d a t r f a t t e n u a t i o n

l e v e l s o f 0 — 3 6 d e c i b e l s ( d b ) i n 4 d b s t e p s . T h e d a t a w e r e g r o u p e d i n t o t w o “ f a m i

l i e s ” o f c u r v e s . F i g u r e 1 p r e s e n t s d a t a f r o m s a m p l e s a t 2 1 . 3 9 % m o i s t u r e a n d b e l o w w h i l e F i g u r e 2 p r e s e n t s s a m p l e s a t 2 9 . 1 6 % m o i s t u r e a n d a b o v e . T h e b o t t o m c u r v e o n F i g u r e s 1 a n d 2 r e p r e s e n t s d i s t i l l e d , f r e e o r c o m p l e t e l y u n b o u n d w a t e r ; i t i s i m p o r t a n t t o n o t e t h a t t h e s i g n a l f r o m f r e e w a t e r f e l l t o z e r o a t 0 d b .

I n t h e f i r s t “ f a m i l y ” ( F i g . 1 ) a n i n c r e a s e i n s i g n a l w a s s h o w n a t t h e h i g h a t t e n u a t i o n l e v e l s w i t h a n i n c r e a s e i n m o i s t u r e c o n t e n t . H o w e v e r , a s t h e a t t e n u a t i o n l e v e l a p p r o a c h e d 0 d b , t h e s i g n a l

f r o m a l l t h e s a m p l e s a p p r o a c h e d a c o m m o n p o i n t , 8 5 N M R u n i t s p e r g w a t e r . T h e s e c o n d “ f a m i l y ” ( F i g . 2 ) , o b t a i n e d

w i t h h i g h m o i s t u r e s a m p l e s , s h o w e d a t a l l r f l e v e l s a d e c r e a s e d s i g n a l w i t h i n c r e a s i n g

m o i s t u r e c o n t e n t ; t h i s w a s o p p o s i t e t o t h a t s h o w n i n F i g u r e 1 f o r t h e l o w m o i s t u r e s a m p l e s . S o m e o f t h e d a t a w e r e n o t

- [ ^ - J O U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

BOUND WATER CA P A C ITY OF CO R N STA R C H - 115

Fig . 1—E ffe c t o f r f a t te n u a t io n o n N M R s igna l o f w a te r in c o rn s ta rch F ig . 2 —E ffe c t o f r f a t te n u a t io n on N M R s igna l o f w a te r in c o rn s ta rch a t lo w m o is tu re c o n te n t. a t h ig h m o is tu re c o n te n t.

p r e s e n t e d i n F i g u r e 1 f o r t h e s a k e o f

c l a r i t y .T h e N M R s i g n a l s a t 2 8 d b f r o m w a t e r

i n c o r n s t a r c h c o n t a i n i n g d i f f e r e n t a -

m o u n t s o f m o i s t u r e w e r e r e c a l c u l a t e d t o a d r y m a t t e r ( D M ) b a s i s , a n d p l o t t e d i n F i g u r e 3 a s t w o s t r a i g h t l i n e s . T h e t w o r e g r e s s i o n l i n e s w e r e c a l c u l a t e d a n d t h e i r i n t e r s e c t i o n w a s f o u n d t o b e a t 0 . 3 0 7 l g

w a t e r p e r g D M , o r 0 . 2 4 g w a t e r p e r g s a m p l e w h i c h g i v e s t h e B W C .

T h e d a t a f r o m a l l t h e 0 d b N M R s i g n a l s a t t h e s e v e r a l m o i s t u r e c o n t e n t s w e r e c a l c u l a t e d o n a p e r g s a m p l e b a s i s a n d p l o t t e d i n F i g u r e 4 . T w o r e g r e s s i o n l i n e s , o n e a s c e n d i n g a n d a n o t h e r d e s c e n d i n g , r e p r e s e n t i n g l o w e r a n d h i g h e r m o i s t u r e s a m p l e s , r e s p e c t i v e l y , w e r e c a l c u l a t e d .

T h e t h r e e s o l i d p o i n t s w e r e n o t i n c l u d e d i n t h e c a l c u l a t i o n f o r r e a s o n s w h i c h w i l l b e d i s c u s s e d u n d e r t h e e q u i l i b r i u m s t u d

i e s . T h e a s c e n d i n g l i n e h a d ‘b ’ a n d ‘a ’ v a l u e s o f 8 3 . 9 N M R u n i t s p e r g w a t e r , a n d + 0 . 2 7 N M R u n i t s p e r g s a m p l e , r e s p e c t i v e l y . T h e ‘r 2 ’ w a s 0 . 9 9 8 , s h o w i n g e x c e l l e n t c o r r e l a t i o n . T h e d e s c e n d i n g l i n e h a d a ‘b ’ v a l u e o f - 2 5 . 9 N M R u n i t s p e r g w a t e r a n d a n ‘a ’ v a l u e o f 2 6 . 5 N M R u n i t s p e r g s a m p l e . T h e ‘r 2 ’ w a s 0 . 9 2 7 .

T h e i r i n t e r s e c t i o n w a s a t a p o i n t c o r r e s p o n d i n g t o 0 . 2 4 l g w a t e r p e r g s a m p l e . T h i s i s e q u a l t o 0 . 3 1 7 g w a t e r p e r g D M ,

w h i c h i s v e r y c l o s e t o t h e v a l u e o b t a i n e d f r o m F i g u r e 3 , 0 . 3 0 7 . F u r t h e r m o r e , F i g u r e 4 i n d i c a t e s t h a t , a s t h e m o i s t u r e c o n

t e n t w a s i n c r e a s e d a b o v e 0 , t h e s i g n a l

i n c r e a s e d l i n e a r l y ( a s c e n d i n g l i n e ) w i t h a s l o p e o f 8 3 . 9 N M R u n i t s p e r g w a t e r u n t i l t h e B W C w a s r e a c h e d . T h i s s l o p e i s w i t h i n

t h e c o n f i d e n c e i n t e r v a l o f t h e “ u n i v e r s a l ” c o n s t a n t o b t a i n e d b y S h a n b h a g e t a l .( 1 9 7 0 ) a n d c l o s e t o t h e c o m m o n p o i n t o f 8 5 i n F i g u r e 1. T h e s i g n a l t h e n d e c r e a s e d l i n e a r l y ( d e s c e n d i n g l i n e ) t o 0 a t 1 0 0 % w a t e r .

Equilibrium at the BWC moisture content

N M R s i g n a l s a t r f 0 o b t a i n e d f r o m s a m p l e s a t o r n e a r t h e B W C m o i s t u r e c o n t e n t w e r e l o w e r t h a n e x p e c t e d ( F i g . 4 ) . S h a n b h a g ( 1 9 7 0 ) a t t r i b u t e d s u c h l o w v a l u e s t o d i f f i c u l t i e s i n d r y i n g a n d i n o b t a i n i n g r e p r e s e n t a t i v e s a m p l e s .

Fig. 3 —R e la tio n be tw e e n N M R s igna l a t lo w r f <evel 128 d b ) a n d m o is tu re c o n te n t o f c o rn

s ta rch .

F ig . 4 —R e la tio n be tw e e n N M R s igna l a t h igh r f le ve l 10 d b ) a n d th e m o is tu re c o n te n t o f c o rn

s ta rch .

F ig . 5 —E ffe c t o f e q u il ib ra t io n t im e o n the N M R s igna l a t h ig h r f le ve l (0 d b ) f ro m co rn s ta rch a t th e b o u n d w a te r c a p a c ity m o is tu re

c o n te n t.

1 1 6 - J O U R N A L OF FOOD SCIENCE— Volume 3 9 (1974)

Fig . 6 —R e la tio n b e tw e e n N M R s igna l a t h ig h r f

le ve l 10 d b ) a n d m o is tu re c o n te n t o f m o d if ie d

starches.

F ig . 7 —R e la tio n b e tw e e n N M R s igna l a t h ig h r f

le ve l (0 d b ) a n d th e m o is tu re c o n te n t o f co rn

s ta rch a n d its de riva tives .

Fig. 8 - R e la t io n be tw e e n b o u n d w a te r a n d to ta l

w a te r in c o rn s ta rc h a n d m a ltose .

A n e x p e r i m e n t w a s c o n d u c t e d t o i n v e s t i g a t e w h e t h e r t h e r e i s a n e q u i l i b r i u m t i m e e l e m e n t i n v o l v e d . A s a m p l e o f c o r n s t a r c h w a s p r e p a r e d t o c o n t a i n 2 4 % m o i s t u r e o n a w e t b a s i s a n d t h e N M R s i g n a l w a s o b t a i n e d a t i n t e r v a l s o f 1 d a y . T h e d a t a , p l o t t e d i n F i g u r e 5 , s h o w t h a t t h e s i g n a l d i d i n c r e a s e w i t h t i m e , a p

p r o a c h i n g a c o n s t a n t v a l u e i n 5 d a y s . T h i s i s a t y p i c a l e q u i l i b r a t i o n c u r v e . T h e e q u i l i b r i u m e n d p o i n t c a l c u l a t e d f r o m t h e s e

d a t a a c c o r d i n g t o U d a n i e t a l . ( 1 9 6 8 ) w a s f o u n d t o b e 2 0 . 5 N M R u n i t s p e r g s a m p l e a s c o m p a r e d t o t h e i n t e r s e c t i o n p o i n t o f

2 0 . 3 N M R u n i t s p e r g s a m p l e i n F i g u r e 4 .I t c a n b e h y p o t h e s i z e d f r o m t h e a b o v e

f i n d i n g t h a t a t o r n e a r t h e B W C m o i s t u r e c o n t e n t , a n a d d i t i o n o f w a t e r t o t h e s y s t e m r e q u i r e s a l o n g p e r i o d o f t i m e f o r e q u i l i b r i u m . M u c h b e l o w t h e B W C t h e f r e e s u r f a c e o f t h e s t a r c h a v a i l a b l e f o r

b i n d i n g i s l a r g e a n d a d d e d w a t e r i s q u i c k l y b o u n d . O n t h e o t h e r h a n d , m u c h a b o v e t h e B W C t h e r e i s s o m u c h e x c e s s w a t e r p r e s e n t t h a t t h e b i n d i n g s i t e s a r e q u i c k l y s a t u r a t e d . H o w e v e r , w h e n t h e a m o u n t o f w a t e r a d d e d t o d r y s t a r c h i s a b o u t e q u a l t o t h e B W C , t h e a m o u n t o f w a t e r i s e q u i v a l e n t t o t h e n u m b e r o f b i n d i n g s i t e s . M o s t o f t h e s i t e s a r e r e a d i l y s a t u r a t e d b u t t h e n a l o n g t i m e i s r e q u i r e d f o r t h e f e w r e m a i n i n g m o l e c u l e s o f f r e e w a t e r t o “ f i n d ” t h e f e w r e m a i n i n g u n b o n d e d s i t e s . A d y n a m i c c o n d i t i o n , i n w h i c h f r e e w a t e r is i n e q u i l i b r i u m w i t h b o u n d w a t e r , p r o b a b l y e x i s t s a t m o i s t u r e c o n t e n t s f r o m j u s t b e l o w t h e B W C t o a l l t h o s e h i g h e r .

S t a r c h d e r i v a t i v e s

T h r e e m o d i f i e d s t a r c h e s , p r e g e l a t i n i z e d w a x y c o m s t a r c h , w a x y m a i z e s t a r c h a n d c r o s s - b o n d e d , p h o s p h a t e d w a x y s o r g h u m ( h y d r o p h i l i c ) s t a r c h , w e r e s t u d i e d .

T h e d a t a o b t a i n e d w e r e p l o t t e d i n F i g u r e

6 a n d t h e B W C w a s 2 4 . 3 , 2 8 . 1 a n d 2 6 . 6 % w a t e r , r e s p e c t i v e l y . P o i n t s o n t h e a s c e n d

i n g l i n e , n o t s h o w n , w e r e c o m m o n t o a l l s a m p l e s .

I n a d d i t i o n , f o u r h y d r o l y t i c d e r i v a t i v e s

o f r e g u l a r c o r n s t a r c h w e r e s t u d i e d : 2 6 . 7D . E . c o r n s y r u p , 6 6 . 0 D . E . c o r n s y r u p ,

m a l t o s e a n d d e x t r o s e . D e x t r o s e g a v e a n e g l i g i b l e s i g n a l a t a l l m o i s t u r e c o n t e n t s , i n d i c a t i n g t h a t i t d o e s n o t b i n d w a t e r . T h e o t h e r t h r e e m a t e r i a l s s h o w e d a s c e n d

i n g r e g r e s s i o n l i n e s w i t h s l o p e s s i m i l a r t o e a c h o t h e r a n d t o t h e l i n e f o r s t a r c h .

D e s c e n d i n g l i n e d a t a f o r m a l t o s e a n d l o wD . E . c o r n s y r u p a r e c o m p a r e d t o t h a t f o r s t a r c h i n F i g u r e 7 . T h e h i g h D . E . c o r n s y r u D d a t a a r e n o t s h o w n ; t h e y w e r e i n t e r m e d i a t e b e t w e e n d a t a f o r t h e l o wD . E . s y r u p a n d m a l t o s e . T h e s e w e r e n e t s t r a i g h t a s f o u n d f o r s t a r c h e s ( F i g . 6 a n d7 ) b u t r a t h e r c u r v i l i n e a r a s o b t a i n e d b y S h a n b h a g ( 1 9 7 0 ) f o r s u c r o s e . T h e B W C w a s 2 3 . 9 % f o r t h e c o r n s y r u p s a n d 2 2 . 8 % f o r m a l t o s e ; t h e s e v a l u e s w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e 2 4 . 0 % o b t a i n e d f o r c o r n s t a r c h .

S h a n b h a g ( 1 9 7 0 ) e x p l a i n e d t h e c u r v i l i n e a r s h a p e b y t h e f a c t t h a t s u c r o s e i s a s o l u b l e m a t e r i a l . A b o v e t h e B W C , t h e f r e e w a t e r p r e s e n t d i s s o l v e s s o m e s u c r o s e a n d

t h e s u g a r t h a t i s d i s s o l v e d c a n n o l o n g e r b i n d w a t e r b e c a u s e o n l y a s o l i d c a n b i n d w a t e r . T h i s f r e e s t h e w a t e r t h a t h a d b e e n b o u n d b y t h e s u g a r t h a t i s n o w s o l u b i l i z e d s o m o r e s u g a r i s d i s s o l v e d .

D a t a o b t a i n e d f r o m c o r n s t a r c h a n d m a l t o s e o n a w e t o r t o t a l s a m p l e b a s i s w e r e r e c a l c u l a t e d o n a D M b a s i s , a n d p l o t t e d i n F i g u r e 8 . B o t h c o r n s t a r c h a n d m a l t o s e s h o w e d a l i n e a r a s c e n d i n g l i n e a t

l o w m o i s t u r e c o n t e n t s . I n c a s e o f c o r n s t a r c h , t h e d e s c e n d i n g l i n e w a s h o r i z o n t a l

b e c a u s e a n y w a t e r a d d e d a b o v e t h e B W C

o n l y d i l u t e d t h e s a m p l e b u t t h e a m o u n t

o f w a t e r b o u n d b y a u n i t q u a n t i t y o f d r y

s t a r c h r e m a i n e d c o n s t a n t . I n c a s e o f t h e s u g a r , t h e s i t u a t i o n i s d i f f e r e n t ; F i g u r e 8

s h o w s t h a t a b o v e t h e B W C t h e r e w a s a d e s c e n d i n g r e g r e s s i o n l i n e w i t h a s l o p e o f — 0 . 0 3 1 2 g B W p e r g t o t a l w a t e r . T h u s ,

a d d i t i o n o f l g f r e e w a t e r , c a u s e d e a c h g D M t o l o s e 0 . 0 3 1 2 g B W . E v i d e n t l y , d i l u t i n g t h e s a m p l e i n c r e a s e d t h e d i s t a n c e s b e t w e e n t h e s u g a r m o l e c u l e s , t h u s d e c r e a s i n g t h e f o r c e s b i n d i n g t h e w a t e r t o t h e s u g a r m o l e c u l e s .

REFERENCESAOAC. 1970. “ Official Methods of Analysis.

Assoc. Off. Agri. Chem., Washington, D.C. CIRF. 1965. “ Standard Analytical M ethods.’’

Corn Industry Research Foundation, Inc., Washington, D.C.

Conway, T.F., Conee, R.F. and Smith, R.J. 1957. NMR m oisture analyzer shows big potential. Food Eng. 29(6): 80.

Dyer, J.R . 1965. “ Application of Absorption Spectroscopy of Organic C om pounds.” Prentice-Hall, Inc., Englwood Cliffs, N.J.

Miller, B.S. and Kaslow, H.D. 1963. Determ ination of m oisture by NMR and oven m ethod in wheat flour, doughs and dried fruits. Food Technol. 17: 142.

Shanbhag, S. 1970. Bound water defined and determined at constant tem perature by wide- line NMR. Ph.D. thesis. University of Illinois, Urbana, 111.

Shanbhag, S., Steinberg, M.P. and Nelson, A.I. 1970. Bound water defined and determined at constant tem perature by wide-line NMR. J. Food Sci. 35: 612.

Toledo, R., Steinberg, M.P. and Nelson, A.I. 1968. Quantitative determ ination of bound water by NMR. J. Food Sci. 33: 315.

Udani, K.H., Nelson, A.I. and Steinberg, M.P. 1968. Rate of m oisture adsorption by wheat flour and its relation to physical, chemical and baking characteristics. Food Technol. 22: 1561.

Varian Associates. 1966. “ Varian PA-7 Process Analyzer an NMR Analyzer for Industry .” Bulletin No. INS 1469. Varian Associates, Palo Alto, Calif.

Ms received 5/4/73; revised 8 /15/73; accepted 8/20/73.

L . F . H O O D , A . S . S E / F R / E D a n d R . M E Y E R

D e p t , o f F o o d S c i e n c e , C o r n e l l U n i v e r s i t y , I th a c a , N Y 1 4 8 5 0

MICROSTRUCTURE OF MODIFIED TAPIOCA STARCH-MILK GELS

INTRODUCTIONM O D I F I E D S T A R C H E S h a v e b e c o m e i m

p o r t a n t f u n c t i o n a l i n g r e d i e n t s i n p r o c e s s e d f o o d s i n r e c e n t y e a r s b e c a u s e o f t h e i r i m p r o v e d f u n c t i o n a l p r o p e r t i e s o v e r

u n m o d i f i e d s t a r c h e s ( W u r z b u r g a n d S z y m a n s k i , 1 9 7 0 ) . T w o t y p e s o f m o d i f i c a t i o n o r d e r i v i t i z a t i o n , c r o s s l i n k i n g a n d s u b s t i t u t i o n , h a v e b e e n p a r t i c u l a r l y i m

p o r t a n t . C r o s s l i n k i n g o f t h e s t a r c h m o l e

c u l e s h e l p s t o m a i n t a i n t h e i n t e g r i t y o f t h e s w o l l e n s t a r c h g r a n u l e d u r i n g c o o k i n g

a n d t h e r e b y p r e v e n t s p a s t e b r e a k d o w n a n d l o s s o f v i s c o s i t y . S u b s t i t u t i o n o n t h e

h y d r o x y l g r o u p s o f t h e s t a r c h m o l e c u l e w i t h b u l k y c h e m i c a l g r o u p s ( i . e . , h y - d r o x y p r o p y l a c e t a t e , p h o s p h a t e ) i n h i b i t s

t h e a s s o c i a t i o n o f t h e a m y l o s e p o l y m e r

d u r i n g r e f r i g e r a t e d o r f r o z e n s t o r a g e a n d h e l p s t o p r e v e n t s y n e r e s i s .

M o d i f i e d s t a r c h e s a r e u s e d i n a v a r i e t y o f f o o d s i n c l u d i n g c a n n e d a n d f r o z e n p u d d i n g s , f r u i t p i e f i l l i n g s , g r a v i e s a n d w h i p p e d t o p p i n g s . A l t h o u g h t h e y p r o v i d e d e s i r a b l e v i s c o s i t y a n d m o u t h f e e l , t h e r e i s l i t t l e k n o w n a b o u t t h e m a c r o m o l e c u l a r s t r u c t u r e a n d m o r p h o l o g y o f t h e m o d i f i e d s t a r c h g r a n u l e , t h e e f f e c t o f g e l a t i n i - z a t i o n o n t h e s t r u c t u r e , a n d t h e p o t e n t i a l i n t e r a c t i o n s b e t w e e n t h e s t a r c h a n d o t h e r

c o m p o n e n t s o f t h e f o o d s y s t e m .

T h e r e h a v e b e e n n o e l e c t r o n m i c r o s c o p i c s t u d i e s o f c h e m i c a l l y m o d i f i e d

s t a r c h e s r e p o r t e d . H a l l a n d S a y r e ( 1 9 6 9 , 1 9 7 0 , 1 9 7 1 , 1 9 7 3 ) h a v e u t i l i z e d s c a n n i n g e l e c t r o n m i c r o s c o p y ( S E M ) t o d e s c r i b e t h e a r c h i t e c t u r e o f s e v e r a l c e r e a l a n d t u b e r s t a r c h e s . J o n e s a n d B e a n ( 1 9 7 2 ) ,

G a l l a n t e t a l . ( 1 9 7 2 , 1 9 7 3 ) , a n d E v e r s a n d

M c D e r m o t t ( 1 9 7 0 ) h a v e a p p l i e d e l e c t r o n m i c r o s c o p i c t e c h n i q u e s t o e v a l u a t e t h e e f f e c t s o f a m y l o l y t i c e n z y m e s o n t h e g r a n u l e s t r u c t u r e . M u s s u l m a n a n d W a g

o n e r ( 1 9 6 8 ) a n d B u t t r o s e ( 1 9 6 3 ) h a v e

d e s c r i b e d t h e m i c r o s t r u c t u r e o f a c i d - m o d i f i e d c o r n s t a r c h e s . S t e r l i n g a n d P a n g -

b o r n ( 1 9 6 0 ) u t i l i z e d t h e r e p l i c a t i o n t e c h n i q u e i n t h e e l e c t r o n m i c r o s c o p i c s t u d y o f p o t a t o s t a r c h .

T h e o b j e c t i v e o f t h i s s t u d y w a s t o e v a l u a t e t h e m i c r o s t r u c t u r e o f m o d i f i e d

t a p i o c a s t a r c h - m i l k g e l s . T h e e f f e c t o f m o d i f i c a t i o n a n d g e l a t i n i z a t i o n c n s t a r c h g r a n u l e s t r u c t u r e w a s a l s o s t u d i e d .

MATERIALS & METHODSPreparation o f gels

Skim m ilk was heated to 7 1 °C . T he starch was dispersed in a small am ount o f unheated m ilk and added to the heated m ilk. T he m ixture was reheated to 7 1 °C and held fo r 15 min. The paste was cooled to 4 °C . This treatm en t

was selected because it sim ulated the processing cond ition s applied to products o f this type (H ood, 1 9 7 1 ). I t was probably n o t su fficient to com pletely gelatinize all o f the granules. T he m odified tapioca starch was a h yd roxypropyl distarch phosphate (S te in Hall C o ., New Y o rk , N Y ). T he co n cen tratio n o f the starch was 7%.

Transm ission e lectron m icroscopy (T E M )

Gel specim ens (ap p rox 1 m m 3) and ungelatinized m odified starch were fixed in unbu ffered 2% 0 s 0 4 for 1 hr, dehydrated through a graded ethan ol series (2 5 and 50% e t h a n o l - 2 changes 30 min each , 70% ethan ol overnight, 80% , 95% , 100% e th a n o l-2 changes 30 m in each ), transferred to propylene oxide fo r 10 m in and infiltrated w ith propylene o x id e :E p o n 8 1 2 m ixtures ( 2 :1 , 1 :1 , 1 :2 , 100% E p on fo r 1 hr each ). Sp ecim ens were allowed to sink through fresh E p on in Beem capsules. P olym erization was carried ou t at 4 0 °C overnight and 6 0 °C for 24 hr. T hin sections (approx 1 0 0 0 A) were cut on a Sorvall MT 2 u ltram icroton e, stained w ith 2% aqueous uranyl acetate and w ith lead citra te (R ey n old s, 1 9 6 3 ) and exam ined in a Philips 3 0 0 electron m icroscope at 80 kV .

Scanning electron m icroscopy (SE M )

Gel specim ens and ungelatinized starch were dehydrated through a graded aceton e series (7 0 , 9 0 and 100% —15 min each ). T he final acetone solution was poured o f f and the starch was allowed to air dry at room tem perature. T he dry starch was sprinkled on to double-backed S c o tch tape w hich had been attached to an SEM stub. T he stubs were coated with gold- palladium , exam ined in a SEM 9 0 0 (Advanced M aterials Research C orp .) and photographed on Polaroid Type 52 or Typ e 55 P/N film . T he tilt angle was 4 5 ° .

RESULTS & DISCUSSIONE L E C T R O N M I C R O G R A P H S o f u n g e l a -

t i n i z e d m o d i f i e d a n d u n m o d i f i e d t a p i o c a s t a r c h a r e s h o w n i n F i g u r e s 1 a n d 2 . T h e

t r u n c a t e d e g g - s h a p e d a n d r o u n d g r a n u l e s w e r e s i m i l a r t o t h o s e t h a t h a v e b e e n o b s e r v e d b y o t h e r s ( W i v i n i s a n d M a y w a l d , 1 9 6 6 ; H a l l a n d S a y r e , 1 9 6 9 ) . G r a n u l e s i z e s r a n g e d f r o m 5 —2 0 ) 4 . T h e s u r f a c e o f t h e g r a n u l e s w a s s m o o t h a n d d i d n o t a p p e a r t o h a v e b e e n a l t e r e d b y t h e c h e m i c a l m o d i f i c a t i o n t r e a t m e n t . T h e r e w a s n o e v i d e n c e t h a t m o d i f i c a t i o n i n d u c e d a n y s w e l l i n g o f t h e g r a n u l e s . A c r o s s s e c t i o n o f t h e m o d i f i e d s t a r c h g r a n u l e r e v e a l e d r a d i a l c h a n n e l s o r z o n e s p a r t i c u l a r l y n e a r t h e g r a n u l e e d g e . T h e s e w e r e a p p a r e n t i n a l m o s t a l l o f t h e g r a n u l e s a n d m a y h a v e b e e n d u e t o t h e m o d i f i c a t i o n t r e a t m e n t o r t h e p r e p a r a t i o n p r o c e d u r e s f o r T E M .

Fig . 1 —S ca n n in g e le c tro n m ic ro g ra p h s o f u n g e la tin iz e d u n m o d if ie d (A ) a n d m o d if ie d (B ) tap ioca

s ta rch . M a rke rs : 10p.

Volume 3 9 f 1974)—JO U R N A L OF FOOD S C 7 F /V C E -1 1 7

^ - J O U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

Fig . 2 —T ransm iss io n e le c tro n m ic ro g ra p h o f u n g e la tin iz e d m o d if ie d

ta p io c a s ta rch . M a rk e r : 1 p.

F ig . 3 —L ig h t (A ) a n d sca n n in g e le c tro n (B ) m ic ro g ra p h s o f g e la t in iz e d

m o d if ie d ta p io c a s ta rch . L ig h t m ic ro s c o p y sp e c im en was s ta in e d w ith

io d in e . N o te th e s ize d if fe re n c e in g ra n u le s f ro m th e tw o p ro ce d u re s .

M a rk e rs : 10 p.

T h i s o b s e r v a t i o n i s c u r r e n t l y b e i n g i n v e s

t i g a t e d .G e l a t i n i z e d m o d i f i e d s t a r c h i s s h o w n

i n F i g u r e s 3 —5 . M o d i f i e d t a p i o c a s t a r c h g r a n u l e s s w e l l e d t o 3 0 - 8 0 / r d u r i n g g e l a - t i n i z a t i o n ( F i g . 3 A ) . T h e r a n g e o f g r a n u l e

s i z e w a s m u c h g r e a t e r i n t h e g e l a t i n i z e d

m o d i f i e d s t a r c h t h a n i n t h e g e l a t i n i z e d u n m o d i f i e d s t a r c h . T h i s p r o b a b l y r e f l e c t s t h e v a r y i n g e f f e c t o f t h e m o d i f i c a t i o n r e

a c t i o n o n i n d i v i d u a l g r a n u l e s a n d d i f f e r

e n t s w e l l i n g r a t e s a m o n g g r a n u l e s . S o m e g r a n u l e s m a y h a v e h a d a h i g h e r d e g r e e o f c r o s s l i n k i n g t h a n o t h e r s a n d t h e r e f o r e w o u l d s w e l l t o a l e s s e r e x t e n t d u r i n g

c o o k i n g . G r a n u l e s o b s e r v e d i n t h e e l e c t r o n m i c r o s c o p e w e r e s m a l l e r t h a n t h o s e

s e e n t h r o u g h t h e l i g h t m i c r o s c o p e ( F i g .3 ) . T h e g e l a t i n i z e d g r a n u l e s a p p e a r e d t o

s h r i n k w h e n d e h y d r a t e d a n d s u b j e c t e d t o t h e h i g h v a c u u m o f t h e S E M c o l u m n . N u m e r o u s f o l d s w e r e e v i d e n t o n t h e s u r f a c e o f t h e g r a n u l e s . T h e s i z e a n d m o r

p h o l o g y o f g e l a t i n i z e d i n d i v i d u a l g r a n u l e s m a y h a v e b e e n d u e t o o n e o r s e v e r a l f a c t o r s : ( a ) v a r i a t i o n i n d e g r e e o f c r o s s l i n k i n g a m o n g g r a n u l e s ; ( b ) v a r i a t i o n i n t h e s i z e o f n a t i v e u n c o o k e d g r a n u l e s w h i c h w o u l d b e r e f l e c t e d i n t h e s i z e o f t h e g e l a t i n i z e d g r a n u l e ; ( c ) s h r i n k a g e c a u s e d b y e l e c t r o n m i c r o s c o p y t e c h n i q u e s ; a n d ( d ) v a r i a t i o n i n t h e i n t e r n a l a r c h i t e c t u r e o f g r a n u l e s .

W h i l e S E M r e v e a l e d g r a n u l e s u r f a c e d e t a i l a n d o v e r a l l g e l m o r p h o l o g y , i t d i d n o t p r o v i d e s u f f i c i e n t l y h i g h r e s o l u t i o n t o e v a l u a t e t h e i n t e r n a l m o l e c u l a r a n d m a c r o m o l e c u l a r s t a r c h s t r u c t u r e . T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y o n t h i n - s e c t i o n e d m a t e r i a l s m a d e t h i s p o s s i b l e . T h e

u l t r a s t r u c t u r e o f g e l a t i n i z e d m o d i f i e d

s t a r c h g r a n u l e s v a r i e d . F i g u r e 4 s h o w s t h e t w o p r e d o m i n a n t t y p e s o f s t r u c t u r e s . T h e g r a n u l e o n t h e r i g h t ( T y p e A ) h a d a

d e n s e , h i g h l y f o l d e d c o a t a n d a d i s p e r s e d c o r e . T h e g r a n u l e o n t h e l e f t ( T y p e B )

h a d a h o m o g e n e o u s a m o r p h o r u s t e x t u r e

w i t h f o l d s e v i d e n t t h r o u g h o u t . T h e c o a t o f t h e T y p e A g r a n u l e h a d a s i m i l a r t e x t u r e t o t h e e n t i r e s u r f a c e o f t h e T y p e B

g r a n u l e . B e c a u s e o f t h e s i m i l a r i t y i n t e x

t u r e a n d t h e f o l d i n g , i t i s p o s s i b l e t h a t

T y p e A g r a n u l e s r e p r e s e n t e d a c r o s s s e c

t i o n o f t h e m o d i f i e d s t a r c h g r a n u l e

w h e r e a s T y p e B g r a n u l e s w e r e r e p r e s e n t a t i v e o f a s u r f a c e v i e w o f t h e o u t s i d e o f

t h e g r a n u l e .B o t h T y p e A a n d B g r a n u l e s w e r e p r e s

e n t i n s i g n i f i c a n t n u m b e r s . R e p r e s e n t a t i v e T y p e A g r a n u l e s a r e s h o w n i n F i g u r e 5 .

T h e b o r d e r w a s d e n s e a n d c o m p r i s e d o f s m a l l o s m i o p h i l i c s u b u n i t s ( 5 0 — 1 0 0 A d i a r n ) e m b e d d e d i n a w e b - l i k e m a t r i x . S o m e o f t h e m a t r i x m a t e r i a l w a s d i s -

F ig . 4 — T r a n s m is s io n e l e c t r o n m i c r o g r a p h o f m o d i f i e d t a p i o c a s t a r c h - m i l k g e l . G r a n u le s h a v e v a r ia

b l e m o r p h o l o g y . C a s e in m i c e l l e s ( m ) , s t a r c h g r a n u l e s ( s ) , n o n g r a n u le s ta r c h ( n ) , M a r k e r : 1 p.

M IC RO STR UC TUR E MODIFIED STARCH-M ILK GELS- 119

Fig. 5 —Transm iss ion e le c tro n m ic ro g ra p h s o f m o d if ie d ta p io c a s ta rc h -m ilk gel. In A a n d B , g ra n u le c o a t is h ig h ly fo ld e d . In C, c o re (c r ) appears to be c o m p o se d o f th e sam e m a te r ia ! as th e c o a t le t ) b u t m o re h ig h ly d ispersed . M a rk e rs : A a n d B , I p ; C, 0. Ip .

1 2 0 -J O U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

Fig. 6 —T ransm iss ion e le c tro n m ic ro g ra p h o f casein m ice lle s . M a rk e r:

0.1 M.

p e r s e d i n t h e c o r e . T h e i d e n t i t y o f t h e s u b u n i t s i s u n k n o w n b u t m a y b e s t a r c h m o l e c u l e s . T h e s u b u r . i t s w e r e o b s e r v e d o n l y i n E p o n e m b e d d e d m a t e r i a l t h a t w a s s t a i n e d w i t h u r a n y l a c e t a t e a n d l e a d c i t r a t e . O t h e r s h a v e o b s e r v e d s i m i l a r p a r t i c l e s i n t h e p o l y s a c c h a r i d e - c o n t a i n i n g c e l l

w a l l s o f p l a n t m a t e r i a l ( P a r t h a s a r a t h y ,1 9 7 3 ) . T h i s p a r t i c u l a r p r e p a r a t i v e m e t h o d f o r e l e c t r o n m i c r o s c o p y m a y s t a i n s p e c i a l l y t h e s t a r c h m o l e c u l e s . W e a r e c u r r e n t l y i n v e s t i g a t i n g t h i s p o s s i b i l i t y . T h e o u t e r e d g e o f t h e g r a n u l e c o a t w a s i r

r e g u l a r b u t w e l l d e f i n e d . T h e r e w a s s o m e e v i d e n c e o f f r a g m e n t s o f t h e g r a n u l e c o a t w h i c h h a d b e e n s l u f f e d o f f i n t o t h e s e r u m ( F i g . 4 a n d 5 b ) . T h e i n n e r e d g e o f t h e

c o a t w a s i r r e g u l a r a n d f r a g m e n t e d ( F i g . 5 c ) . T h e m a t e r i a l i n t h e c o a t w a s s l u f f e d o f f i n t o t h e c o r e a s d i s p e r s e d a g g r e g a t e s .

T h e m o r p h o l o g i c a l d i f f e r e n c e b e t w e e n t h e c o a t a n d t h e c o r e m a y h a v e b e e n d u e t o t h e c h e m i c a l m o d i f i c a t i o n o f t h e g r a n u l e . I t h a s b e e n s u g g e s t e d t h a t t h e c r o s s - l i n k i n g m o d i f i c a t i o n o c c u r s o n l y i n t h e o u t e r r e g i o n s o f t h e g r a n u l e ( G l i c k s m a n ,1 9 6 9 ) . T h e t h i c k n e s s o f t h e c o a t m a y r e p r e s e n t t h e d e p t h t o w h i c h t h e g r a n u l e h a s b e e n m o d i f i e d . N o c o a t - c o r e s t r u c t u r e h a s b e e n o b s e r v e d i n u n g e l a t i n i z e d m o d i f i e d g r a n u l e s ( F i g . 2 ) .

T h e f o l d e d a n d c o n v o l u t e d c h a r a c t e r o f t h e c o a t m a y r e f l e c t t h e g r a n u l e s h r i n k a g e w h i c h o c c u r s d u r i n g p r e p a r a t i o n f o r m i c r o s c o p y . I f t h i s i n t e r p r e t a t i o n

i s c o r r e c t , t h e a b s e n c e o f f o l d s i n t h e g r a n u l e c o r e w o u l d s u g g e s t t h a t s h r i n k a g e o c c u r r e d o n l y i n t h e o u t e r r e g i o n s o f t h e

g r a n u l e . T h e r e w a s n o e v i d e n c e o f g r a n u l e s h r i n k a g e i n u n g e l a t i n i z e d g r a n u l e s o r i n

g e l s t h a t h a d n o t b e e n f i x e d , d e h y d r a t e d o r e m b e d d e d ( F i g . 3 A ) . L i n e t a l . ( 1 9 7 1 ) h a v e n o t e d s i m i l a r d i f f e r e n c e s i n e l e c t r o n

m i c r o s c o p i c a n d l i g h t s c a t t e r i n g o b s e r v a t i o n s o n c a s e i n m i c e l l e s . E f f o r t s t o d e v e l o p e l e c t r o n m i c r o s c o p y t e c h n i q u e s w h i c h a v o i d s h r i n k a g e a r e i n p r o g r e s s .

T h e m o r p h o l o g i c a l c h a r a c t e r i s t i c s a n d s i z e ( 4 0 0 — 3 0 0 0 A ) o f t h e c a s e i n m i c e l l e s ( F i g . 6 ) w e r e s i m i l a r t o t h o s e t h a t h a v e b e e n r e p o r t e d f o r m i l k a n d m i l k g e l s

( H o o d , 1 9 7 1 ; S c h m i d t a n d B u c h h e i m ,1 9 6 8 ) . M i c e l l a r s u b u n i t s w e r e e v i d e n t . T h e a p p e a r a n c e o f t h o s e s u b u n i t s m a y b e e n h a n c e d o r a f f e c t e d b y t h e e l e c t r o n m i c r o s c o p y t e c h n i q u e s . M i c e l l e s w e r e n o t a g g r e g a t e d t o g e t h e r n o r w a s t h e r e a n y e v i d e n c e o f a c o n t i n u o u s n e t w o r k b e t w e e n t h e m i c e l l e s . I n s o m e t y p e s o f m i l k g e l s , c a s e i n m i c e l l e s h a v e a g g r e g a t e d ( C a r - r o l l e t a l . , 1 9 7 1 ; S c h m i d t , 1 9 6 8 ) . T h i s d o e s n o t a p p e a r t o h a p p e n i n s t a r c h - m i l k

REFERENCESButtrose, M.S. 1963. Electron microscopy of

acid-degraded starch granules. Die Starke 15: 85.

Carroll, R .J., Thompson, M.P. and Melnychyn,P. 1971. Gelation of concentrated shimmilk. Electron microscopic study. J. Dairy Sei. 54: 1245.

Evers, A.D. and M cDermott, E.E. 1970. SEM of wheat starch. 2. Structure of granules m odified by alpha amylases. Preliminary report. Die Starke 22: 23.

Gallant, D., Derrien, A., Aum aitre, A. and Guil- bot, A. Degradation in vitro de l’amidon par le suc pancréatique. Die Stärke 25:56.

Gallant, D., Mercier, C. and Guilbot, A. 1972. Electron microscopy of starch granules modified by bacterial a-amylase. Cereal Chem. 49: 354.

Glicksman, M. 1969. “ Gum Technology in the Food Industry,” p 303. Academic Press. New York.

Hall, D.M. and Sayre, J.G. 1969. A scanning electron-microscope study of starches. PartI . R oot and tuber starches. Text. Res. J. 39: 1044.

Hall, D.M. and Sayre, J.G. 1970. Internal architecture of potato and canna starch. Part 1. Crushing studies. Text. Res. J. 40: 147.

Hall, D.M. and Sayre, J.G. 1971. Internal architecture of potato and canna starch. Part 2. Swelling studies. Text. Res. J . 41: 404.

Hall, D.M. and Sayre, J.G. 1973. A comparison of starch granules as seen by both scanning electron and ordinary light m icroscopy. Die Starke 25: 119.

Hood, L.F. 1971. Milk-egg custard: A new and improved dairy food. Amer. Dairy Rev. 33(10): 28L.

Hood, L.F. 1971. Electron microscopy of carrageenan-milk gels. J. Dairy Sei. 54: 767.

Jones, F.T. and Bean, M.M. 1972. A light and SEM look at enzyme-damaged wheat starch. Microscope 20: 333.

Lin, S.H.C., Dewan, R.K., Bloomfield, V.A. and Morr, C.V. 1971. Inelastic light scattering study of the size distribution of bovine casein micelles. Biochem. 10: 4788.

Mussulman, W.C. and Wagoner, J.A. 1968. Electron microscopy of unm odified and acid- modified corn starches. Cereal Chem. 45: 162.

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Ms received 6/12/73; revised 9 /17 /73 ; accepted9/19/73.

gels . M ic e l le s w e r e d i s p e r s e d in t h e s e r u ma n d a p p a r e n t l y h a d n o t i n t e r a c t e d w i t ht h e s t a r c h g r a n u le s .

L . F . H O O D a n d A . S . S E I F R l E D

D e p t , o f F o o d S c i e n c e , C o r n e l l U n i v e r s i t y , I th a c a , N Y 1 4 8 5 0

EFFECT OF FROZEN STORAGE ON THE MICROSTRUCTURE AND SYNERESIS OF MODIFIED TAPIOCA STARCH-MILK GELS

INTRODUCTION

T H E S T R U C T U R E o f m o d ifie d tap io c a s ta rch (h y d ro x y p ro p y l d is ta rc h phos- p h a te ) - m i lk gels has b e en d esc rib e d (H o o d et a l., 1 9 7 4 ). M o rp h o lo g ica l d iffe ren ces w ere e x p la in e d b y v a r ia tio n s in th e deg ree o f m o d if ic a t io n an d in th e in te rn a l a rc h ite c tu re o f th e g ran u le s . A c o a t a n d a co re w ere e v id en t in so m e g ran u le s . T h e co re a p p e a re d to be c o m p o se d o f th e sam e m a te ria l as th e c o a t b u t w as m o re d isp e rse d . C ase in m ice lles w e re osm io - p h ilic a n d m ad e u p o f g ra n u la r su b u n its . T h ere w as n o in te ra c t io n e v id e n t a m o n g m ice lles a n d g ran u les .

C ro ss lin k e d -su b s titu te d ta p io c a s ta rch e s a re u til iz e d in f ro z e n a n d re fr ig e ra te d p u d d in g s , f ru it p ies an d g rav ies b ecau se th e y fa c ili ta te p ro cess in g an d in h ib it sy n eres is d u rin g s to rag e . A lth o u g h th e y a re re sp o n sib le p r in c ip a lly fo r th e e x te n d e d sh e lf life o f th e se p ro d u c ts , gel s tru c tu re s w ill d e te r io ra te a f te r e x te n d e d s to rag e p e rio d s . T h e e x te n t o f d e te r io ra t io n is re la te d to th e f ro z e n s to ra g e c o n d it io n s (S c h o c h , 1 9 6 8 ).

T h e re is l it t le in fo rm a t io n av ailab le o n th e e f fe c t o f f ro z e n s to rag e o n th e m o le c u la r an d m a c ro m o le c u la r s tru c tu re o f s ta rc h gels. T hese c o n s id e ra tio n s a re im p o r ta n t n o t o n ly in u n d e rs ta n d in g h o w free z in g a f fe c ts s ta rc h g ra n u le s a n d case in m ice lles b u t in th e d e v e lo p m e n t o f im p ro v ed m o d if ie d s ta rc h e s fo r f ro z e n fo o d s .

T h is s tu d y w as u n d e r ta k e n to ev a lu a te th e e ffe c t o f c e r ta in c o n d it io n s o f f ro z e n s to ra g e , a n d cy c lic freez in g a n d th aw in g o n th e m ic ro s tru c tu re an d deg ree o f sy n e res is o f m o d if ie d ta p io c a s ta rch -m ilk gels.

MATERIALS & METHODS

P r e p a r a t i o n o f g e l s a n d

e l e c t r o n m i c r o s c o p y

M o d i f i e d t a p i o c a s t a r c h ( 7 % ) - s k im m i l k g e l s

w e r e p r e p a r e d a n d p r o c e s s e d f o r e l e c t r o n m i c r o s c o p y a s d e s c r i b e d p r e v i o u s l y ( H o o d e t a l . ,

1 9 7 4 ) .

S t o r a g e c o n d i t i o n s

G e l s w e r e p a c k a g e d in 1 - q t p l a s t i c c o n t a i n e r s w i t h l i d s a n d s u b j e c t e d t o o n e o f f o u r d i f f e r e n t f r o z e n s t o r a g e t r e a t m e n t s :1 . S t o r e d in a - 3 2 ° C f r e e z e r .2 . S t o r e d i n a 2 2 f t 3 u p r i g h t a u t o m a t i c d e f r o s t

f r e e z e r . T h e d e f r o s t c y c l e o c c u r r e d f o u r

t i m e s / d a y .

3 . S t o r e d i n s a m e a u t o m a t i c d e f r o s t f r e e z e r d u r i n g t h e d a y . O n c e e a c h w e e k , g e l s w e r e t r a n s f e r r e d t o a 4 ° C r e f r i g e r a t o r a t 2 p m .

A t 8 a m o f t h e f o l l o w i n g d a y , t h e y w e r e r e t u r n e d t o t h e f r e e z e r .

4 . S a m e a s s t o r a g e t r e a t m e n t 3 e x c e p t g e l s w e r e t h a w e d a n d r e f r o z e n f o u r t i m e s e a c h w e e k .

F r e e z i n g t r e a t m e n t s ( t r t ) a r e l i s t e d i n o r d e r o f

i n c r e a s i n g d e g r e e o f h e a t s h o c k . C h a n g e s in i n t e r n a l p r o d u c t t e m p e r a t u r e d u r i n g t r t 2 , 3 a n d 4 a r e s h o w n in F i g u r e 1 . T h e r m o c o u p l e s w e r e

i n s e r t e d i n t h e c e n t e r o f a n d o n t h e o u t s i d e e d g e o f t h e p r o d u c t .

S a m p l e s w e r e s t o r e d r a n d o m l y o n t h e t o p a n d b o t t o m s h e l v e s o f t h e p a r t i a l l y - f u l l f r e e z e r

i n t r t 2 , 3 , a n d 4 . I n a d d i t i o n , t h e y w e r e p o s i t i o n e d r a n d o m l y in t h e c e n t e r o f t h e s h e lv e s a n d a t t h e e d g e o f t h e s h e l v e s a d j a c e n t t o t h e i n s i d e w a l l o f t h e f r e e z e r . P o s i t i o n i n g o n a s h e l f

h a d n o e f f e c t o n t h e p r o d u c t t e m p e r a t u r e .

H o w e v e r , t h e r e w a s a d i f f e r e n c e i n p r o d u c t t e m p e r a t u r e b e t w e e n t h e b o t t o m a n d t o p s h e lv e s . S a m p l e s o n t h e b o t t o m s h e l f w e r e 4 ° C h i g h e r t h a n t h e t o p s h e l f a t t h e b e g i n n i n g a n d

e n d o f t h e 6 - h r c y c l e b u t w e r e n o t s u b j e c t e d t o a s m u c h t e m p e r a t u r e v a r i a t i o n d u r i n g t h e c y c l e . O n t h e b o t t o m s h e l f , p r o d u c t t e m p e r a t u r e o n l y v a r i e d 1 ° C w h i l e o n t h e t o p s h e l f i t v a r i e d5 - 6 ° C . I n a d d i t i o n t o t h e t e m p e r a t u r e v a r i a t i o n b e t w e e n s h e l v e s , t h e t e m p e r a t u r e o n t h e

o u t s i d e e d g e o f t h e p r o d u c t w a s a l w a y s 1 - 2 ° C h i g h e r t h a n in t h e c e n t e r o f t h e p r o d u c t . T h e s e t e m p e r a t u r e v a r i a t i o n s w i t h i n t h e f r e e z e r a r e i n d i c a t i v e o f t h e e x t e n s i v e h e a t s h o c k t h a t p r o d u c t s u n d e r g o w h e n s t o r e d in a n a u t o m a t i c d e f r o s t f r e e z e r .

F i g u r e 2 s h o w s t h e i n t e r n a l t e m p e r a t u r e c h a n g e i n t h e p r o d u c t w h e n i t w a s d e f r o s t e d a n d r e f r o z e n ( t r t 3 a n d 4 ) . T h e p r o d u c t w a r m e d

u p t o - 3 ° C w i t h i n 7 h r i n a 4 ° C r e f r i g e r a t o r . I t d i d n o t c o m p l e t e l y t h a w d u r i n g t h e 1 8 h r in t h e

F i g . 1 — P r o d u c t t e m p e r a t u r e v a r i a t i o n i n a u t o m a t i c d e f r o s t f r e e z e r . ( A )

o u t s i d e o f p r o d u c t , e d g e o f b o t t o m s h e l f ; ( B ) c e n t e r o f p r o d u c t , e d g e o f

b o t t o m s h e l f ; I C I o u t s i d e o f p r o d u c t , e d g e o f t o p s h e l f ; ( D ) c e n t e r o f

p r o d u c t , e d g e o f t o p s h e l f .

Volume 39 C974)-JOURNAL OF FOOD SCIENCE-'\2'\

M2—JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

r e f r i g e r a t o r . A f t e r t r a n s f e r r i n g i t b a c k t o t h e f r e e z e r , i t r e q u i r e d 6 h r t o c o o l d o w n t o

- 2 2 ° C .

S y n e r e s i s d e t e r m i n a t i o n

T h e m e t h o d w a s a d a p t e d f r o m S c h o c h

( 1 9 6 8 ) . 1 0 m l a l i q u o t s w e r e s t o r e d i n 1 5 m l g r a d u a t e d p o l y c a r b o n a t e c o n i c a l c e n t r i f u g e t u b e s . T u b e s w e r e s t o r e d in c o v e r e d 1 - q t c o n t a i n e r s a n d s u b j e c t e d t o t h e f r o z e n c o n d i t i o n s d e s c r i b e d a b o v e . I n o r d e r t o d e t e r m i n e t h e d e

g r e e o f s y n e r e s i s t h e t u b e s w e r e t h a w e d a n d c e n t r i f u g e d a t 1 0 0 x G f o r 1 5 m i n . T h e a m o u n t o f f l u i d r e l e a s e d w a s r e p o r t e d a s a p e r c e n t a g e o f

t h e o r i g i n a l g e l v o l u m e .


T H E M IC R O S T R U C T U R E o f s ta rch -m ilk gels h a s b e e n d e sc rib e d e a rlie r (H o o d et a l., 1 9 7 4 ). H ere w e w ill lim it o u r d iscu ssio n to sy n e res is an d u l tra s tru c tu ra l c h an g es in gel s t ru c tu re , s ta rch g ran u le s a n d case in m ice lles d u rin g f ro z e n s to rag e .

S y n e re s is is a c h a ra c te ris tic o f m o st s ta rc h gels. It is a m a n ife s ta t io n o f th e r é tro g ra d a t io n o f th e am y lo se m o lecu le s a n d p e rh a p s to a sligh t d eg ree , th e assoc ia tio n o f th e b ra n c h e d m o le c u le s (S c h o c h ,1 9 6 8 ). As th e m o le c u le s a sso c ia te , th e w a te r m o le c u le s are e x p e lled fro m b e tw e e n th e m a n d sy n e rsis b e c o m e s a p p a r e n t. C h em ica l m o d if ic a t io n , p a r tic u la r ly th e a d d it io n o f b u lk y e s te r o r e th e r g ro u p s o n th e s ta rc h m o le c u le s , re d u ce s th e d eg ree o f sy n e res is d u rin g re fr ig e ra te d o r f ro z e n s to rag e .

T h e e ffe c t o f len g th an d te m p e ra tu re o f f ro z e n s to ra g e a n d n u m b e r o f freeze- th a w cy cles o n th e deg ree o f sy n e res is is sh o w n in T ab le 1. G els s to re d a t —3 2 °C ( t r t 1 ) sh o w ed neg lig ib le sy n eres is . T h e c o n s is te n c y o f th e gels ch an g e d very l it t le d u rin g th e 60 d ay s to ra g e p e r io d e x c e p t b y th e en d o f th e s to ra g e p e r io d , th e y h ad d e v e lo p ed a g ra in y a p p e a ra n c e . S to r age in a n a u to m a tic d e f ro s t f re e z e r ( t r t 2 ) c au sed m o re sy n e res is b u t th e a m o u n t o f sy n e res is d id n o t a p p ea r to in c rease b e y o n d 20 d ay s s to rag e . T h is ty p e o f f ree z e r h e a t sh o c k s b u t d o es n o t th a w th e p ro d u c t e ach tim e it goes th ro u g h a de f ro s t c y c le (F ig . 1). H eat sh o c k has a d e tr im e n ta l e ffe c t o n gel te x tu re w h ich re su lts in p rogressive in c reases in g ra in iness a n d lo ss o f v isco sity .

S u b je c tin g th e gels to c y c lic freez in g an d th a w in g ( t r t 3 a n d 4 ) m a rk e d ly in c reased th e e x te n t o f sy n e res is and d e c reased th e sm o o th n e s s an d te x tu re u n ifo rm ity . T h e a m o u n t o f sy n e res is re ac h ed a m a x im u m w ith in 2 0 d a y s fo r th o se sam ples cy c led d a ily w h e rea s it re q u ire d 4 0 d ay s o f s to ra g e w ith w e ek ly cy clin g s to a tta in th e sam e level. T h e deg ree o f sy n e resis a p p ea rs to d e p e n d p r in c ip a lly o n th e n u m b e r o f f re e z e - th a w c y c les a n d n o t o n th e le n g th o f s to ra g e t im e in th e a u to m a tic d e f ro s t f ree z e r .

E le c tro n m ic ro g rap h s o f s ta rch -m ilk gels h e ld u n d e r d if fe re n t c o n d it io n s o f f ro z e n s to ra g e a re sh o w n in F ig u re s 3 - 8 .

F i g . 2 — V a r i a t i o n i n p r o d u c t t e m p e r a t u r e a f t e r

t r a n s f e r f r o m f r e e z e r t o r e f r i g e r a t o r a n d b a c k t o

f r e e z e r . T h e r m o c o u p l e w a s p o s i t i o n e d i n t h e

c e n t e r o f t h e p r o d u c t .

T h e m o rp h o lo g y o f th e g ra n u le f ro m th e gel s to re d 6 0 d ay s a t —3 2 °C (F ig . 3 ) d id n o t d if fe r m a rk e d ly fro m th e g ra n u le in th e u n f ro z e n gel e x c e p t th a t th e re w ere no g ran u le s w ith th e c o a t-c o re s tru c tu re (H o o d e t a l ., 1 9 7 4 ). T h e su rfa ce o f all th e g ran u le s w as h o m o g e n e o u s , g ra in y and h ig h ly fo ld e d . C ase in m ice lle s sh o w ed so m e slig h t ev id en ce o f d is in te g ra tio n . T h e su rface o f so m e m ice lles w as ro u g h o r f ra g m e n te d a n d th e su b u n its w ere m o re c lea rly d e f in e d th a n in th e u n f ro z e n gel. T h ere w as so m e e v id en ce o f o p e n spaces b e tw e e n su b u n its w ith in m ice lles .

G els s to re d fo r 6 0 d a y s in th e a u to m a tic d e f ro s t f re e z e r a re sh o w n in F ig u re4 . G ra n u le su rfa ce s w e re s im ila r to th o se s to re d a t —3 2 °C . In g e n e ra l, m ice lle s w ere sim ila r to th o se in th e u n f ro z e n an d —3 2 °C f ro z e n e x c e p t th e re w as so m e m ice lle ag g reg a tio n . T h is h e lp s to e x p la in th e sy n eres is n o te d in th e se sam p le s b u t n o t in th e u n f ro z e n o r th e fro z e n 6 0 d a y s a t —3 2 °C . S y n e re s is m ay b e cau sed by s ta rc h r é tro g ra d a t io n as w ell as in c re a s in g ly t ig h t c o m p le x in g a m o n g c ase in m icelles. C a rro ll e t al. (1 9 7 1 ) a n d S c h m id t(1 9 6 8 ) have sh o w n th a t th e m ice lle s agg reg a ted d u rin g s to ra g e o f c o n c e n tr a te d m ilk a n d th a t su b s e q u e n tly g e la tio n o c c u rre d .

G els th a t w ere f ro z e n - th a w e d w ere very d if fe re n t m o rp h o lo g ic a lly f ro m th o se th a t h ad n o t b e en th a w e d . T h o se gels th a t w ere th a w e d a n d re fro z e n o n c e a w e ek fo r 6 0 d ay s (F ig . 5 ) h a d large z o n e s d e v o id o f m ice lles a d ja c e n t to th e g ra n u les . T h is w as p ro b a b ly se ru m w h ic h h a d t e e n e x p e lled fro m th e g ran u le s . G ra n u le s d id n o t a p p e a r to d if fe r g re a tly f ro m th o se d e sc rib e d a b o v e e x c e p t th e y h a d fe w er fo ld s a n d w ere s lig h tly m o re g ra n u la r . T h ey w ere m o re irreg u la r in sh a p e th a n th o se w h ic h h a d n o t b e e n fro z e n - th a w e d .

G els th a t w ere f ro z e n - th a w e d five tim e s in 8 d ay s a re sh o w n in F ig u re 6. T h ere w as so m e in d ic a tio n th a t th e g ra n u le h a d b e en r u p tu re d in so m e a rea s a n d th a t th e y w ere co a lesc in g . C ase in m ice lles w ere s ig n ific a n tly ch an g ed b y th e cy c lic freez in g a n d th a w in g . S u b u n its w e re n o t as h ig h ly ag g reg a ted as in th e u n c y c le d sam p le s . T h is is d if f ic u lt to e x p la in in th e

F i g . 3 - E l e c t r o n m i c r o g r a p h o f g e l s t o r e d f o r 6 0 d a y s a t - 3 2 ° C . S t a r c h g r a n u l e ( s ) ; c a s e i n m i c e l l e

( m ) ; M a r k e r : A , I p ; B , 0 . 1 p .

F i g . 4 — E l e c t r o n m i c r o g r a p h o f g e l s t o r e d f o r 6 0 d a y s i n a n a u t o m a t i c d e f r o s t f r e e z e r . M a r k e r : A ,

1 p ; B , 0 . 1 p .

F i g . 6 — E l e c t r o n m i c r o g r a p h o f g e l s t o r e d f o r 8 d a y s i n a n a u t o m a t i c d e f r o s t f r e e z e r a n d t h a w e d

a n d r e f r o z e n f i v e t i m e s . M a r k e r : A , 1 p ; B , 0 . 1 p .

F i g . 7 — E l e c t r o n m i c r o g r a p h o f g e l s t o r e d f o r 6 0 d a y s i n a n a u t o m a t i c d e f r o s t f r e e z e r a n d t h a w e d

a n d r e f r o z e n f o u r t i m e s e a c h w e e k . M a r k e r : A , 1 p ; B , 0 . 1 p .

F i g . 5 — E l e c t r o n m i c r o g r a p h o f g e l s t o r e d f o r 6 0 d a y s i n a n a u t o m a t i c d e f r o s t f r e e z e r a n d t h a w e d

a n d r e f r o z e n o n c e a w e e k . M a r k e r : A , I p ; B , 0 . 1 p .

MIC

RO

ST

RU

CT

UR

E F

RO

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N S

TA

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H-M

ILK G

ELS

—M

2

“\24—JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

Table 1-Effect of frozen storage on syneresis of modified tapioca starch-milk gels _____

TreatmentStorage time

(days)Freeze-

thaw cyclesSyneresis

(% of gel vol)

1 20 1 040 1 160 1 0

2 20 1 1660 1 17

3 8 1 2420 3 2240 6 4960 9 52

4 8 5 3820 13 5640 27 5960 42 52

F i g . 8 — E l e c t r o n m i c r o g r a p h o f g e l s t o r e d f o r 2 d a y s i n a n a u t o m a t i c

d e f r o s t f r e e z e r a n d t h a w e d a n d r e f r o z e n o n c e . M a r k e r : I p .

c o n te x t o f th e sy n e res is d a ta un less it is a n in d ic a tio n o f th e p a r tia l so lu b iliz a tio n o f th e m ice lles a n d a c o n c o m itte n t lo ss in th e ir w a te rb in d in g c a p a c ity . D a ta are n e ed e d o n m ice lle h y d ra t io n an d calci- u m :p h o s p h o ru s ra tio s to s u b s ta n t ia te th is h y p o th e s is .

A fte r th e gels h a d b e en cy c led 42 tim e s in 60 d ay s , th e re w ere n o in ta c t g ra n u le s ev id e n t (F ig . 7 ). S ta rch a p p ea red to be in th e c o n tin u o u s p h ase . T h is w o u ld a c c o u n t fo r th e a lm o s t c o m p le te ab sen ce

o f w a te r-h o ld in g c a p a c ity by th e gel. Micelles w ere d is to r te d an d so m e w ere su b s ta n tia lly d isag g reg a ted . M icelle f ra g m e n ts a p p e a re d to be in c lo se a sso c ia tio n w ith e ac h o th e r .

We w ere p u z z led b y th e lac k o f th e c o a t-c o re ty p e g ran u le s w h ic h w e o b served in th e u n f ro z e n gels (H o o d e t a l.,1 9 7 4 ). T h e re fo re , gels w h ich h ad b een fro z e n a n d th a w e d fo r less th a n e igh t t im e s an d s to re d less th a n a w e ek w ere e x a m in e d . T h e c o a t-c o re ty p e g ra n u le w as

e v id e n t in gels cy c led o n c e b u t n o t in th o se cy c led five t im e s (F ig . 8 ). T h e p r o p o r t io n o f c o a t-c o re to th e h o m o g e n e o u s g ran u le s w as lo w er in th e sa m p le s th a t h ad b e e n fro z e n - th a w e d o n ce th a n in th o se th a t h ad n o t b e e n f ro z e n . T h e d is a p p e a ra n c e o f th e c o a t-c o re g ra n u le m ay b e re la te d to p h y s ic a l c h an g e s a p p a re n t in th e gels a f te r freez in g . C o a t-c o re g ra n u le s c o u ld p ro v id e u se fu l in d ic a to rs fo r th e d e v e lo p m e n t o f im p ro v e d m o d if ie d s ta rc h e s fo r f ro z e n p ro d u c ts .

We have d e m o n s tra te d th a t u l tra s t ru c - tu ra l c h an g es o c c u r in m o d if ie d s ta rc h - m ilk gels d u rin g f ro z e n s to ra g e a n d th a t th e m a g n itu d e o f th e s e c h an g es is d e p e n d e n t m o re on th e e x te n t o f te m p e ra tu re f lu c tu a tio n s th a n o n th e d u ra t io n o f f ro z en s to rag e . T h aw in g a n d re fre e z in g h a d a m o re d ra s tic e f fe c t th a n th e h e a t sh o c k c au sed b y th e d e fro s t cy c le in f ro s t-fre e free z e rs . O n th e o th e r h a n d , f ro s t- f re e f ree z e rs cause m o re u l tr a s t ru c tu ra l a lte ra t io n s th a n c o n s ta n t te m p e ra tu re f ro z e n s to rag e . A p p a re n tly s ta rc h g ra n u le s agg lo m e ra te , an d m ice lle s u b u n its d isaggreg a te a f te r c y c lic free z in g a n d th aw in g .

T h e d eg ree o f sy n e re s is re p o r te d w o u ld b e u n a c c e p ta b le in m o s t fo o d p ro d u c ts . S c h o c h (1 9 6 8 ) h a s su g g ested 10% as th e m a x im u m a c c e p ta b le level o f sy n e res is . T h is w o u ld v a ry w ith c o m m o d it ie s b u t i t w o u ld seem th a t 20% c o u ld be a c c e p ta b le in so m e p ro d u c ts . E x p e r im e n ts a re c u r re n tly in p ro g ress in o u r la b o ra to ry to d e f in e u l tr a s t ru c tu ra l a n d p h y s ic o c h e m ic a l c h an g es v /h ich o c c u r in m o d if ie d s ta rch -m ilk gels w h e n th e deg ree o f sy n e res is is less th a n 10% .

O u r o b se rv a tio n s p ro v id e m ac ro - m o le c u la r in d ic a to rs o f th e h e a t sh o c k - in d u c e d s t ru c tu ra l d e te r io ra t io n o f m o d ified s ta rch -m ilk gels. S u b s e q u e n tly h igh re so lu tio n m ic ro sc o p y w ill be ap p lie d to d isc e rn m o le c u la r ch an g es w ith in a n d o n th e su rface o f th e s ta rc h g ra n u le d u rin g freez in g

REFERENCES

Carroll, R .J., Thompson, M.P. and Melnychyn,P. 1971. Gelation of concentrated skim milk. Electron microscopic study. J. Dairy Sci. 54: 1245.

Hood. L .F., Seifried, A.S. and Meyer, R. 1974. M icrostructure of m odified tapioca starch- milk gels. J. Food Sci. 39: 117.

Schm idt, D.G. 1968. Electron-microscopic studies of the gelation of UHTST sterilized concentrated skim milk. Neth. Milk & Dairy J. 22: 40.

Schoch, T.J. 1968. Effects of freezing and cold storage on pasted starches. In “ The Freezing Preservation of Foods,” Vol 4, p.44. Avi Publishing Co., W estport, Conn.


APPLIED SCIENCE and ENGINEERING

J O S E P H M . M E N D E L S O H N

U S D C N a t i o n a l M a r i n e F i s h e r i e s S e r v i c e

A t l a n t i c F i s h e r y P r o d u c t s T e c h n o l o g y C e n t e r , E m e r s o n A v e n u e , G l o u c e s t e r , M A 0 1 9 3 0

RAPID TECHNIQUES FOR SALT-CURING FISH: A Review

INTRODUCTION

W O R L D P R O D U C T IO N o f sa lted fish p re p a re d b y t r a d it io n a l m e th o d s fro m co d a n d o th e r le a n sp ec ies a n d fro m h e r ring h as b e e n d ecreas in g o v e r re c e n t y ears. T h e p ro d u c tio n o f sa lte d fish fro m lean sp ec ie s d e c re a sed fro m 4 4 6 ,0 0 0 m e tr ic to n s in 1967 to 4 2 5 ,0 0 0 m e tr ic to n s in 19 7 0 , a n d p ro d u c tio n o f sa lte d or d ried h e rrin g w e n t d o w n fro m 8 9 1 ,0 0 0 m etric to n s in 195 8 to 4 8 8 ,0 0 0 m e tric to n s in 1970 . T h is r e d u c t io n in th e p ro d u c tio n o f sa lte d fish is d u e to several causes, th e p rin c ip a l o n e b e in g an in su ffic ien t su p p ly o f th e fish tra d it io n a lly used fo r sa ltin g . T h e fish n o rm a lly used fo r sa ltin g a re b e in g so ld in th e fresh o r f ro ze n m a rk e ts , w h ic h is in n e ed o f an ev er-in creas in g su p p ly . A n o th e r d e te r r a n t p re v en tin g e x p a n s io n o f th e p ro d u c tio n o f sa lte d fish is th e lo n g p ro c ess in g tim e an d h igh la b o r c o s ts a sso c ia ted w ith th e sa ltin g p rocess .

E ven th o u g h sa lte d fish is b e co m in g scarce , th e n eed fo r th is v a lu ab le m arin e p ro te in is in creas in g . In m an y u n d e rd e v e lo p ed c o u n tr ie s , fish is th e m ain so u rce o f d ie ta ry p ro te in , a n d sa lted fish is an in teg ra l p a r t o f th e d ie t. In E u ro p e an d C anada th e re is a large t r a d it io n a l m a rk e t fo r sa lted fish fo r u se in th e m a n u fa c tu re o f sp e c ia lty p ro d u c ts . In th e U n ited S ta te s a b o u t 0 .2 m e tr ic to n o f sa lte d fish is p ro d u c e d so th a t m o s t o f th e 1 7 ,0 0 0 m etric to n s n e ed e d fo r th e p ro d u c tio n o f fish cak es m u s t b e im p o r te d . W ith in c reasin g d e m a n d s in m o st c o u n tr ie s fo r h ig h -q u a lity sa lted fish , a p o te n tia l ly large c o m m erc ia l m a rk e t is a lre ad y e s tab lish e d . T o sa tis fy th is m a rk e t, p ra c tic a l w ay s to increase th e p ro d u c tio n o f sa lte d fish are n eed ed . N o t o n ly are m o re e ff ic ie n t p ro d u c tio n te c h n iq u e s n ecessa ry , b u t also th e se te c h n iq u e s sh o u ld be a d a p ta b le to a v a r ie ty o f fish species to b ro a d e n th e so u rce o f raw m ate ria l.

P re se n tly , th e re a re tw o c o n v e n tio n a l c o m m e rc ia l te c h n iq u e s fo r sa lt-cu rin g fish : d ry -sa ltin g a n d w e t-sa ltin g . T hese p ro cesses re q u ire c o n s ta n t h a n d lin g o f th e fish a n d th e p ro c ess c o u ld ta k e up to 3 m o n th s to c o m p le te . P ro d u c t q u a li ty is u su a lly q u ite low . In a d d it io n , m o s t o f th e sa lte d p ro d u c ts re q u ire re fr ig e ra tio n and hav e a very l im ite d sh e lf life , a d ist in c t d isad v an tag e fo r u n d e rd e v e lo p e d c o u n tr ie s w h ich are lo c a te d in th e tro p ic s o r su b tro p ic s . T h e l i te ra tu re a b o u n d s w ith a rtic le s o n all p h ases o f th e se c o n v e n tio n a l sa ltin g te c h n iq u e s , an d m an y g o o d rev iew s are av ailab le (Ja rv is, 1 9 5 0 ; T ressle r a n d L em o n , 1 9 5 1 ; R agulin , 1 9 5 8 ; S ta n sb y , 1 9 6 3 ; B urgess e t a l. , 1 9 6 5 ; In g ram a n d K itch e ll, 1 9 6 7 ).

M any re sea rc h e rs have s tu d ie d c o n v en t io n a l sa ltin g te c h n iq u e s an d suggested im p ro v e m e n ts , b u t th e o b v io u s d isad v an tag es o f c o n v e n tio n a l sa ltin g p ro c e d u re s have p ro v id e d an im p e tu s to dev elo p ra p id sa ltin g te c h n iq u e s . T h is a rtic le is w r i t te n in o rd e r to b rin g to g e th e r all o f th e ra p id sa ltin g te c h n iq u e s to p ro v id e th e w id est p o ss ib le c h o ic e o f m e th o d s to th o se in te re s te d in rap id sa ltin g . T h e th e o ry , a d v an tag e s a n d d isad v an tag es o f each o f th e ra p id sa ltin g m e th o d s w ill be d iscussed .

REVIEW OF TECHNIQUES

B o i l e d s a l t e d f i s h - p i n d a n g

P i n d a n g o r b o i l e d s a l t f i s h is p o p u l a r in I n d o n e s i a , t h e P h i l i p p i n e s a n d T h a i l a n d . I t is m a d e b y p l a c i n g l a y e r s o f e v i s c e r a t e d f i s h a n d s a l t a l t e r n a t e l y i n s u i t a b l e c o o k i n g c o n t a i n e r s . T h e c o n t a i n e r s a r e c o v e r e d a n d h e l d o v e r a f i r e . I n a r a p i d p r o c e d u r e f o r p r o d u c i n g b o i l e d s a l t e d f i s h ( I l j a s a n d R o n s i v a l l i , 1 9 6 9 ) , t h e s a l t a n d f i s h f i l l e t s a r e s t e a m e d in p l a s t i c p o u c h e s . A f t e r c o o k i n g o r s t e a m i n g f o r a b o u t 2 h r , t h e d r i p is d i s c a r d e d a n d m o r e s a l t i s a d d e d . T h e p o u c h e s a r e t h e n s t e a m e d f o r a n a d d i t i o n a l 2 h r a n d t h e n e w l y f o r m e d d r i p 'd i s c a r d e d . T h e p o u c h e s a r e t h e n s e a l e d a n d s t o r e d a t r o o m t e m p e r a t u r e .

T h e a d v a n t a g e s o f t h i s p r o c e s s a r e t h a t t h e s a l t e d p r o d u c t i s p r e p a r e d w i t h i n 1 d a y a n d c a n b e s t o r e d a t r o o m t e m p e r a t u r e p r o v i d e d t h e c o n c e n t r a t i o n o f s a l t in t h e f i n i s h e d p i n d a n g is

a b o v e 1 0 % t o i n h i b i t g r o w t h o f p a t h o g e n i c o r g a n i s m s . M o s t o f t h i s p r o c e s s c o u l d b e a u t o m a t e d a n d t h e s a l t a n d w a t e r c o n t e n t o f t h e f i n i s h e d p r o d u c t c o u l d b e c o n t r o l l e d .

A d i s a d v a n t a g e o f t h e c o n v e n t i o n a l p i n d a n g

m e t h o d is t h a t i t t a k e s a b o u t a d a y t o p r o d u c e a r e l a t i v e l y s m a l l a m o u n t o f f i n i s h e d p r o d u c t . T h e p r o c e s s m a y b e l i m i t e d t o t h o s e s p e c i e s o f f i s h t h a t d o n o t f a l l a p a r t d u r i n g p r o l o n g e d b o i l i n g . C o m m e r c i a l e q u i p m e n t n e e d e d t o b o i l o r s t e a m l a r g e q u a n t i t i e s o f f i s h c o u l d p r e s e n t a p r o b l e m in r e g a r d s t o s p a c e a n d p o w e r r e q u i r e

m e n t s . H a n d l i n g t h e p o u c h e s t o r e m o v e d r i p b e t w e e n s t e a m i n g s is t i m e - c o n s u m i n g a n d c o u l d b e d a n g e r o u s t o t h e w o r k e r s u n l e s s d o n e a u t o m a t i c a l l y . I n a d d i t i o n , p i n d a n g h a s a l i m i t e d

r o o m t e m p e r a t u r e s t o r a g e l i f e o f a b o u t 3 m o n t h s . C o o k i n g t h e f i s h l i m i t s i t s p r o d u c t u s a g e a n d i t s p o t e n t i a l , b e i n g p o p u l a r i n o n l y a f e w c o u n t r i e s .

B a g g e d s a l t e d f i s h —B r i t i s h m e t h o d

A r e c e n t l y d e v e l o p e d t e c h n i q u e ( O r r , 1 9 6 7 )

c o n s i s t s o f p l a c i n g r a w f i s h i n p l a s t i c b a g s w i t h s a l t a n d w a t e r i n t h e r a t i o o f 1 6 : 6 : 2 . T h e a i r in t h e b a g s is e x p e l l e d , t h e b a g s a r e s e a l e d a n d p a c k e d in s u i t a b l e c a r t o n s . E a c h c a r t o n is l a b e l e d w i t h i n s t r u c t i o n s n o t t o u s e b e f o r e a s p e c i f i e d d a t e t o i n s u r e c o m p l e t e s a l t i n g . A 20- d a y p e r i o d is r e c o m m e n d e d a s a s a f e t y f a c t o r a n d t o c o m p e n s a t e f o r v a r i a b l e s . I n t h i s p r o c e s s , t h e s a l t p e n e t r a t e s t h r o u g h t h e f i s h f l e s h d u r i n g p r e p a r a t i o n a n d s h i p m e n t .

T h e a d v a n t a g e s o f t h i s p r o c e s s a r e s i m p l i c i t y a n d t h e r e q u i r e m e n t o f v e r y l i t t l e p r o c e s s i n g s p a c e a n d e q u i p m e n t . I t i s a d a p t a b l e t o m a c k e r e l , c o d , h e r r i n g , h a k e , p o l l o c k a n d p r o b a b l y t o m o s t s p e c i e s o f f i s h . S a l t i n g t a k e s p l a c e d u r in g p r o c e s s i n g a n d t r a n s i t a n d t h e s a l t e d p r o d u c t is r e a d y f o r c o n s u m p t i o n a f t e r t h e m i n i m u m s p e c i f i e d p e r i o d . I t c a n b e s t o r e d a t r o o m t e m p e r a t u r e i f s a l t e d u n i f o r m l y a n d c a n b e m a d e i n t o a v a r i e t y o f p r o d u c t s b e c a u s e t h e f is h r e m a i n s r a w a n d i n t a c t d u r i n g t h e p r o c e s s i n g p e r i o d .

T h e d i s a d v a n t a g e s in t h i s t e c h n i q u e a r e t h a t t h e d i s t r i b u t i o n o f s a l t i n t h e f i s h is u n p r e d i c t a b l e a n d i t c o u l d t a k e u p t o 3 w k f o r s a l t i n g

Volume 39 (1974)-JOURNAL OF FOOD SCIENCE- 1 2 5


t o b e c o m p l e t e . I f a p a c k a g e r e m a i n s i m m o b i l e a n d t h e s a l t d o e s n o t c o m e in c o n t a c t w i t h t h e e n t i r e c o n t e n t s o f t h e p o u c h a n d p e r m e a t e t h r o u g h t h e f i s h f l e s h s o o n e n o u g h , t h e r e m a y b e s o m e b a c t e r i a l g r o w t h . A e r o b i c b a c t e r i a a s w e l l a s a n a e r o b i c b a c t e r i a m a y g r o w a n d c o u l d c a u s e s p o i l a g e . T h e f a t c o n t e n t ( s a l t d i f f u s e s s l o w e r i n o i l t h a n in w a t e r ) , t h i c k n e s s , s i z e a n d o r i e n t a t i o n o f t h e f i l l e t s d e t e r m i n e t h e s a l t p e r m e a t i o n . A l a r g e t h i c k f i l l e t m a y n o t b e t h o r o u g h l y s a l t e d b y t h e t i m e i t i s d e l i v e r e d to i t s d e s t i n a t i o n . S h i p p i n g w a t e r i n p o u c h e s n o t o n l y i n c r e a s e s t r a n s p o r t a t i o n c o s t s , b u t a l s o t h e r i s k t h a t a n e n t i r e s h i p m e n t c o u l d b e r u i n e d i f

t h e p o u c h e s w e r e t o b r e a k o r l e a k .

D e h y d r a t i o n - i n j e c t i o n s a l t e d f i s h

A t t e m p t s h a v e b e e n m a d e t o d e v e l o p a r a p i d

s a l t i n g m e t h o d f o r f i s h f i l l e t s b y p e r s o n n e l a t t h e A t l a n t i c F i s h e r y P r o d u c t s T e c h n o l o g y C e n t e r . F i s h f i l l e t s w e r e s a l t e d b y a f r e e z e - d r y i n g t e c h n i q u e in w h i c h t h e w a t e r w a s r e m o v e d a n d r e p l a c e d b y s a t u r a t e d b r i n e ( C a r v e r , 1 9 6 9 ) . T h e p r o c e s s c o n s i s t s o f p l a c i n g f r o z e n f i l l e t s in a

l a r g e c h a m b e r a n d t h e n d r a w i n g a v a c u u m t o r e m o v e t h e m o i s t u r e . A t a p r e d e t e r m i n e d t i m e ,

t h e w i t h d r a w n w a t e r w a s r e p l a c e d b y a s a t u r a t e d s a l t s o l u t i o n . W h i l e u n d e r v a c u u m , t h e

s a l t s o l u t i o n w a s i n j e c t e d s l o w l y t h r o u g h a

s m a l l p o r t i n t h e c h a m b e r .T h e b i g a d v a n t a g e o f t h i s r a p i d s a l t i n g m e t h

o d is t h a t i t r e d u c e s t h e t i m e n e e d e d f o r s a l t i n g . I t is t h e m o s t r a p i d m e t h o d k n o w n t o t h i s a u t h o r f o r s a l t i n g w h o l e f i l l e t s . A s e c o n d a d v a n t a g e is t h a t t h e a m o u n t o f s a l t i m p a r t e d t o t h e f i l l e t s c a n b e c o n t r o l l e d w i t h a r e a s o n a b l e d e g r e e o f p r e c i s i o n s in c e i t is d e p e n d e n t o n t h e

a m o u n t o f w a t e r r e m o v e d b y f r e e z e d r y i n g .S e v e r a l p r o b l e m a r e a s a r e s u g g e s t e d in t h i s

t e c h n i q u e . A s e e m i n g l y g r e a t d i s a d v a n t a g e o f t h i s m e t h o d o f s a l t i n g is t h e e x p e n s e o f f r e e z e d r y i n g . A l s o , s u r f a c e w a t e r is r e m o v e d q u i c k l y w h i l e s u b s u r f a c e w a t e r r e m o v a l r e q u i r e s m o r e t i m e a n d v e r y s p e c i a l i z e d h i g h v a c u u m e q u i p m e n t . I n j e c t i o n o f t h e s a t u r a t e d s a l t s o l u t i o n i n t o t h e s a l t i n g c h a m b e r m u s t b e d o n e a t p r e c i s e ly t h e e x a c t t i m e f o r m a x i m u m e f f i c i e n c y a n d w i t h n o l o s s o f v a c u u m . A n i n j e c t i o n o f d r y s a l t a l s o m a y b e n e c e s s a r y t o k e e p a h ig h s a l t c o n c e n t r a t i o n i n t h e b r i n e . A g a in , i f t h e f i l l e t s a r e n o t c o m p l e t e l y c o v e r e d w i t h t h e s a l t s o l u t i o n , t h e y w i l l s a l t i r r e g u l a r l y . A n a e r o b i c b a c t e r i a m a y s t a r t t o g r o w , t h u s c a u s i n g l o w e r p r o d u c t q u a l i t y . A l s o , i t m a y b e n e c e s s a r y t o i n c l u d e a f i n a l d r y i n g s t e p .

T h e s a l t p r o p u l s i o n m e t h o d

A n o t h e r m e t h o d t e s t e d a t t h e A t l a n t i c F i s h e r y P r o d u c t s T e c h n o l o g y C e n t e r ( A F P T C ) in v o l v e d t h e p r o p u l s i o n o f s a l t c r y s t a l s i n t o f i s h f i l l e t s a s p e l l e t - p r o j e c t i l e s ( L e e , 1 9 6 9 ) . I t w a s f o u n d t h a t s m a l l p r o j e c t i l e s a b o u t 1 /8 in . d i a m c o u l d b e d r i v e n i n t o f i l l e t s w i t h o u t s i g n i f i c a n t e v i d e n c e o f t h e e n t r y o f t h e p r o j e c t i l e s . T h e t h e o r e t i c a l a d v a n t a g e s a r e t h a t d r y s a l t c o u l d q u i c k l y b e d i s t r i b u t e d t h r o u g h o u t t h e f i l l e t ( t h e

d e p t h t o w h i c h t h e p r o j e c t i l e s p e n e t r a t e a n d t h e i r d i s t r i b u t i o n m u s t b e c o n t r o l l e d ) . I n n a t e w a t e r o f t h e f i s h o r f i l l e t s e r v e s a s t h e v e h i c l e f o r s a l t d i s t r i b u t i o n . T h e r a p i d i t y o f s a l t i n g is d e p e n d e n t m a i n l y o n t h e d i s t a n c e b e t w e e n t h e p e l l e t s w h i c h m u s t b e s m a l l d u e t o s lo w d i f f u s i o n in f i s h m u s c l e .

T h i s m e t h o d is i n c l u d e d in t h i s a r t i c l e o n l y t o c i t e i t , b e c a u s e n o t e n o u g h w o r k h a s b e e n d o n e o n i t t o p e r m i t u s t o d i s c u s s i t i n d e t a i l . O n e p r o b a b l e d i s a d v a n t a g e is t h a t t h e r e h a s b e e n n o t e d a d i s c o l o r a t i o n a t t h e s i t e w h e r e t h e

s a l t p e l l e t s l o d g e d . A n o t h e r m i g h t b e t h e n o n -

u n i f o r m d i s t r i b u t i o n o f t h e s a l t c r y s t a l s i n f i l l e t s o f v a r i o u s s h a p e a n d t h i c k n e s s .

T h e D e l V a l l e / N i c k e r s o n m e t h o d

T h e n e e d f o r a m o r e e f f i c i e n t t e c h n i q u e t o r a p i d l y s a l t a n d d r y f is h w a s r e c o g n i z e d b y D e l V a l l e a n d N i c k e r s o n , a n d t h e y s t u d i e d t h e c o n v e n t i o n a l p r o c e s s e s f o r s a l t i n g a n d d r y i n g a n d d e t e r m i n e d t h e e q u i l i b r i u m c o n s i d e r a t i o n s in s a l t i n g ( D e l V a l l e a n d N i c k e r s o n , 1 9 6 7 A ) , t h e d y n a m i c a s p e c t s ( D e l V a l l e a n d N i c k e r s o n , 1 9 6 7 B ) a n d t h e d i f f u s i o n o f w a t e r ( D e l V a l l e

a n d N i c k e r s o n , 1 9 6 8 B ) in t h e p r o c e s s . I n 1 9 6 8 ,

D e l V a l l e e t a l . p u b l i s h e d a s e r i e s o f a r t i c l e s o n a q u i c k d r y - s a l t i n g p r o c e s s f o r f i s h ( D e l V a l l e

a n d N i c k e r s o n , 1 9 6 8 A ; D e l V a l l e a n d G o n - z a l e z - I n i g o , 1 9 6 8 ; D e l V a l l e a n d N i c k e r s o n ,

1 9 6 8 B ) .T h e p r o c e d u r e i n v o l v e s ( 1 ) G r i n d i n g t h e

s k i n n e d f i s h f l e s h w i t h s i m u l t a n e o u s a d d i t i o n o f

s a l t ( 2 0 - 1 0 0 % o f f i s h w e i g h t ) . T h i s i n c r e a s e s t h e s u r f a c e a r e a o f t h e f i s h a n d d e c r e a s e s t h e d i f f u s i o n d i s t a n c e , t h u s i n c r e a s i n g t h e r a t e o f s a l t p e n e t r a t i o n ; ( 2 ) F u r t h e r m i x i n g o f t h e

g r o u n d p r o d u c t t o d i s t r i b u t e t h e s a l t a n d f i s h u n i f o r m l y ; ( 3 ) P r e s s i n g t h e f i s h a n d s a l t m i x a t

h i g h p r e s s u r e s t o r e m o v e w a t e r a n d f o r m c a k e s . P r e s s i n g is n e c e s s a r y t o p r o d u c e c o h e r e n t c a k e s t h a t w i l l n o t d i s i n t e g r a t e u p o n s u b s e q u e n t

h a n d l i n g a n d d u r i n g l e a c h i n g in b o i l i n g w a t e r t o r e m o v e s a l t p r i o r t o e a t i n g ; a n d ( 4 ) D r y i n g t h e c a k e s . S u n - d r y i n g is r e c o m m e n d e d t o k e e p p r o c e s s i n g c o s t s l o w .

T h i s t e c h n i q u e w a s d e s i g n e d f o r u s e in u n d e r d e v e l o p e d c o u n t r i e s a n d , a s s u c h , r e q u i r e s l i t t l e p r o c e s s i n g e q u i p m e n t . A c c o r d i n g t o t h e a u t h o r s i t r e q u i r e s a r e l a t i v e l y s h o r t s a l t i n g t i m e a n d p r o d u c e s l o w - c o s t i n g s a l t e d f i s h i n d e f i n i t e l y s t a b l e w i t h o u t r e f r i g e r a t i o n i f p r o t e c t e d a g a i n s t r o d e n t i n f e s t a t i o n . T h e m e t h o d w o r k s

w e l l w i t h m o s t s p e c i e s o f f i s h i n c l u d i n g t r a s h f i s h , t r i m m i n g s a n d r e j e c t s f r o m f i l l e t i n g p l a n t s ( D e l V a l l e e t a l . , 1 9 7 3 ) .

F o r i t s i n t e n d e d u s e , t h e m e t h o d is a n e x c e l l e n t o n e ; b u t a s a p r o c e s s f o r i n d u s t r i a l i z e d c o u n t r i e s , i t i s n o t s u i t a b l e . O n e o f t h e p r o b l e m s w i t h t h e p r o c e s s i s t h a t i t d o e s n o t p r e v e n t o x i d a t i v e c h a n g e s w h i c h c a n o c c u r i n a l l p h a s e s o f t h e p r o c e s s . A ls o d u r i n g p r e s s i n g t o f o r m c a k e s , s o m e o f t h e m i x e x u d e s f r o m t h e p l a t e s . T h e d r y i n g t i m e is t o o l o n g f o r a n e f f i c i e n t i n d u s t r i a l p r o c e s s c a p a b l e o f h a n d l i n g s e a s o n a l

a b u n d a n c e s o f f i s h . F r e s h e n i n g o r d e s a l t i n g t h e c a k e s p r i o r t o e a t i n g r e q u i r e s b o i l i n g f o r 1 0 m i n in l a r g e a m o u n t s o f w a t e r . E x p o s u r e t o m e t a l c a t a l y s t s d u r i n g p r e s s i n g a n d m e c h a n i c a l d r y i n g i n a i r t o r e d u c e p r o c e s s i n g t i m e m a y a l s o c a u s e d e t e r i o r a t i o n o f t h e p r o d u c t .

T h e A n d e r s o n - M e n d e l s o h n m e t h o d

T h i s t e c h n i q u e w a s d e s i g n e d t o r e d u c e e v e n f u r t h e r t h e p r o c e s s i n g t i m e f o r s a l t - c u r i n g f is h a n d p r o d u c e a h i g h - q u a l i t y p r o d u c t s t a b l e a t r o o m t e m p e r a t u r e . T h e t h e o r y o f r a p i d w e t

s a l t i n g b r i n g s t o g e t h e r t h e m o s t a d v a n t a g e o u s c o n d i t i o n s f o r r a p i d s a l t - c u r i n g o f f i s h ( A n d e r s o n a n d M e n d e l s o h n , 1 9 7 2 ) . T h e t h e o r y w a s

v e r i f i e d in t h e l a b o r a t o r y b y u s i n g i d e a l c o n d i t i o n s ; h o w e v e r , s o m e c o n d i t i o n s w e r e a l t e r e d t o i n c r e a s e t h e t h r o u g h p u t t o c o m m e r c i a l s c a l e .

I n t h e s m a l l s c a l e l a b o r a t o r y e x p e r i m e n t s , s k in l e s s f i l l e t s w e r e p l a c e d in a b l e n d e r a n d g r o u n d u n d e r a s a t u r a t e d s a l t s o l u t i o n ( l g r : 1 m l r a t i o o f f i s h t o b r i n e ) t o w h i c h e x c e s s s a l t ( 4 g : l g r a t i o o f f i s h - t o - s a l t ) w a s a d d e d . T h e m i x w a s a l l o w e d t o s t a n d f o r 5 m i n . F r e e f l u i d w a s d r a i n e d t h r o u g h c h e e s e c l o t h t o r e m o v e e x c e s s

m o i s t u r e , a n d t h e w e t s a l t e d f i s h w a s v a c u u m - d r u m d r i e d . A f t e r d r y i n g ( a b c u t 3 0 m i n f o r

s m a l l b a t c h e s ) , t h e p r o d u c t w a s p a c k e d in l i g h t i m p e r m e a b l e p a c k a g e s a n d s t o r e d a t r o o m t e m

p e r a t u r e .I n e x p e r i m e n t s t o s i m u l a t e c o m m e r c i a l

p r o d u c t i o n , m i n c e d f i s h c o l l e c t e d f r o m a m e a t / b o n e s e p a r a t o r w a s m i x e d w i t h e n o u g h s a t u r a t e d s a l t s o l u t i o n t o c o v e r t h e f i s h f l e s h c o m p l e t e l y . E x c e s s s a l t w a s a d d e d in t h e r a t i o s o f 4 : 1 ( f l e s h t o s a l t ) . T h e s l u r r y w a s m i x e d c o n

t i n u o u s l y o v e r a s h o r t p e r i o d ( l e s s t h a n 5 m i n ) .

F r e e l i q u i d w a s t h e n p r e s s e d o u t i n a c o n t i n u o u s p r e s s f o l l o w e d b y d r y i n g A c o n t i n u o u s v a c u u m - d r u m d r y e r w o u l d b e m o s t e f f i c i e n t r e

q u i r i n g o n l y a s h o r t d w e l l - t i m e a n d w i t h o u t t h e d e l e t e r i o u s e f f e c t o f a i r . H o w e v e r , a w a t e r - j a c k e t e d r o t a r y r i b b o n v a c u u m d r y e r w a s u s e d

in t h e s c a l e d - u p p r o c e s s b e c a u s e n o l a r g e v a c u u m - d r u m d r y e r w a s a v a i l a b l e . A t w o - s t a g e v a c u

u m s y s t e m w a s u s e d in t h e r i b b o n d r y e r ( C a r v e r , 1 9 7 3 ) . T h e f i r s t s t a g e c o n s i s t e d o f a l a r g e w a t e r j e t a s p i r a t o r p u m p a n d t h e s e c o n d

s t a g e a s t e a m j e t p u m p c a p a b l e o f p r o d u c i n g

a b s o l u t e p r e s s u r e s d o w n t o 5 m m H g . D e p e n d in g u p o n t h e a m b i e n t w a t e r t e m p e r a t u r e ( t h e h i g h e r t h e t e m p e r a t u r e , t h e h i g h e r t h e v a p o r

p r e s s u r e ) t h e s a l t e d f i s h w a s a l l o w e d t o r e m a i n in t h e d r y e r f o r a b o u t 4 h r . T h e s e c o n d i t i o n s

h o l d o n l y a s l o n g a s t h e r e a r e n o l e a k s in t h e s y s t e m . D u r i n g t h e d r y i n g c y c l e , a t h e r m o c o u p l e p l a c e d i n s i d e t h e d r y e r w a s m o n i t o r e d to m a k e c e r t a i n t h a t t h e t e m p e r a t u r e o f t h e s a l t e d p r o d u c t n e v e r r o s e a b o v e 1 2 5 ° F t o m a i n t a i n t h e r a w f i s h c h a r a c t e r i s t i c s . F r o m t h e d r y e r , t h e s a l t e d f i s h w a s v a c u u m - p a c k e d in l i g h t i m p e r m e a b l e p o u c h e s a n d s t o r e d a t r o o m t e m p e r a t u r e .

A s i n t h e D e l V a l l e / N i c k e r s o n m e t h o d , t h e s u r f a c e a r e a is e n l a r g e d t o d e c r e a s e t h e d i f f u s io n d i s t a n c e , t h e r e b y i n c r e a s i n g t h e r a t e o f s a l t p e n e t r a t i o n . T h e f i s h is c o v e r e d w i t h b r i n e d u r in g s a l t i n g s o t h a t e x p o s u r e t o o x y g e n is v i r t u a l ly e x c l u d e d , a n d a l l s u r f a c e s a r e b r o u g h t i n t o c o n t a c t w i t h t h e b r i n e i m m e d i a t e l y . T h e a d d i t i o n o f e x c e s s s a l t t o m a i n t a i n s a t u r a t i o n w h e n t h e b r i n e is d i l u t e d w i t h t h e f l u i d o f t h e f is h

p r o v i d e s a l a r g e s a l t c o n c e n t r a t i o n g r a d i e n t u n t i l s a l t i n g is c o m p l e t e w i t h i n t h e f i s h f l e s h ( l e s s t h a n 5 m i n ) . S y n e r e s i s r e s u l t s w h e n t h e m u s c l e p r o t e i n is d e h y d r a t e d in t h e p r e s e n c e o f h ig h

c o n c e n t r a t i o n o f s a l t . I t s o c c u r r e n c e i n d i c a t e s t h a t t h e s a t u r a t e d s a l t s o l u t i o n d i f f u s e s s o

q u i c k l y t h a t “ s a l t i n g i n ” o f t h e m u s c l e p r o t e i n is f o l l o w e d i m m e d i a t e l y b y “ s a l t i n g o u t ” w i t h

n o v i s i b l e g e l f o r m a t i o n ; t h e r e f o r e , c o m p l e t e s a l t i n g o f t h e m u s c l e t a k e s p l a c e a t t h i s p o i n t . S i n c e t h e p a r t i c l e s i z e o f t h e m i n c e d f i s h is s m a l l , d r y i n g t i m e is s h o r t a s : s r e c o n s t i t u t i o n

( i n s t a n t a n e o u s l y i n b o i l i n g w a t e r ) o f t h e d r i e d p r o d u c t , t h u s m a k i n g i t a c o n v e n i e n c e i t e m .

S t o r a g e s t u d i e s i n p r o g r e s s h a v e s h o w n t h a t f i s h c a k e s o f a c c e p t a b l e q u a l i t y c a n b e m a d e f r o m s a l t e d w h i t i n g s t o r e d a t r o o m t e m p e r a t u r e f o r 11 m o n t h s .

A s w i t h t h e D e l V a l l e / N i c k e r s o n t e c h n i q u e , a l o s s i n p r o d u c t q u a l i t y m a y r e s u l t f r o m e x p o s u r e t o a i r a n d m e t a l c a t a l y s t s d u r i n g c o m m i n u t i o n . W h e n a n t i o x i d a n t s w e r e u s e d , p r o d u c t q u a l i t y i m p r o v e d . H o w e v e r , t h i s e f f e c t w a s n o t t h o r o u g h l y i n v e s t i g a t e d . A l s o , t h e f i n a l p r o d u c t s a r e l i m i t e d t o t h o s e t h a t c a n b e m a d e f r o m s a l t e d f i s h o f s m a l l p a r t i c l e s iz e .

DISCUSSION

E A C H R A P ID S A L T IN G te c h n iq u e has its ad v an tag e s a n d d isa d v an tag e s . T h e

SALT-CURING FISH TECHNIQUES-127

ch o ice o f a te c h n iq u e m u st be g o v ern ed by th e p ro d u c t d e s ired , th e e q u ip m e n t availab le an d th e t im e av ailab le fo r p re paring th e p ro d u c t. In th e “ b o ile d salt f ish ,” “ bagged sa lt f ish ” an d sa lt p ro p u lsion te c h n iq u e s , v ery l it t le e q u ip m e n t is n eed ed an d th e fin a l p ro d u c t is n o t d ried . T hese te c h n iq u e s a p p e a r su ita b le fo r u n d e rd ev e lo p e d c o u n tr ie s . In th e d e h y d ra tio n -in je c tio n an d ra p id w e t-sa ltin g te c h n iq u es , m u ch m o re e q u ip m e n t is n eed e d , and th e p ro cess tim e d e p e n d s o n th e d ry ing stage.

In th e Del V a lle -N ick e rso n sa ltin g te c h n iq u e , if a ir d ry in g is u se d , e q u ip m en t c o s ts can b e lo w e red if so m e q u a lity loss is a c c e p ta b le . S u n d ry in g re q u ire s m o re tim e th a n m ec h an ic a l d ry in g , th u s lim itin g th e o u tp u t o f sa lte d fish. P ilo t p lan t p ro d u c tio n an d large scale a c c e p tance tr ia ls o f th e q u ick -sa lted fish cak es m ad e by th e ra p id d ry -sa ltin g m e th o d are no w in p ro g ress (D el V alle e t a l., 197 3 ).

A lth o u g h th e A n d e rso n -M en d e lso h n sa ltin g m e th o d re q u ire s c o s tly p ieces o f e q u ip m e n t, it c an b e u sed to p ro cess tre m e n d o u s q u a n ti t ie s o f sa lted fish. Large scale c o m m e rc ia l e q u ip m e n t is av ailab le , and th e th ro u g h p u t is lim ite d

o n ly b y th e e ffic ie n c y o f th e d ry e r . It p ro d u c e s a h igh q u a lity p ro d u c t cap ab le o f lo n g -te rm ro o m te m p e ra tu re s to rag e . T his p ro d u c t is a c o n v en ien c e ty p e ite m and m ay have m o re c o m m e rc ia l a p p ea l p ro v id ed e n o u g h uses fo r m in ced sa lted fish can b e fo u n d .

REFERENCES

Anderson, M.L. and Mendelsohn, J.M. 1972. A rapid salt-curing technique. J. Food Sci. 37: 627.

Burgess, G.H.D., Cutting, C.L., Lovern, J.A. and Waterman, J.J. Ed. 1965. Salt-curing. “ In Fish Handling and Processing.” Her Majesty’s Stationary Office, Edinburgh, Scotland.

Carver, J.H. 1969, 1973. Private comm unication. National Marine Fisheries Service, A tlantic Fishery Products Technology Center, Emerson Ave., Gloucester, MA 01930.

Del Valle, F.R. and Gonzalez-Inigo, J.L. 1968. A quick-salting process for fish. 2. Behavior of different species of fish with respect to the process. Food Technol. 22(9): 85.

Del Valle, F.R. and Nickerson, J.T.R. 1967A. Studies on salting and drying fish. 1. Equilibrium considerations in salting. J. Food Sci. 32: 173.

Del Valle, F.R. and Nickerson, J.T .R . 1967B. Studies on salting and drying fish. 2. Dynamic aspects of the salting of fish J. Food Sci. 32: 218.

Del Valle, F.R. and Nickerson, J.T.R. 1968a . A quick-salting process for fish. 1. Evolution of the process. Food Technol. 22(8): 104.

Del Valle, F.R. and Nickerson, J.T.R. 1968B. Salting and drying fish. 3. Diffusion of water. J. Food Sci. 33: 499.

Del Valle, F.R ., Padilla, M., Ruz, A. and Rodriguez, R. 1973. Pilot plant production of large-scale acceptance trials with quick- salted fish cakes. J. Food Sci. 38: 246.

Iljas, S. and Ronsivalli, L.J. 1969. Improved m ethod for producing pindang. Fishery Ind. Res. 5(1), U.S. Fish & Wildlife Service, Bureau of Commercial Fisheries.

Ingram, J. and Kitchell, A.G. 1967. Salt as a preservative for foods. J. Fd. Technol. 2: 1.

Jarvis, N.D. 1950. Curing of fishery products. Research Report 18. BCF Fish and Wildlife Service, U.S. Dept, of the Interior.

Lee, J.M. 1969. Private com m unication. National Marine Fisheries Service, Atlantic Fishery Products Technology Center, Emerson Avenue, Gloucester, MA 01930.

Orr, A.T. 1967. Method and product for curing an d p a c k a g in g f ish . British Patent 1.077.644.

Ragulin, A.E. 1958. The comparative characteristics of anchovy salting using dry salt and salt solutions. In “ Israel Program for Scientific Translations” (translated from Russian by Styr, J.). Technol. of Fish Processing, p. 46. Office of Technical Services,U.S. Dept, of Commerce, Washington, D.C.

Stansby, M.E. 1963. Cured fishery products. In “ Industrial Fishery Technology,” p. 323. Reinhold Publishing Corp., New York.

Tressler, D.K. and Lemon, J.McW. 1951. The principles of salting—The salting of cod and other groundfish. In “ Marine Products of Commerce.” Reinhold Publishing Corp., New York.

Ms received 8/3 /73; revised 10/13/73; accepted10/15/73.

J . H . v o n E L B E , J . T . K L E M E N T , C . H . A M U N D S O N , R . G . C A S S E N S a n d R . C . L I N D S A Y

D e p a r t m e n t s o f F o o d S c i e n c e a n d M e a t & A n i m a t S c i e n c e

U n i v e r s i t y o f W i s c o n s i n - M a d i s o n , M a d i s o n , W t 5 3 7 0 6

EVALUATION OF BETALAIN PIGMENTS AS SAUSAGE COLORANTS

INTRODUCTION

SO D IU M N IT R IT E a n d n i tr a te have b een u se d fo r m an y y ears to d ev elo p th e c h a ra c te r is tic c o lo r o f c u re d m ea t p ro d u c ts . In a d d it io n to th e c o lo r f ix a t io n p ro p e r ty , th e se sa lts a re b e liev ed to c o n tr ib u te to flav o r d e v e lo p m e n t ar.d p re v en t b a c te r io lo g ic a l sp o ilag e o f c u re d m e a t p ro d u c ts (W asserm an a n d T alley , 1 9 7 2 ; C h ris tia n sen e t a l., 1 9 7 3 ). R e c e n tly , th e use o f so d iu m n i tr i te has b e en q u e s tio n e d b e cau se o f th e p o ss ib le fo rm a t io n o f c a rc in o g en ic N -n itro sa m in es u n d e r a c id ic c o n d it io n s (F id d le r e t al., 1 9 7 2 ). A n ex te n siv e rev iew o f th e fo rm a tio n o f N -n itro sa m in es in fo o d s has b e e n p u b lish ed re c e n tly b e S e b ra n ek a n d C assens (1 9 7 3 ) . S ince n i tra te s an d n i tr i te s c o u ld b e d e em ed a p o te n t ia l p u b lic h e a lth h a z a rd , a n d th e ir u se m ig h t b e p ro h ib ite d , a new c o lo r sy s te m to p ro d u c e th e c h a ra c te ris tic c u re d m ea t c o lo r w o u ld b e in g rea t d e m an d . T he u se o f n a tu ra l p ig m e n ts sh o u ld b e in v es tig a te d to d e te rm in e th e ir a p p lic a b ili ty to c u re d m ea t sy s tem s.

T he red b e e t ro o t (B e t a v u l g a r i s ) is a r ic h so u rc e o f p ig m e n ts k n o w n as b e ta - la in s (M ab ry a n d D re id ing , 1 9 6 8 ). T h is class o f p ig m e n ts c o n ta in s b o th th e b e ta - c y a n in (re d ) a n d b e ta x a n th in (y e llo w ) p ig m e n ts . T h e b e ta c y a n in s fo u n d in b e e ts in c lu d e b e ta n in ( th e m a jo r p ig m e n t) , iso- b e ta n in , p re b e ta n in , iso p re b e ta n in , and th e b e ta x a n th in s , v u lg a x a n th in I an d II (N ilsso n , 1 9 7 0 ). T h e c h e m is try o f th e se p ig m e n ts w as re c e n tly rev iew ed b y M abry (1 9 7 0 ) . A lth o u g h sim ila r p ig m e n ts a re fo u n d in o th e r p la n ts , th e b e e t is th e o n ly fo o d p ro d u c t c o n ta in in g th is g ro u p o f p ig m e n ts . S ince b e e t p o w d e r is p e rm it te d as a c o lo r a d d itiv e u n d e r th e 196 0 C o lo r A d d itiv e A m e n d m e n t, th e se p ig m e n ts w ere u sed to s im u la te c u re d m e a t c o lo r in c o o k e d , sm o k e d (b o lo g n a ) a n d se m id ry , fe rm e n te d (su m m er sau sage) sausage p ro d u c ts .

MATERIALS & METHODSPreparation of betalain powder

B e e t p o w d e r w a s p r e p a r e d u s i n g t h e f o l l o w i n g p r o c e d u r e : f r e s h b e e t s w e r e b l a n c h e d a t lO C P C f o r 1 5 m i n , d i c e d ( 6 m m ) a n d m i l l e d w i t h a F i t z p a t r i c k m o d e l D m i l l , e q u i p p e d w i t h a 1 / 4 - i n . ( m e s h s iz e ) s c r e e n . T h e p u l p w a s p r e s s e d w i t h a h y d r a u l i c p r e s s a t a p r e s s u r e o f 3 0 0 p s i , a n d t h e j u i c e ( 1 0 % s o l u b l e s o l id s ) w a s

c o l l e c t e d , d e s l u d g e d ( S e l f C l e a n i n g C l a r i f i e r , D e

L a v a l , P X 3 0 9 ) , a n d c o n c e n t r a t e d i n a s in g le e f f e c t f a l l i n g f i l m e v a p o r a t o r t o a s o l u b l e s o l id s c o n t e n t o f 2 0 % . T h e e v a p o r a t o r w a s o p e r a t e d w i t h a n i n l e t t e m p e r a t u r e o f 3 3 ° C a n d a n o u t l e t t e m p e r a t u r e o f 4 4 ° C . T h e c o n c e n t r a t e w a s i m m e d i a t e l y c o o l e d t o 2 8 ° C b y p a s s i n g i t o v e r a

t u b u l a r s u r f a c e c o o l e r . B e e t p o w d e r w a s o b t a i n e d b y s p r a y d r y i n g t h e c o n c e n t r a t e i n a l a r g e e x p e r i m e n t a l d r y e r l o c a t e d in t h e U n i v e r s i t y o f W i s c o n s i n F o o d P r o c e s s i n g P i l o t P l a n t . D r y i n g c o n d i t i o n s w e r e : a i r i n l e t t e m p e r a t u r e , 1 7 5 ° C ; a i r o u t l e t t e m p e r a t u r e , 1 2 3 ° C ; p r e s s u r e , 1 5 0 0 p s i ; n o z z l e s i z e , S X 6 4 / 2 1 .

T h e r e s u l t i n g p o w d e r w a s p a c k e d i n N o . 3 0 3 o r N o . 1 0 c a n s . T h e c a n s w e r e f l u s h e d w i t h n i t r o g e n , s e a l e d u n d e r v a c u u m a n d s t o r e d a t — 2 3 ° C . T h e b e t a n i n c o n t e n t w a s d e t e r m i n e d b y t h e m e t h o d d e s c r i b e d b y v o n E l b e e t a l . ( 1 9 7 2 ) . T h e m o i s t u r e c o n t e n t o f t h e p o w d e r w a s d e t e r m i n e d b y t h e s t a n d a r d A O A C m e t h o d ( A O A C ,

1 9 6 0 ) .

Preparation of pure betaninP u r e b e t a n i n w a s p r e p a r e d f o l l o w i n g t h e

m e t h o d b y v o n E l b e e t a l . ( 1 9 7 2 ) . T h i s m e t h o d i n v o l v e s t h e p r e p a r a t i o n o f a n a q u e o u s p i g m e n t e x t r a c t , a n d s e p a r a t i o n a n d p u r i f i c a t i o n o f t h e p i g m e n t s b y c h r o m a t o g r a p h y .

Preparation of sausagesC o o k e d s m o k e d bologna. 1 0 - lb l o t s o f

b o l o g n a e m u l s i o n s c o n t a i n i n g b e e f a n d p o r k in a p p r o x i m a t e l y e q u a l a m o u n t s w e r e f o r m u l a t e d

t o r e s u l t i n p r o d u c t s c o n t a i n i n g 3 0 % f a t a n d 1 0 % ( w / w ) a d d e d w a t e r . S a l t , c o m m e r c i a l s p i c e m i x t u r e c o n s i s t i n g o f w h i t e p e p p e r , g i n g e r , e x t r a c t o f c o r i a n d e r , a n d n u t m e g , a n d ic e w e r e a d d e d t o t h e l e a n m e a t ( b e e f ) a n d t h e m i x t u r e

w a s c h o p p e d f o r a p p r o x i m a t e l y 4 m i n . P o r k w a s t h e n a d d e d a n d c h o p p i n g w a s c o n t i n u e d f o r a p p r o x i m a t e l y 3 m i n u n t i l a n e m u l s i o n w a s a c h i e v e d . S a m p l e s p r e p a r e d i n c l u d e d t h e f o l l o w i n g : c o n t r o l ( n o c o l o r a g e n t s ) ; b o l o g n a c o n t a i n i n g t h e m a x i m u m l e g a l l e v e l s o f s o d i u m n i t r i t e a n d s o d i u m n i t r a t e ( h e r e a f t e r r e f e r r e d t o a s N 0 2 a n d N 0 3 , r e s p e c t i v e l y ) ; 1 / 4 o z N a N 0 2 , 2 - 3 / 4 o z N a N O 3 / 1 0 0 lb m e a t ( r e f e r r e d t o a s 1 5 6 p p m N 0 2 ) ; b o l o g n a c o n t a i n i n g b e e t p o w d e r t o g iv e c o n c e n t r a t i o n s o f 3 3 , 4 5 a n d 5 6 p p m b e t a n i n , r e s p e c t i v e l y ; a n d b o l o g n a c o n t a i n i n g 4 5 p p m p u r i f i e d b e t a n i n . T h e b e e t p o w d e r a d d e d c o n t a i n e d 6 0 0 m g b e t a n i n p e r lO O g . T h e a d d i t i v e s w e r e i n c o r p o r a t e d i n t h e d r y s t a t e w i t h t h e s p i c e . T h e t e m p e r a t u r e o f t h e m e a t m i x t u r e n e v e r e x c e e d e d 1 3 ° C d u r i n g c h o p p i n g . T h e f i n i s h e d e m u l s i o n w a s s u b s e q u e n t l y s t u f f e d i n t o a 2 - in . d i a m s a u s a g e c a s i n g ( B r e c h t e e n C a s in g N o . 1 6 8 5 3 , B r e c h t e e n C a s in g C o . , M t . C l e m e n s , M i c h . ) , a n d p r o c e s s e d w i t h a s t e p w i s e h e a t i n g c y c l e ( a m b i e n t t o 8 0 ° C ) w i t h s m o k e u n t i l a n i n t e r n a l t e m p e r a t u r e o f 6 7 —6 9 ° C w a s r e a c h e d ( P r i c e a n d S c h w e i g e r t , 1 9 7 1 ) . T h e s a u s a g e w a s s u b s e q u e n t l y s h o w e r e d w i t h c o l d w a t e r t o 3 0 ° C .

Semidry ferm ented summer sausage. 10-lbl o t s o f s u m m e r s a u s a g e c o n t a i n i n g a m i x t u r e o f 6 5 % g r o u n d b e e f c h u c k a n d 3 5 % g r o u n d r e g u l a r p o r k t r i m m i n g s w e r e f o r m u l a t e d t o r e s u l t in

p r o d u c t s c o n t a i n i n g a p p r o x i m a t e l y 3 0 % f a t . A f t e r t h e a d d i t i o n o f s a l t , s u g a r a n d c o m m e r c i a l s p i c e ( m o n o s o d i u m g l u t a m a t e , b l a c k p e p p e r ) , t h e m i x t u r e w a s c h o p p e d in a s i l e n t c u t t e r t o a p p r o p r i a t e p a r t i c l e s iz e . T h e c h o p p e d m e a t

w a s t r a n s f e r r e d t o a m i x e r w h e r e a c u l t u r e o f P e d io c o c c u s c e r e v is ia e ( A C - 1 ; C h r i s H a n s e n L a b o r a t o r y , M i l w a u k e e ) w a s a d d e d a n d s u b

s e q u e n t l y m i x e d f o r 1 m i n . S a m p l e s p r e p a r e d i n c l u d e d : c o n t r o l ( n o c o l o r a g e n t s ) ; s u m m e r s a u s a g e c o n t a i n i n g m a x i m u m l e g a l l e v e l s o f n i t r i t e - n i t r a t e ; 1 / 4 o z N 0 2 , 2 - 3 / 4 o z N O 3 / 1 0 0 lb m e a t ( r e f e r r e d t o a s 1 5 6 p p m N 0 2 ) , s u m m e r s a u s a g e c o n t a i n i n g b e e t p o w d e r ( 6 0 0 m g b e t a n i n / l O O g ) t o g iv e c o n c e n t r a t i o n s o f 2 5 a n d 3 3 p p m b e t a n i n , r e s p e c t i v e l y . T h e a d d i t i v e s w e r e i n c o r p o r a t e d in t h e d r y s t a t e w i t h t h e s p i c e .

T h e f i n i s h e d m i x t u r e s w e r e s u b s e q u e n t l y s t u f f e d i n t o 2 - in . d i a m c a s i n g ( B r e c h t e e n N o . 1 6 8 5 3 ) , p l a c e d in a s m o k e h o u s e , a n d h e l d a t

3 5 ° C w i t h s m o k e u n t i l a p H o f 4 . 8 w a s a c h i e v e d . T h e s a u s a g e w a s t h e n h e a t e d ( s m o k e h o u s e t e m p e r a t u r e 6 5 ° C ) t o a n i n t e r n a l t e m p e r a t u r e o f 6 0 ° C .

A t t h e c o n c l u s i o n o f h e a t i n g , a l l s a u s a g e s w e r e s h o w e r e d w i t h c o l d w a t e r u n t i l t h e y r e a c h e d 5 0 ° C , a n d t h e n w e r e s t o r e d a t 4 ° C . A f t e r c o o l i n g , a l l s a m p l e s w e r e w r a p p e d l o o s e l y i n S a r a n w r a p t o m i n i m i z e d e h y d r a t i o n .

Microbiological analysesT h e b a c t e r i a l f l o r a w e r e e n u m e r a t e d f o r

t o t a l a e r o b i c m i c r o o r g a n i s m s , a n d a e r o b i c a n d a n a e r o b i c s p o r e f o r m e r t y p e s . S a u s a g e s w e r e

a s e p t i c a l l y c u t , a n d d u p l i c a t e 1 1 -g s a m p l e s w e r e r e m o v e d . E q u a l p o r t i o n s o f i n t e r n a l a n d e x t e r n a l s a u s a g e w e r e p l a c e d in a s t e r i l e b l e n d e r j a r . S t e r i l e d i s t i l l e d w a t e r w a s a d d e d t o m a k e a 1 : 1 0 d i l u t i o n a n d t h e m i x t u r e w a s b l e n d e d f o r 2

m i n . S u b s e q u e n t d i l u t i o n s w e r e m a d e w i t h 9 9 m l s t e r i l e d i s t i l l e d w a t e r b l a n k s .

T h e t o t a l b a c t e r i a l c o u n t s w e r e e n u m e r a t e d o n d u p l i c a t e p o u r p l a t e s o n s t a n d a r d p l a t e

c o u n t ( S P C ) a g a r . T h e p l a t e s w e r e c o u n t e d a f t e r a 3 - d a y i n c u b a t i o n p e r i o d a t 3 0 ° C . T o t a l s p o r e s w e r e d e t e r m i n e d a f t e r h e a t s h o c k i n g b y a3 - t u b e m o s t p r o b a b l e n u m b e r ( M P N ) m e t h o d in f l u i d t h i o g l y c o l a t e b r o t h . T h i s m e t h o d is s i m i l a r t o t h a t e m p l o y e d f o r s p i c e s . T h e t u b e s w e r e i n c u b a t e d a t 3 7 ° C f o r 2 d a y s a n d t h e n o b s e r v e d f o r g r o w t h .

ColorC o l o r m e a s u r e m e n t s w e r e m a d e w i t h a

H u n t e r C o l o r D i f f e r e n c e M e t e r ( H u n t e r A s s o c i a t e s L a b , F a i r f a x , V a . ) . T h e i n s t r u m e n t w a s s t a n d a r d i z e d w i t h t h e p i n k s t a n d a r d p l a t e ( N o . D 2 5 - 1 3 0 2 ; L = 4 . 5 , a L = 1 2 . 6 , b L = 7 . 3 ) . R e s u l t s w e r e e x p r e s s e d a s L , a L , b L , a n d w h e r e a p p l i c a b l e t h e q u a n t i t y A E = [ ( L , — L 2 ) 2 + ( a , - a 2 ) 2 + ( b , - b , ) 2 ] 1/2 w a s c a l c u l a t e d .


B E TALAIN 4 S S A U S A G E C O L O R A N T S - 1 2 9

Sensory evaluation

The bologna and sum m er sausage samples were evaluated for co lor and flavor using a sem itrained 35-m em ber panel. F o r flavor evaluations each series o f sam ples was presented sim ultaneously using full or subdued lighting in isolated taste panel bo oth s . Sim ilar co lor evaluations were cond ucted separately using a standard M acBeth daylight-type lam p system (M acBeth D aylight C orp., Newburgh, N .Y .) for illum ination. T he sam ples were scored on a hedonic scale o f e ith er 7 or 9 poin ts as indicated . Mean scores, analysis o f variance and least sign ificant d ifferen ces were determ ined using a Univac 1 1 0 8 com pu ter (A ST M , 1 9 6 8 ) . In addition, a small five-m em ber exp ert panel critically evaluated the product characteristics o f each sample.

Chem ical analyses

M oisture and fat were determ ined with vacuum oven and S o xh let m eth od s, respectively (A O AC, 1 9 6 0 ) . T hiob arb itu ric acid (T B A ) num bers w ere determ ined by the m ethod o f Tarladgis e t al. ( 1 9 5 9 ) to estim ate degree o f lipid oxid ation.

Table 1—In itia l levels of percent fa t, percent moisture and TBA numbers of bologna and summer sausage

SampleFat%

Moisture%

TBAno.

BolognaControl — no additives 34.2 49.0 0.624N itrite

1 56 ppm 35.6 46.6 0.3121 ppm 33.1 52.0 0.205

Beet powder33 ppm 32.0 49.0 0.31245 ppm 32.9 50.6 0.27856 ppm 31.3 50.0 0.312

Purified beet color45 ,pm 32.0 51.9 0.278

Summer sausageControl — no additives 28.7 48.0 0.468N itrite

1 56 ppm 33.1 42.0 0.312Beet powder

25 ppm 30.0 49.0 0.31233 ppm 32.7 46.1 0.312

RESULTS & DISCUSSIOND A T A f o r t h e i n i t i a l l e v e l s o f p e r c e n t m o i s t u r e , f a t a n d T B A n u m b e r s a r e g i v e n i n T a b l e 1. C o n s i d e r i n g t h e l i m i t a t i o n s

i m p o s e d i n t h e p r e p a r a t i o n o f 1 0 - l b l o t s ,

t h e c o m p o s i t i o n d a t a i n d i c a t e t h a t r e l a

t i v e l y g o o d p r o d u c t u n i f o r m i t y w a s a c c o m p l i s h e d . A l t h o u g h t h e l e g a l l i m i t o f

f a t w a s e x c e e d e d i n s o m e l o t s , a l l p r o d u c t s s i m u l a t e d t h o s e c o m m e r c i a l l y a v a i l

a b l e . G r e a t e r T B A n u m b e r s w e r e o b s e r v e d i n t h e c o n t r o l l o t s c o m p a r e d t o

l o t s c o n t a i n i n g c o l o r a g e n t s . D u r i n g s t o r a g e s o m e f l u c t u a t i o n i n T B A n u m b e r s

w a s o b s e r v e d . T h e s e r e s u l t s a r e s i m i l a r t o t h o s e r e p o r t e d b y M a c N e i l a n d M a s t

( 1 9 7 3 ) i n a s t u d y f o r f r a n k f u r t e r s .S a u s a g e s p r e p a r e d w i t h o u t s o d i u m

n i t r i t e - n i t r a t e o r b e e t p i g m e n t e x h i b i t e d a d e f i n i t e g r e y c o l o r . H o w e v e r , s a u s a g e s c o n t a i n i n g b e e t p i g m e n t o r p u r e b e t a n i n

i n t h e f o r m u l a t i o n e x h i b i t e d a h u e c l o s e l y m a t c h i n g t h a t o f s a u s a g e c o n t a i n i n g s o d i u m n i t r i t e - n i t r a t e i n t h e f o r m u l a t i o n . T h e s a m p l e s w i t h b e e t p i g m e n t a p p e a r e d t o h a v e a l o w e r v a l u e ( d a r k e r ) . D i s t r i b u t i o n o f t h e b e e t p i g m e n t t h r o u g h o u t t h e w a t e r p h a s e o f t h e s a u s a g e m i x t u r e s w a s e x c e l l e n t , a n d b e c a u s e o f t h e e x t r e m e i n s o l u b i l i t y o f t h e p i g m e n t i n l i p i d s t h e f a t p a r t i c l e s r e m a i n e d u n c o l o r e d .

I n b o t h t y p e s o f s a u s a g e s s e n s o r y e v a l u a t i o n s c o r e s f o r c o l o r a l o n e w e r e h i g h e r f o r s a u s a g e s c o n t a i n i n g n i t r i t e - n i t r a t e a t 0 d a y s o f s t o r a g e . H o w e v e r , t h e c o l o r s c o r e s o f t h e s a u s a g e s w i t h o p t i m u m b e e t p i g

m e n t l e v e l ( 3 3 p p m b e t a n i n ) a f t e r 2 w k o f s t o r a g e s h o w e d n o s t a t i s t i c a l d i f f e r

e n c e s c o m p a r e d t o s a u s a g e s c o n t a i n i n g n i t r i t e - n i t r a t e ( T a b l e 2 ) . T a b l e 2 f u r t h e r p o i n t s o u t t h a t c o l o r s t a b i l i t y w h e n e x p o s e d t o l o w i n t e n s i t y l i g h t ( 2 5 c a n d l e - p o w e r ) w a s g r e a t e r i n s a u s a g e s c o n t a i n i n g

b e e t p i g m e n t , a n d a f t e r 2 w k o f l i g h t e x

Table 2—Sensory evaluation color scores fo r bologna and summer sausage samples before and after storage w ith and w ithou t exposure®

Stored w ithou t Stored w ithexposure to light exposure to light

Sample 0 7 days 14 days 7 days 14 days

BolognaControl — no additives 2 .00a 2.47a 2.33a 3.06a 2.33a,bN itrite

1 56 ppm 4.67c 4.72c 4.28b 3.42a ,b 2.48a,bBeet powder

33 ppm 3.75b,c 3.66b 3.56b 4.29b 3.90c56 ppm 3.31b 2 .66a 2.06a 3.55a,b 3.48b,c

Summer sausageControl — no additives 1.78a 1.84a 1.89a 2.26a 1.90aN itrite

156 ppm 5.24c 4.81c 4.17c 3.06a,b 2 .00aBeet powder

25 ppm 3.05b 3 31b 2.56b 3.48a,b 3.10b33 ppm 3.62b 3.50b,c 3.00b 3.55b 3.25b

a Scale is a 7 point hedonic — 1 = dislike extremely to 7 = like extremely. Values that are followed by the same letter In any one column are not significantly different from each other at the 5% level.

p o s u r e s a u s a g e s c o n t a i n i n g t h e p i g m e n t w e r e j u d g e d s i g n i f i c a n t l y h i g h e r t h a n s a u

s a g e s c o n t a i n i n g s o d i u m n i t r i t e - n i t r a t e . T h e c o l o r s c o r e s o b t a i n e d f r o m p a n e l s c o r r e l a t e d w e l l w i t h c o l o r v a l u e s o b t a i n e d w i t h t h e H u n t e r C o l o r D i f f e r e n c e M e t e r ( T a b l e 3 ) . S i m i l a r ‘a^’ a n d ‘b L ’ v a l u e s w e r e n o t e d i n s a u s a g e s c o n t a i n i n g s o d i u m n i t r i t e - n i t r a t e a n d s a u s a g e s c o n t a i n i n g t h e o p t i m u m l e v e l o f b e e t p i g m e n t . S a u s a g e s f o r m u l a t e d w i t h p i g m e n t , h o w e v e r , h a d l o w e r L v a l u e a n d t h e r e f o r e a p p e a r e d d a r k e r . O n e x p o s u r e t o l i g h t a g r e a t e r

c h a n g e i n ‘a i / v a l u e w a s n o t e d i n s a u s a g e s

c o n t a i n i n g s o d i u m n i t r i t e - n i t r a t e . T h a t t h e c o l o r c h a n g e s i n p i g m e n t - c o n t a i n i n g s a u s a g e s i s l e s s a f t e r s t o r a g e a n d e x p o s u r e t o l i g h t i s i n d i c a t e d b y t h e s m a l l e r A E v a l u e ( T a b l e 3 ) .

S e n s o r y e v a l u a t i o n s c o r e s f o r b o l o g n a a n d s u m m e r s a u s a g e s f o r f l a v o r , t e x t u r e , c o l o r a n d o v e r a l l a c c e p t a n c e a r e s h o w n i n T a b l e 4 . I n g e n e r a l , t h e f l a v o r s c o r e s f o r t h e n i t r i t e - n i t r a t e - c o n t a i n i n g p r o d u c t s w e r e h i g h e r t h a n f o r t h o s e p r o d u c t s c o n t a i n i n g b e e t p i g m e n t s ; h o w e v e r , t h e r e w a s n o s t a t i s t i c a l d i f f e r e n c e b e t w e e n t h e n i t r i t e - n i t r a t e - c o n t a i n i n g s a u s a g e s a n d t h e

1 3 0 - J O U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

Table 3—Hunter color reflectance values of bologna and summer sausage stored at 4°C w ith and w ith o u t exposure to light3

Sample L Sample a\_ Sample b[_ AE

Stored 7 days Stored 7 days Stored 7 days Stored 7 days0 NL WL 0 NL WL 0 NL WL N L WL

BolongnaControl — no additives 55.5 57.1 52.3 5.3 7.0 6.4 10.6 1 1 .2 10.5 2.4 3.3N itrite

156 ppm 53.4 53.6 56.9 11.4 11.5 4.6 9.8 9.5 10.5 0.2 7.7Beet powder

33 ppm 46.1 50.4 50.7 9.5 10.0 8.0 11 .0 11.7 11.7 4.4 4.956 ppm 47.9 46.2 46.2 11.7 10.7 10.1 10.7 1 1 .1 11.7 2.0 2.5

Summer sausage

Control — no additives 44.1 45.4 45.7 3.8 6.0 5.1 7.3 8.1 8.7 2.7 2.5N itrite

1 56 ppm 46.2 48.6 54.2 11.5 11.7 2.2 7.5 7.8 9.0 2.4 12.4Beet powder

25 ppm 43.1 46.3 47.8 8.4 8.7 6.3 6.8 7.3 8.7 3.2 5.533 ppm 42.7 45.2 49.2 10.1 1 1 .1 7.4 7.2 7.3 8.3 2.7 7.1

a Average of triplicate observations. NL = no light; WL = with light

s a u s a g e s c o n t a i n i n g t h e o p t i m u m l e v e l o f

b e e t p i g m e n t ( 3 3 p p m b e t a n i n ) . R e s u l t s f o r c o l o r w e r e s i m i l a r t o t h e p r e v i o u s r e s u l t s w h e n c o l o r o n l y w a s e v a l u a t e d . I n i t i a l l y c o l o r s c o r e s w e r e h i g h e r f o r

n i t r i t e - n i t r a t e - c o n t a i n i n g s a u s a g e s , b u t a f t e r 1 o r 2 w k o f s t o r a g e t h e r e w a s n o s t a t i s t i c a l d i f f e r e n c e b e t w e e n t h e s e t r e a t

m e n t s . H o w e v e r , t h e r e s u l t s o n s u m m e r s a u s a g e i n d i c a t e h i g h e r f l a v o r , t e x t u r e a n d

a c c e p t a n c e a b s o l u t e s c o r e s i n t h o s e s a m

p l e s c o n t a i n i n g n i t r i t e t h a n t h o s e w i t h o u t a d d e d n i t r i t e . A l l c o l o r e d s a m p l e s h a d a s i g n i f i c a n t d i f f e r e n c e i n o v e r a l l a c c e p t

a n c e a n d w e r e p r e f e r r e d o v e r t h e c o n t r o l .

I n a s e c o n d e x p e r i m e n t t h e s a m e p r o d

u c t s w e r e s e r v e d t o t h e j u d g e s i n a d a r k e n e d r o o m , e l i m i n a t i n g c o l o r d i f f e r e n c e s . I t i s o f i n t e r e s t t o n o t e t h a t w h e n c o l o r d i f f e r e n c e s w e r e e l i m i n a t e d , a g a i n n o f l a

v o r p r e f e r e n c e s w e r e f o u n d , a n d t h e a b

s o l u t e f l a v o r s c o r e s w e r e h i g h e r ( T a b l e 5 ) . T h e s e r e s u l t s i n d i c a t e t h a t t h e f l a v o r o f

e a c h p r o d u c t c a t e g o r y b e h a v e s s o m e w h a t d i f f e r e n t l y w h e n n i t r i t e i s r e m o v e d .

B o l o g n a w a s n o t m a r k e d l y a f f e c t e d , w h i l e s u m m e r s a u s a g e w a s a f f e c t e d .

U n d e r t h e s a m e c o n d i t i o n s t h e e x p e r t p a n e l i s t s w e r e a b l e t o d e t e c t s u b t l e d i f f e r e n c e s . T o t h e e x p e r t p a n e l t h e m o s t

n o t i c e a b l e d i f f e r e n c e b e t w e e n n i t r i t e -

n i t r a t e - c o n t a i n i n g p r o d u c t s a n d c o n t r o l

o r b e e t p i g m e n t c o l o r e d p r o d u c t s w a s t h e o c c u r r e n c e o f s l i g h t “ f r e s h m e a t ” o r

“ p o r k y ” f l a v o r s i n t h o s e n o t c o n t a i n i n g

n i t r i t e - n i t r a t e . A p p a r e n t l y , l o w o r h i g h l e v e l s o f n i t r i t e - n i t r a t e d o n o t a d d a n y

h i g h i m p a c t f l a v o r n o t e s , b u t b y s o m e

m e a n s s u b t r a c t m u c h o f t h e c h a r a c t e r i s t i c

f r e s h m e a t f l a v o r . T h e e x p e r t p a n e l a l s o o b s e r v e d t h e d e v e l o p m e n t o f o x i d i z e d

a n d / o r s t a l e f l a v o r s i n t h e c o n t r o l , b u t

t h i s c h a r a c t e r i s t i c w a s n o t n o t e d i n t h e

n i t r i t e - n i t r a t e o r b e e t p i g m e n t - c o n t a i n i n g

s a m p l e s . T h e s e o b s e r v a t i o n s w e r e s u p

p o r t e d b y T B A n u m b e r s ( T a b l e 1 ) . T h e

l a r g e r s e m i t r a i n e d p a n e l m e m b e r s d i d n o t r e a c t s t r o n g l y t o s u b t l e f l a v o r a n d c o l o r

Table 4 —Sensory evaluation scores o f bologna and summer sausage w ith color differences apparent to judges3

Sample

Bologna

Control — no additives N itrite

1 56 ppm Beet powder

33 ppm 56 ppm

Summer sausageControl — no additives N itrite

1 56 ppm Beet powder

25 ppm 33 ppm

FlavorStorage tim e (days)

0 7 14

5.50a 5.81a 5.56a

6.72c 6.28a 5.78a

6.31 be 5.86ab

6.53a5.97a

5.72a5.44a

4.70a 4.41a 3.72a

6.51b 5.47b 5.44b

4.68a4.92ab

4.78ab5.44b

4.78b5.06b

TextureStorage tim e (days)

0 7 14

6.1 1 a 6.31a 5.89a

6.39a 6.31a 6 .00a

6.47a6.25a

6.56a6.34a

5.94a5.72a

4.39a 4.47a 4.06a

5.58b 5.25b 5.28b

4.81a4.58a

4.69ab5.03ab

4.33a4.61a

ColorStorage tim e (days)

0 7 14

3.36b 3.78a 4.67a

6.81c 6.50c 5.83b

5.75c5.44b

5.56bc4.78b

5.50b3.89a

2.78a 3.09a 3.22a

7.32c 6 .88c 6.06c

4.65b4.86b

4.53b5.47b

4.78abc5.22bc

Overall acceptance Storage tim e (days)

0 7 14

4.6 I a 5.16a 5.18a

6.56b 6.34c 5.47a

6.14bc5.78c

6.00bc5.44ab

5.88a4.59a

3.84a 3.97a 3.72a

6.38b 5.75b 5.44c

4.43a4.57a

4.69a5.31b

4.50abc4.94bc

3 Scale: 9 point hedonic with 1 - dislike extremely to 9 - like extremely. Values that are followed by the same letter In any one column are not significantly different from each other at the 5% level.

BE TALAIN A S S A U SA G E C O L O R A N T S - 131

Table 5—Sensory evaluation scores fo r flavor and texture of bologna and summer sausage w ith color differences elim inated3

Flavor TextureStorage tim e (days) Storage tim e (days)

Sample 7 14 7 14

Bologna

Control — no additives 5.90a 6.48a 6.55a 6.43aN itrite

156 ppm 5.77a 6.29a 6.48a 6 .10 aBeet powder

33 ppm 6.26a 6.29a 6.52a 6.38a

Summer sausageControl — no additives 4.56a 3.59a 4.34a 4.05aN itrite

156 ppm 5.59b 5.25b 5.09b 5.20cBeet powder

25 ppm 4.91b 4.80b 4.56ab 4.55ab33 ppm 4.91b 4.90b 5.00b 4.85bc

Scale: 7 point hedonic with 1 = dislike extremely to 7 = like extremely. Values that are followed by the same letter in any one column are not significantly different from each other at the 5% level.

Table 6—Standard plate and spore counts of bologna and summer sausage samples at 3 days and after 14 days o f storage at 4°C

Standard plate count Spore countStorage time (days) Storage tim e (days)

Sample 0 14 0 14

BolognaControl — no additive 3.3 x 104 5.2 x 106 4.3 x 104 9.3 x 106N itrite

1 56 ppm 3.4 x 10“ 1.9 x 104 9.3 x 104 7.3 x 102Beet powder

56 ppm 2.2 x 10 4 3.1 x 104 9.3 x 102 9.3 x 103

Summer sausageControl — no additives 2.9 x 10s 2.2 x 1 0 5 2.5 x 10s 9.3 x 103N itrite

1 56 ppm 9.1 x 107 2.6 x 1 0 7 4.3 x 10" 2.4 x 104Beet powder

33 ppm 1.8 x 1 0 7 4.6 x 106 2.0 x 10 4 9.3 x 104

d i f f e r e n c e s i n t h e s a m p l e s , p o s s i b l y b e c a u s e t h e y w e r e s o m e w h a t a c c u s t o m e d t o t h e w i d e r a n g e o f s a u s a g e f l a v o r s n o r m a l l y e n c o u n t e r e d i n d i f f e r e n t b r a n d s o f c o m m e r c i a l l y a v a i l a b l e s a u s a g e s . T h e

s e m i t r a i n e d p a n e l r e s u l t s i n d i c a t e t h e g r e a t i n f l u e n c e o f c o l o r o n f l a v o r s c o r e s

a n d o v e r a l l a c c e p t a n c e , a n d a l s o p o i n t o u t t h a t t h e f l a v o r a c c e p t a n c e w a s l i t t l e a f

f e c t e d b y t h e p r e s e n c e o f s o d i u m n i t r i t e - n i t r a t e . T h e s e r e s u l t s s u p p o r t t h o s e o f M a c N e i l a n d M a s t ( 1 9 7 3 ) w h o h a v e s t a t e d

t h a t c o l o r , a n d n o t f l a v o r , w a s t h e m a j o r p r o b l e m t h a t m u s t b e o v e r c o m e s h o u l d

n i t r i t e s a n d n i t r a t e s b e e l i m i n a t e d a s a c o l o r - f i x i n g a g e n t . S i n c e b o t h s a u s a g e

p r o d u c t s w e r e s m o k e d , t h e r e s u l t s a l s o a g r e e w e l l w i t h t h o s e r e p o r t e d b y W a s s e r -

m a n a n d T a l l e y ( 1 9 7 2 ) w h o r e p o r t e d t h a t t h e f l a v o r o f f r a n k f u r t e r s w a s s i g n i f i c a n t

l y a f f e c t e d b y s m o k e , a n d t h a t f e w d e

t e c t a b l e d i f f e r e n c e s w e r e o b s e r v e d a s a r e s u l t o f t h e p r e s e n c e o f n i t r i t e . F r o m t h e s e s t u d i e s i t w o u l d a p p e a r t h a t i n b o l o g n a a n d s u m m e r s a u s a g e s t h e a d d i

t i o n o f s o d i u m n i t r i t e a n d n i t r a t e i s n o t e s s e n t i a l f o r a c h i e v i n g a n a c c e p t a b l e f l a v o r s c o r e . H o w e v e r , s u b t l e f l a v o r d i f f e r e n c e s w e r e n o t e d b y t h e e x p e r t p a n e l w h e n n i t r i t e - n i t r a t e s a l t s w e r e e l i m i n a t e d .

T a b l e 6 g i v e s s t a n d a r d p l a t e c o u n t s a n d s p o r e c o u n t s f o r b o t h t y p e s o f s a u s a g e s a t 0 d a y a n d a f t e r 1 4 d a y s o f s t o r a g e a t 4 ° C f o r t h e i n i t i a l s t u d y . G e n e r a l l y , t h e c o u n t s i n c r e a s e d d u r i n g s t o r a g e i n t h e c o n t r o l s a m p l e s , a n d r e m a i n c o n s t a n t i n t h e n i t r i t e - n i t r a t e a n d b e e t p i g m e n t - c o n t a i n i n g s a u s a g e s . T h e r e l a t i v e l y c o n s t a n t m i c r o b i a l c o u n t s i n t h e b e e t p i g m e n t - c o n t a i n i n g s a u s a g e s a r e d i f f i c u l t

t o e x p l a i n , a n d i n a n a t t e m p t t o e x p l a i n t h e s t a b i l i t y , t h e n a t u r a l n i t r a t e c o n t e n t ( 1 3 3 0 p p m ) o f f r e s h b e e t s ( W i l s o n , 1 9 4 9 ) w a s c o n s i d e r e d . T h i s a m o u n t o f n i t r a t e c o u l d b e a p o t e n t i a l s o u r c e o f n i t r i t e i n

c u r e d m e a t p r o d u c t s . T h e r e f o r e , t h e b e e t p i g m e n t p r e p a r a t i o n a d d e d w a s a n a l y z e d

f o r r e s i d u a l n i t r a t e c o n t e n t b y t h e D e v a r d a m e t h o d ( B r e m m e r a n d K e e n e y ,

1 9 6 5 ) . R e s u l t s i n d i c a t e d t h a t t h e p i g m e n t p r e p a r a t i o n c o n t a i n e d a l e v e l o f n i t r a t e ( 8 m g n i t r a t e p e r l O O g b e e t p o w d e r ) t h a t

c o u l d r e s u l t i n a c o n c e n t r a t i o n o f l e s s t h a n 1 p p m n i t r i t e i n t h e f i n i s h e d p r o d u c t

i f a l l n i t r a t e w e r e r e d u c e d t o n i t r i t e .T o o b t a i n s o m e i n d i c a t i o n w h e t h e r o r

n o t 1 p p m n i t r i t e w o u l d h a v e a n y e f f e c t o n f l a v o r , c o l o r o r b a c t e r i a l c o u n t , a n e x p e r i m e n t w a s c o n d u c t e d w i t h b o l o g n a s a u s a g e s . S a m p l e s c o n t a i n i n g m a x i m u m

Table 8—Standard plate counts o f bologna samples at 0 days and after 14 days of storage at 4°C plus elevated temperature treatment

Standard plate count

StoredSample 0 days 14 days

BolognaControl — no additives 7 x 103 2 x 10 "N itrite

1 56 ppm 8 x 10 3 1.6 x 10 "1 ppm 5 x 103 1 x 10 4

Pure beet color 45 ppm 1.6 x 10 4 1 x 10 4

Beet powder 45 ppm 8 x 1 0 3 8 x 10 4

Table 7—Sensory evaluation scores o f bologna3

FlavorStorage tim e (days)

0 7 14

ColorStorage tim e (days) 0 7 14

Colorb Storage time

7 days

BolognaControl — no additives 4.59a 4.72a 3.88a 2.06a 2.46a 2.53a 2.36aN itrite

156 ppm 4.52a 4.20a 4.18a 5.52c 5.28b 3.95ab 3.16b1 ppm 4.19a 4.20a 4.12a 2.35a 2.71a 2.63a 2.44ab

Pure beet color 45 ppm 4.19a 4.48a 4.24a 4.45b 4.58a 4.16ab 4.72c

Beet powder 45 ppm 4.22a 4.56a 4.06a 4.35b - 5.32b 4.68c

a Scale: 7 point hedonic where 1 = dislike extremely to 9 = like extremely. Values that a'efollowed by same letter in any one column are not significantly different from each other at the 5% level.

b Color evaluation scores after exposure to light for 7 days at 4°C.


l e g a l l e v e l s o f n i t r i t e a n d n i t r a t e a n d s a m p l e s c o n t a i n i n g 1 p p m n i t r i t e a n d b e e t p o w d e r t o g i v e a c o n c e n t r a t i o n o f 4 5 p p m b e t a n i n , a n d 4 5 p p m p u r e b e t a n i n w e r e p r e p a r e d . T h e 4 5 p p m b e e t p i g m e n t

l e v e l w a s c h o s e n b e c a u s e t h i s a m o u n t o f b e e t p o w d e r c o n t a i n e d a l e v e l o f n i t r a t e t h a t c o u l d r e s u l t i n a 1 p p m n i t r i t e m a x i m u m l e v e l i n t h e f i n i s h e d p r o d u c t i f t h e

n i t r a t e w e r e r e d u c e d t o n i t r i t e . S e n s o r y s c o r e s f r o m t h e s e m i r r a i n e d p a n e l f o r c o l o r o f s a u s a g e c o n t a i n i n g 1 p p m n i t r i t e l e v e l y i e l d e d c o l o r s c o r e s s i m i l a r t o t h e c o n t r o l , a n d t h i s i n d i c a t e d t h a t t h e c o l o r

p r e s e n t i n s a u s a g e s c o n t a i n i n g b e e t p i g m e n t w a s d u e t o t h e b e t a n i n c o n t e n t . T h e

c o l o r s c o r e s f o r s a u s a g e s c o n t a i n i n g e i t h e r b e e t p o w d e r o r p u r e b e t a n i n w e r e a s p r e

v i o u s l y s h o w n n o t s i g n i f i c a n t l y d i f f e r e n t f r o m s a u s a g e s c o n t a i n i n g m a x i m u m l e g a l l e v e l s o f n i t r i t e - n i t r a t e a f t e r 1 w k o f s t o r

a g e . A g a i n , t h e c o l o r w a s m o r e s t a b l e i n s a u s a g e s p r o d u c e d w i t h b e e t p i g m e n t t h a n t h a t p r o d u c e d b y c o m m e r c i a l l e v e l s

o f n i t r i t e - n i t r a t e . N o d i f f e r e n c e w a s o b s e r v e d i n t h e c o l o r s c o r e s f o r s a u s a g e s

c o n t a i n i n g b e e t p o w d e r o r p u r e b e t a n i n . A s i n t h e p r e v i o u s e x p e r i m e n t n o f l a v o r

p r e f e r e n c e s w e r e o b s e r v e d f o r b e e t p i g m e n t a n d n i t r i t e - c o n t a i n i n g s a u s a g e s ( T a b l e 7 ) .

S i n c e t h e e a r l i e r s t u d y h a d s h o w n t h a t t h e m i c r o b i o l o g i c a l c o u n t s w e r e q u i t e s t a b l e d u r i n g 1 4 d a y s o f r e f r i g e r a t e d s t o r a g e , b o l o g n a s a m p l e s a f t e r 1 4 d a y s ’ s t o r a g e w e r e p l a c e d u n d e r a c c e l e r a t e d s t o r a g e c o n d i t i o n s t o s t i m u l a t e g r o w t h p r i o r t o a n a l y s i s i n t h e s e c o n d s t u d y . T h i s i n v o l v e d p l a c i n g t h e i n t a c t s a u s a g e s a t 2 1 ° C

f o r 2 4 h r i m m e d i a t e l y p r i o r t o p l a t i n g .

T h e r e s u l t s o f t h e c o u n t s a r e s h o w n i n T a b l e 8 . W h i l e s o m e i n c r e a s e s i n c o u n t s w e r e o b s e r v e d f o r m o s t p r o d u c t s s t o r e d f o r 1 4 d a y s ( p l u s e l e v a t e d t e m p e r a t u r e

t r e a t m e n t ) o v e r f r e s h l y m a d e p r o d u c t s ( n o e l e v a t e d t e m p e r a t u r e t r e a t m e n t ) , t h e i n c r e a s e s w e r e o n l y m o d e r a t e . I t i s i n t e r

e s t i n g t o n o t e t h a t o n l y t h e s a m p l e c o n

t a i n i n g p u r i f i e d b e e t p i g m e n t r e m a i n e d a t t h e s a m e o r l o w e r c o u n t . T h e s e r e s u l t s a r e

i n c o n c l u s i v e , b u t d o i n d i c a t e t h a t u n d e r

t h e c o n d i t i o n s o f t h i s s t u d y c o m m e r c i a l l e v e l s o f n i t r i t e - n i t r a t e h a v e l i t t l e e f f e c t

o n t h e t o t a l a e r o b i c p l a t e c o u n t .

CONCLUSION

S H O U L D N I T R I T E S a n d n i t r a t e s b e e l i m i n a t e d a s a n a d d i t i v e f o r s a u s a g e s , t h e a p p l i c a t i o n f o r b e t a l a i n s a s a c o l o r a n t i n s o m e s a u s a g e s i s f e a s i b l e . C u r e d m e a t

c o l o r s c a n b e s i m u l a t e d t o a h i g h d e g r e e w i t h s o m e l e v e l s o f b e t a l a i n p i g m e n t s . T h e c o l o r o f b e t a l a i n - c o n t a i n i n g s a u s a g e s p r o v e d t o b e m o r e s t a b l e t o l i g h t e x p o s u r e d u r i n g s t o r a g e t h a n t h e c o l o r o f t h o s e c o n t a i n i n g n i t r i t e - n i t r a t e s a l t s . S e n s o r y e v a l u a t i o n i n d i c a t e d n o s i g n i f i c a n t o v e r a l l p r e f e r e n c e f o r s a m p l e s p r e p a r e d w i t h n i t r i t e - n i t r a t e s a l t s o v e r s a m p l e s p r e p a r e d w i t h o p t i m u m l e v e l s o f b e t a l a i n p i g m e n t . H o w e v e r , t h e f l a v o r s c o r e f o r s u m m e r s a u s a g e w a s h i g h e r w h e n n i t r i t e - n i t r a t e s a l t s w e r e p r e s e n t . E x p e r t t a s t e r s

w e r e a b l e t o d e t e c t s u b t l e f l a v o r a n d c o l o r d i f f e r e n c e s i n b e t a l a i n , a n d n i t r i t e - c o n t a i n i n g s a m p l e s . T h e a p p l i c a t i o n o f b e t a l a i n p i g m e n t s a s c o l o r a n t s i n r e a d y - t o - e a t s a u s a g e s a s s h o w n i n t h i s s t u d y

c o u l d p o s s i b l y b e e x t e n d e d t o o t h e r s a u s a g e p r o d u c t s ( f r a n k f u r t e r s ) w i t h t h e

u t i l i z a t i o n o f b e t a l a i n p i g m e n t s a s l a k e s .

REFERENCESAOAC. 1960. “ Official Methods of Analysis.”

Association of Official Analytical Chemists, Washington, D.C.

ASTM. 1968. “ Manual on Sensory Testing M ethods.” Tech. Publ. 434. ASTM, Philadelphia.

Bremmer, J.M. and Keeney, D.R. 1965. Steam distillation m ethods for determ ination of am m onium, n itrate and nitrite. Anal. Chem. Acta 32: 485.

Christiansen, L.N., Johnson, R.W., K autter,D.A., Howard, J.W. and Aunan, W.J. 1973. Effect of nitrite and n itrate on tox in production by Clostridium botulinum and on nitrosamine form ation in perishable canned comm inuted cured meat. Appl. Microb. 25: 357.

Fiddler, W., Piotrowski, E.G., Pensabene, J.W., Doerr, R.C. and Wasserman, A.E. 197 2. Effect of sodium nitrite concentration on N-nitrosodimethylamine form ation in frankfurters. J. Food Sci. 37: 668.

Mabry, T.J. 1970. Betalains, red-violet and yellow alkaloids of Centrospermae. In “ Chemistry of the Alkaloids,” p. 367. van Nostrand Reinhold Co., New York.

Mabry, T.J. and Dreiding, A.S. 1968. The be talains. In “ Recent Advances in Phytochem istry ,” Vol 1, p. 145. Appleton-Century- Crofts, New York.

MacNeil, J.H. and Mast, M.G. 1973. F rankfurters w ithout nitrates and nitrites. Food Prod. Develop. 7(2): 36.

Nilsson, T. 1970. Studies into the pigments in beet root. Lantbrukshoegskolans Annal. 36: 179.

Price, J.F . and Schweigert, B.S. 1971. “ The Science of Meat and Meat Products.” Freeman Publishing Co., San Francisco.

Sebranek, J.G. and Cassens, R.G. 1973. Nitrosa- mines: A review. J. Milk Food Tech. 36: 76.

Tarladgis, B.G., W atts, B.M., Y ounathan, M.T. and Dugan, L. Jr. 1959. A distillation m ethod for the quantitative determ ination of m alonaldehyde in rapid foods. J. Am. Oil Chem. Soc. 37: 44.

von Elbe, J.H ., Sy, S.H., Maing, I-Y. and Gable- man, W.H. 1972. Quantitative analysis of betacyanins in red table beets (Beta vulgaris). J. Food Sci. 37: 932.

Wasserman, A.E. and Talley, F. 1972. The effect of sodium nitrite on the flavor of frankfurters. J. Food Sci. 37: 536.

Wilson, J.K. 1949. N itrate in foods and its relation to health. Agron. J. 41: 20.

Ms received 6/28/73; revised 8 /31 /73 ; accepted 9/6/73.____________ _______________________


Research supported by the College of Agricultural & Life Sciences and the Graduate School, University of Wisconsin, Madison. The authors acknowledge the cooperation of Elem ent Sausage Co., Milwaukee, for assisting with methods and materials.

J. E. W EB B , C. C. B R U N S O N a n d J. D. Y A T E S

C a m p b e ll In s t itu te fo r A g r ic u ltu ra l Research, C a m p b e ll S oup Co., F a y e tte v ille , A R 72 701

EFFECTS OF DIETARY FAT AND dl-a-TOCOPHERYL ON STABILITY CHARACTERISTICSOF PRECOOKED FROZEN BROILER PARTS

INTRODUCTIONR E S U L T S o f e x p e r i m e n t s t o e v a l u a t e t h e

u s e o f t o c o p h e r o l s a s a n t i o x i d a n t s i n s t a b i l i z a t i o n o f p o u l t r y p r o d u c t s h a v e b e e n

v a r i e d b u t p r o m i s i n g . A l s o , n u m e r o u s r e s e a r c h e r s h a v e s t u d i e d d i e t a r y f a t s a n d

t h e i r e f f e c t o n c a r c a s s c o m p o s i t i o n a n d s t a b i l i t y .

E a r l y r e s e a r c h b y K u m m e r o w e t a l .( 1 9 4 8 ) , H o o d e t a l . ( 1 9 5 0 ) , C r i d d l e a n d M o r g a n ( 1 9 5 1 ) a n d M e c c h i e t a l . ( 1 9 5 3 ) i n d i c a t e d t h a t t h e i n c l u s i o n o f h i g h l e v e l s o f v i t a m i n E i n t h e d i e t s o f p o u l t r y p r o d u c e d d e p o t f a t s a n d t i s s u e s o f s u p e r i o r s t a b i l i t y . M e c c h i e t a l . ( 1 9 5 6 a , b ) f e d c h i c k e n s a n d t u r k e y s d i e t s c o n t a i n i n g a d d e d t o c o p h e r o l f o r v a r i o u s p e r i o d s o f t i m e a n d r e p o r t e d t h a t t o c o p h e r o l c o n t e n t a n d f a t s t a b i l i t y w e r e i m p r o v e d i n

b o t h s p e c i e s . T h e s e w o r k e r s c o n c l u d e d t h a t t o c o p h e r o l s p l a y a m a j o r r o l e i n s t a b i l i z i n g p o u l t r y f a t s .

I n m o r e r e c e n t w o r k w i t h r e l a t i v e l y

l o w l e v e l s o f t o c o p h e r y l s u p p l e m e n t a t i o n , W e b b e t a l . ( 1 9 7 2 a ) f o u n d t h a t f e e d i n g a s l i t t l e a s 1 1 I . U . v i t a m i n E / k g o f f e e d f o r

3 6 d a y s p r e - s l a u g h t e r o r 2 2 0 I . U . v i t a m i n

E / k g o f f e e d f o r 1 2 d a y s p r e - s l a u g h t e r h e l d

T B A n u m b e r s f o r p r e - f r i e d , f r o z e n b r o i l e r p a r t s s i g n i f i c a n t l y b e l o w t h o s e o f p a r t s

f r o m b i r d s w h i c h r e c e i v e d c o n t r o l d i e t s . P a n e l s c o r e s i n d i c a t e d t h a t a m o r e s t a b l e

p r o d u c t w a s o b t a i n e d w h e n v i t a m i n E w a s a d d e d i n e i t h e r o f t h e t w o f e e d i n g r e g i

m e n s . A l s o , W e b b e t a l . ( 1 9 7 2 b ) a n d W e b b e t a l . ( 1 9 7 3 ) , w o r k i n g w i t h t u r k e y s , w e r e

a b l e t o s h o w m a r k e d i m p r o v e m e n t s i n p r o d u c t s t o r a g e s t a b i l i t y f r o m t h e i n c l u s i o n o f 2 2 I . U . v i t a m i n E / k g o f d i e t .

C r u i c k s h a n k ( 1 9 3 4 ) a n d H i l d i t c h e t a l . ( 1 9 3 4 ) w e r e a b l e t o s h o w t h a t t h e f a t c o m p o s i t i o n o f p o u l t r y d e p e n d e d t o a g r e a t e x t e n t o n t h e d i e t a r y f a t c o n s u m e d .

L a t e r , K l o s e e t a l . ( 1 9 5 2 ) , F i n g e n b a u m a n d F i s h e r ( 1 9 5 9 ) , M a r i o n a n d W o o d r o o f( 1 9 6 2 ) , M a r i o n a n d E d w a r d s ( 1 9 6 3 ) a n d M a r i o n e t a l . ( 1 9 6 7 ) a l l i n d i c a t e d t h a t t h e d i e t a r y f a t a f f e c t s b o d y f a t c o m p o s i t i o n . T h r e e s t u d i e s r e p o r t e d i n 1 9 7 1 i n d i c a t e d t h e d e f i n i t e i n f l u e n c e o f d i e t a r y f a t o n

d e p o s i t e d c a r c a s s f a t o f b r o i l e r s ( E d w a r d s a n d H a r t , 1 9 7 1 ; J e n e t a l . , 1 9 7 1 ; a n d S c h u l e r a n d E s s a r y , 1 9 7 1 ) . T h e s e t h r e e g r o u p s a l l s t a t e d t h a t t h e d e g r e e o f s a t u r a t i o n o f f a t t y a c i d s i n t i s s u e s a n d d e p o t

f a t s w a s i n f l u e n c e d b y t h e d e g r e e o f u n s a t u r a t i o n o f t h e d i e t a r y f a t t y a c i d s a n d

t e n d e d t o a s s u m e t h e f a t t y a c i d c o m p o s i t i o n o f t h e d i e t .

W i t h t h e k n o w l e d g e t h a t d i e t a r y f a t s

d o a f f e c t c a r c a s s f a t c o m p o s i t i o n a n d t h a t v i t a m i n E w i l l h e l p t o s t a b i l i z e c a r c a s s f a t s w h e n f e d i n t h e d i e t , i t w a s d e e m e d

i m p o r t a n t t o e v a l u a t e a g a m u t o f c o m m e r c i a l l y a v a i l a b l e f a t s a n d o i l s , a n d t o d e t e r m i n e t h e b e n e f i t s o f s u p p l e m e n t i n g t h e s e d i e t a r y f a t s a n d o i l s w i t h v i t a m i n E f o r p r o d u c t s t a b i l i t y .

EXPERIMENTALTWO EXPERIMENTS, Exp 1 and 2, were ou tlined. Exp 1 was designed to evaluate the effects o f dietary fat sources and characteristics on product flavor and stability. Exp 2 was designed to evaluate the beneficial effect on product stability obtained from the addition of dl-a-tocopheryl acetate to the dietary variables evaluated in Exp 1.

Sexed chicks from a commercial broiler strain were maintained in battery brooders in a controlled environment. At 1 day of age, they were placed on a commercial broiler starter ration for a pre-experimental adjusting period. At 4 days of age, each sex was divided into weight

Table 1 —Dietary variables utilized in Experiments 1 and 2a

Diet Variables

1 No added fa t (sucrose used as a fille r)2 Animal fa t (tallow)3 Poultry oil4 Corn oil (Mazóla)5 Feed grade fa t (animal + vegetable)6 Vegetable oil — U/Sb ratio of 1.07 Vegetable oil — U/S ratio of 1.58 Vegetable oil — U/S ratio of 2.09 Vegetable oil — U/S ratio of 2.5

10 Vegetable oil — U/S ratio of 3.011 Vegetable oil — w ith CSOc — U/S ratio of 1.012 Vegetable oil — w ith CSO — U/S ratio of 1.513 Vegetable oil — w ith CSO — U/S ratio of 2.014 Vegetable oil — w ith CSO — U/S ratio of 2.515 Vegetable oil — w ith CSO — U/S ratio of 3.016 Animal fa t — 50% maximum ffad17 Animal + vegetable fa t — 50% max ffa18 Animal + vegetable fa t — 50% max ffa

20% efae

a Exp 2 supplemented diets were formed by adding 11.2 I.U. vitamin E/kg of basal finisher ration,

b Unsaturated fatty acids to saturated fatty acids ratio c Cottonseed oil d Free fatty acids e Essential fatty acids

Table 2—Composition o f basal broiler rations

Percent o f ration

Ingredient Starter Grower Finisher

Corn, yellow 34.000 20.000 20.000Grain sorghum 20.000 44.690 45.490Soybean meal (50% protein) 30.750 15.400 15.400Corn gluten meal 0.000 1.000 1.000Poultry by-product meal 5.250 6.500 6.500Fish meal (Peruvian) 2.500 5.000 4.000A lfa lfa meal 0.000 0.500 0.500Defluorinated phosphate 1.350 0.670 0.790Limestone (ground) 0 .210 0.360 0.360Methionine hydroxy analog 0.200 0.200 0.200Salt 0.320 0.270 0.250Trace minerals 0.025 0.025 0.025Vitam in m ixture3 0.128 0.130 0.130A ntib io tic supplement 0.025 0.010 0.010

Fat, stabilized 0.000 0.250 0.250Fat (variable types & sources

added to form treatment diets)5.240 5.000 5.000

a Vitamin mixture contained vitamins A, B12, niacin, pantothenic acid and riboflavin.

D, K, choline, folacin,

Volume 3 9 (1 9 7 4 )-J O U R N A L OF FOOD SCIENCE- 1 3 3


groups and distributed equally in to each experim ental pen. Ten m ales were placed in each o f two pens for each ration variable utilized in E xp 1 (T ab le 1). T en fem ales were placed in each o f tw o pens for each ration variable u tilized in E xp 2 (Table 1).

T he basal starter ration (Tab le 2) being used for the starter phase o f growth (1 0 days) was m ixed with the fa t variables excluded. T he ration was pelleted and crum bled before addition o f the fat variables (5 .2 5 % by w eight). All basal starter rations for b o th E xp 1 and 2 contained 4 .4 4 I.U . supplem ental vitam in E /kg o f diet, and natural sources provided approxim ately 1 .7 6 I.U . vitam in E /kg diet.

During the 22-day grower and 13-day finisher phases, grower and finisher rations (Table 2) were pelleted prior to the fat variable additions (5 .0 0 % by w eight). A small am ount (0 .2 5 % ) o f tallow was added to all rations as an aid to pelleting. All basal grower rations for bo th E xp 1 and 2 contained 3 .7 4 I.U . supplem ental vitam in E /k g o f diet, and natural sources provided approxim ately 1 .76 I.U . vitamin E /k g diet.

Basal finisher rations used for E xp 1 and the nonsupplem ented diets o f E xp 2 contain ed no added vitam in E , and the natural sources provided about 1 .7 6 I.U . vitam in E /kg. Supplem ented finisher diets for E xp 2 were augm ented with 1 1 .2 I.U . vitam in E/kg.

All birds were processed at 4 9 days o f age using standard processing procedures. D epot fat samples (abdom inal and visceral com bined) were rem oved during evisceration for percent depot fat and iodine num ber determ inations (A O A C , 1 9 6 0 ) . Birds were aged in ice slush for 20 hr. The thigh-drum stick (T -D ) parts were then rem oved from each bird and frozen in the raw state at - 4 0 ° C . A fter 20 days o f frozen storage, T-D parts were thawed in tap w ater, battered w ith a bland batter and deep-fat fried for 12 min at 16 3 °C . Parts were rem oved from the deep-fat fryer, individually wrapped in alum inum foil and frozen in a plate freezer at 4 0 ° C. A fter thoroughly freezing, E xp 1 samples were divided into Studies 1, 2 and 3 (Table 3) and stored at - 2 3 ° C. E xp 2 samples were divided into Studies 1, 2, 3 and 4 as outlined in Tables 4 - 7 .

Table 3 -T B A numbers and taste panel scores-Experiment 1

Diet Variables

Mean TBA number3

Rancidflavor3

O ff-flavo r3

Study 1 2 Animal fa t (tallow) 1.55 B 7.4 a 6.8 a

5 Feed grade fa t (animal + vegetable) 1.73 B 7.0 a 6.3 a

6 Vegetable oil — U/S ratio of 1.0 1.56 B 7.2 a 6.6 a

10 Vegetable oil — U/S ratio of 3.0 1.10 A 7.2 a 6.6 a

11 Vegetable oil — w ith CSO — U/S ratio o f 1.0 1.32 AB 7.3 a 6.9 a

15 Vegetable oil — w ith CSO — U/S ratio of 3.0 1.12 A 7.2 a 6.8 a

Study 2 1 No added fat 2.17 B 7.0 a 6.5 ab

16 Animal fa t — 50% max. ffa 1.90 B 7.5 a 6.7 b

17 Animal + vegetable fa t — 50% max ffa 2.15 B 7.0 a 6.3 ab

18 Animal + vegetable fa t — 50% max ffa — 20% efa 2.12 B 7.1 a 6.4 ab

8 Vegetable oil — U/S ratio of 2.0 1.72 B 7.4 a 7.0 b


Study 3 3 Poultry oil 1.78 D 7.0 a 6.2 b

4 Corn oil 1.16 BC 7.2 a 7.0 c

9 Vegetable o il — U/S ratio o f 2.5 1.04 BC 7.4 a 6.5 be


7 Vegetable oil — U/S ratio of 1.5 1.32 C 7.0 a 6.6 be12 Vegetable oil — w ith CSO — U/S ratio of 1.5 0.81 AB 6.8 a 6.0 b

3 In each study, means within a column followed by different small or capital letters differ significantly: (P « 0.05) or (P « 0.01), respectively.

Table 4 —TBA numbers and taste panel scores—Short storage. Study 1, Experiment 2.

Diet VariablesMean TBA number3

Rancidflavor3

O ffflavor3

2 Animal fa t (tallow) 1.82 ab 7.2 a 6.8 a3 Poultry oil 2.34 c 6.9 a 6.6 a5 Feed grade fa t 1.67 ab 7.0 a 6.4 a2E Diet 2 + 11.2 I.U. v it. E/kg 1.64 ab 7.2 a 6.9 a3E Diet 3 + 11.2 I.U. v it. E/kg 2.02 be 7.3 a 6.9 a5E Diet 5 + 11.2 I.U. v it. E/kg 1.44 a 7.2 a 6.3 a

Supplementation

No added v it. E 1.94 a 7.0 a 6.6 aV it. E added 1.70 b 7.2 a 6.7 a

a Means within a column followed by different small letters differ significantly (P « 0.05).

A fter 8 days o f frozen storage, evaluation o f E xp 1 samples was started , and 13 days were required for com pletion . Evaluation o f E xp 2 samples was started after 25 days o f frozen storage and com pleted in a 22-day period.

O ne rep licate o f a study was rem oved from the freezer, and the thigh and drum stick portions o f each T-D part were separated while fro zen through the use o f pruning shears. The drum stick portions were heated for 30 min at 2 3 2 °C in an oven, the skin and batter were rem oved, and duplicate 10-g samples o f diced- m ixed tissue were taken from each part for

Table 5—TBA numbers and taste panel scores- 2, Experiment 2

Short storage. Study

Diet VariablesMean TBA Rancid number3 flavor3

Off-flavor3

6 Vegetable oil — U/S ratio 1.0 1.65 o 7.1 a 6.8 aT B A num ber d eterm inations (Tarladgis et ah. 8 Vegetable oil — U/S ratio 2.0 1.65 o 7.3 a 7.0 a1 9 6 0 ). T he thigh parts were then heated as 10 Vegetable oil — U/S ratio 3.5 1.43 ab 7.5 a 7.3 awere the drum sticks. S ix samples o f tissue were 6E Diet 6 + 1 1 .2 I.U. v it. E/kg 1.42 ab 7.1 a 6.8 aremoved from each part and presented to a panel o f six m em bers o f rancid flavor and off-

8E Diet 8 + 11.2 I.U. v it. E/kg 1.13 a 7.4 a 6.8 a

flavor scores. Panel m em bers were asked to rate 10E Diet 10 + 11.2 I.U. vit. E/kg 1.26 ab 7.3 a 6.8 asam ples for rancid flavor and off-flavor on a rating scale o f one (least desirable) to ten (m ost desirable).

Supplementation

No added v it. E 1.58 a 7.3 a 7.0 a

The data were analyzed using analysis o f V it. E added 1.27 b 7.3 a 6.8 a

variance, correlation and m ultiple-range tech- a iy|eans withln a column followed by different small letters differ signiques as ou tlined by S tee l and Torrie (1 9 6 0 ) . nificantly: (P «0.05).

BROILER DIETS AND PRODUCT S TA Bl LI TY- 135

Table 6—TBA numbers and taste panel scores—Short storage. Study 3, Experiment 2


Rancidflavora

Off-flavora

11 Vegetable/CSO — U/S ratio 1.0 2 .18c 7.1 a 6.9 c13 Vegetable/CSO — U/S ratio 2.0 1.20 ab 6.8 a 5.2 a15 Vegetable/CSO — U/S ratio 3.5 1.50 b 7.3 a 6.8 c11 E Diet 11 + 11 .2 I.U. v it. E/kg 1.97 c 6.8 a 6.2 be13E Diet 13 + 11.2 I.U. vit. E/kg 0.99 a 6.8 a 5.9 ab15E Diet 15 + 11.2 I.U. vit. E/kg 1 . 1 2 ab 7.4 a 6.9 c

Supplementation

No added vit. t 1.63 a 7.1 a 6.3 aV it. E added 1.36 b 7.0 a 6.3 a

a Means within a column followed by different small letters differ significantly : (P < 0.05).

Table 7—TBA numbers and taste panel scores—Short storage. Study 4, Experiment 2


Rancidflavor3

O ffflavor3

16 Animal fa t — 50% max ffa 2.08 b 7.0 a 6.5 a17 Animal + veg. 50% max ffa 2 .10 b 7.1 a 6.4 a18 Animal + veg. 50% max ffa- 2.06 b 7.1 a 6.7 a

20% efa16E Diet 16 + 11.2 I.U. v it. E/kg 1.73 a 7.2 a 6.8 a17E Diet 17 + 11.2 I.U. vit. E/kg 1.60 a 7.4 a 7.3 a18E Diet 18 + 11.2 I.U. v it. E/kg 1.52 a 7.4 a 6.9 a

Supplementation

No added vit. E 2.08 a 7.1 a 6.5 aV it. E added 1.61 b 7.4 a 7.0 b

a Means within a column followed by different small letters differ significantly: (P < 0.05).

Table 8—Iodine numbers and percent depot fa t from birds that received fa t variables as 5% of rations—Experiment 1

DietPercent

depot fa ta

Iodine numbers

depot fa t Feed fa t Bird depot fa tb

1 1.31 0 .62-2 .13 — 81.22 2.04 1 .06-3 .23 54.0 76.23 1.99 0 .71-2 .27 87.7 84.74 1.77 0 .80-2 .19 125.7 10 1.05 2.05 1 .09-2 .29 70.1 82.46 1.67 0 .90-3 .53 47.9 75.67 1.54 0 .64-2 .59 74.5 83.08 1.88 0 .84 -2 .72 81.5 85.89 1.71 1.01-3.21 96.6 92.1

10 1.56 1 .17-3 .00 1 2 1 .1 100.311 1.79 0 .82 -2 .70 49.8 76.912 2.01 0.93-2 .77 75.1 84.813 2.01 0 .7 7 -2 .7 5 85.9 85.314 1.62 0 .92-2 .27 100.0 91.615 1.83 0 .71-2 .27 119.8 100.616 2 .1 2 0 .91 -2 .7 6 62.1 79.717 1.53 0 .84 -2 .3 0 90.5 89.218 1.86 1.15-2 .36 79.8 85.3

a Percent depot fat was obtained by dividing live bird weight into the depot fat weight and multiplying by 100.

15 Each value is the mean of samples from eight birds.

RESULTS & DISCUSSIOND E P O T F A T a n d i o d i n e n u m b e r d a t a f o r t h e 1 8 d i e t s o f E x p 1 a r e p r e s e n t e d i n T a b l e 8 . N o s i g n i f i c a n t d i f f e r e n c e s e x i s t e d d u e t o t h e d i f f e r e n t v a r i a b l e s a d d e d t o

t h e d i e t s , a s m e a s u r e d b y p e r c e n t d e p o t f a t . I o d i n e - n u m b e r m e a n s f o r t h e s u p p l e

m e n t a l d i e t a r y f a t s r a n g e d f r o m a l o w o f4 7 . 9 t o a h i g h o f 1 2 5 . 7 . T h e i o d i n e - n u m b e r m e a n s f o r d e p o t - f a t s a m p l e s e n c o m p a s s e d a s m a l l e r r a n g e t h a n d i d t h e s u p p l e m e n t a r y f a t s w h i c h t h e b i r d s w e r e f e d . A s i g n i f i c a n t ( P < 0 . 0 1 ) c o r r e l a t i o n c o e f f i c i e n t ( 0 . 9 9 ) w a s o b t a i n e d f o r t h e d i e t a r y - f a t i o d i n e n u m b e r s v s . t h e d e p o t - f a t i o d i n e n u m b e r s .

T a b l e 3 c o n t a i n s T B A n u m b e r a n d t a s t e p a n e l d a t a o b t a i n e d f o r E x p 1 , S t u d i e s 1 — 3 . T B A n u m b e r r e s u l t s f o r S t u d y 1 r e v e a l e d a s i g n i f i c a n t d i f f e r e n c e ( P <

0 . 0 1 ) b e t w e e n d r u m s t i c k s o b t a i n e d f r o m b i r d s f e d t h e v a r i o u s d i e t s . T h e t w o h i g h -

l y - u n s a t u r a t e d , v e g e t a b l e - o i l d i e t s p r o d u c e d b r o i l e r d r u m s t i c k s w i t h l o w e r T B A n u m b e r s t h a n d i d t h e a n i m a l f a t , f e e d - g r a d e f a t a n d l o w - s a t u r a t i o n - r a t i o v e g e t a b l e o i l s . T h e d i f f e r e n c e s w e r e r e v e r s e d f r o m w h a t m i g h t h a v e b e e n e x p e c t e d

b a s e d o n f a t u n s a t u r a t i o n l e v e l s ( T a b l e 8 ) . T h e p a n e l w a s u n a b l e t o d e t e c t d i f f e r

e n c e s f r o m t h i g h s a m o n g b i r d s f e d d i f f e r e n t v a r i a b l e s .

T B A - n u m b e r m e a n s f r o m S t u d y 2 ( T a b l e 3 ) f o r d r u m s t i c k s f r o m t h e v e g e t a b l e o i l - C S O d i e t d i f f e r e d s i g n i f i c a n t l y ( P ^ 0 . 0 1 ) f r o m t h e n u m b e r s f o r d r u m s t i c k s f r o m t h e r e m a i n i n g f i v e d i e t s o f t h a t s t u d y . A g a i n , b a s e d o n u n s a t u r a t i o n l e v e l s ( T a b l e 8 ) , t h i s d i r e c t i o n o f d i f f e r e n c e w a s n o t e x p e c t e d . T h e p a n e l d e t e c t e d n o s i g

n i f i c a n t r a n c i d i t y d i f f e r e n c e s d u e t o d i e t a r y v a r i a b l e s i n S t u d y 2 . T h e o f f - f l a v o r s c o r e s f o r t h i g h s f r o m b i r d s r e c e i v i n g t h e v e g e t a b l e o i l - C S O d i e t w e r e s i g n i f i c a n t l y

l o w e r ( P < 0 . 0 5 ) t h a n s c o r e s f o r a s i m i l a r d i e t w i t h o u t C S O ( 8 ) a n d a n a n i m a l f a t c o n t a i n i n g d i e t ( 1 6 ) .

I n S t u d y 3 , t h e p o u l t r y - o i l d i e t p r o d u c e d d r u m s t i c k s w i t h s i g n i f i c a n t l y h i g h e r T B A n u m b e r s t h a n t h o s e f o r t h e o t h e r f i v e d i e t s . T h e v e g e t a b l e o i l - C S O d i e t ( 1 4 ) p r o d u c e d d r u m s t i c k s w i t h a T B A n u m b e r m e a n s i g n i f i c a n t l y l o w e r ( P < 0 . 0 1 ) t h a n m e a n s f o r t h e o t h e r f i v e d i e t s o f S t u d y 3 . H o w e v e r , n u m e r i c a l l y t h i s v a l u e ( 1 . 7 8 ) w a s l o w e r t h a n v a l u e s f o r f o u r o f t h e d i e t s i n S t u d y 2 .

R a n c i d f l a v o r s c o r e s d i d n o t d i f f e r f o r t h i g h s f r o m t h e v a r i o u s d i e t s . H o w e v e r , p a n e l o f f - f l a v o r s c o r e s f o r t h i g h s f r o m b i r d s f e d c o r n o i l w e r e s i g n i f i c a n t l y h i g h e r ( P < 0 . 0 5 ) t h a n t h o s e f o r t h i g h s f r o m b i r d s f e d p o u l t r y o i l o r v e g e t a b l eo i l - C S O c o n t a i n i n g d i e t s .

I n b o t h S t u d i e s 1 a n d 3 , T B A n u m b e r s f o r d r u m s t i c k s f r o m t h e m o r e h i g h l y - u n s a t u r a t e d , v e g e t a b l e - o i l d i e t s w e r e l o w e r t h a n n u m b e r s f o r d r u m s t i c k s f r o m t h e m o r e h i g h l y - s a t u r a t e d , v e g e t a b l e - o i l


d i e t s . B l e n d i n g f o r m u l a s f o r t h e v a r i o u s U / S - r a t i o n e d v e g e t a b l e o i l s w e r e n o t a v a i l a b l e ; s o , t h e c o n c e n t r a t i o n o f s u c h i t e m s a s C S O i n t h e v e g e t a b l e o i l - C S O b l e n d s w e r e n o t k n o w n a n d m a y h a v e v a r i e d . T h e a d d e d s t a b i l i z i n g a n t i o x i d a n t s i n t h e

f a t m i x t u r e s o r n a t u r a l a n t i o x i d a n t s i n t h e d i f f e r e n t f e e d s w e r e n o t k n o w n ; t h e r e f o r e , i t w a s h y p o t h e s i z e d t h a t i n

c r e a s i n g l y l a r g e r a m o u n t s o f a n t i o x i d a n t s w e r e p r e s e n t a s u n s a t u r a t i o n r a t i o s i n c r e a s e d , a n d t h a t t h e s e a n t i o x i d a n t s w e r e d e p o s i t e d i n t h e b r o i l e r p a r t s , t h u s s t a b i l i z i n g t h e m . N o o t h e r e x p l a n a t i o n c a n p r e s e n t l y b e g i v e n f o r t h e u n e x p e c t e d

r e v e r s e d r e s u l t s .

R e s u l t s f r o m t h e t h r e e s t u d i e s o f E x p 1 r e v e a l e d t h a t t h e v a r i o u s d i e t a r y f a t v a r i a b l e s d i d p r o d u c e s a m p l e s w i t h T B A

n u m b e r m e a n s a n d o f f - f l a v o r s c o r e s w h i c h d i f f e r e d s i g n i f i c a n t l y . H o w e v e r , i n a l l c a s e s , t h e r e s u l t s w e r e n o t w h a t m i g h t h a v e b e e n e x p e c t e d b a s e d o n d i e t a r y f a t a n d o i l s a t u r a t i o n l e v e l s .

T h e c o r r e s p o n d i n g T - L s a m p l e s ( f r o m t h e s a m e b i r d s ) w e r e e v a l u a t e d a f t e r 3 m o n t h s o f f r o z e n s t o r a g e . T h e r e s u l t s w e r e s o s i m i l a r t o t h o s e r e p o r t e d h e r e f o r s h o r t s t o r a g e t h a t i t w a s n o t d e e m e d n e c e s s a r y t o c o n s u m e s p a c e f o r t h e r e p e t i t i v e p r e s e n t a t i o n o f t h o s e r e s u l t s .

D a t a o b t a i n e d f r o m E x p 2 a r e p r e s e n t e d i n T a b l e s 4 - 7 . A s i n E x p 1 , S t u d y 3 , t h e p o u l t r y - o i l d i e t p r o d u c e d d r u m s t i c k s w i t h a h i g h T B A - n u m b e r m e a n ( T a b l e 4 ) . V i t a m i n E s u p p l e m e n t a t i o n o f

t h e p o u l t r y - o i l d i e t d i d r e d u c e t h e d r u m s t i c k T B A - n u m b e r m e a n s o m e w h a t ; a n d f o r e a c h o f t h e o t h e r p a i r e d d i e t s o f S t u d y 1 , v i t a m i n E - s u p p l e m e n t e d d i e t s y i e l d e d d r u m s t i c k s w i t h n u m e r i c a l l y

l o w e r T B A - n u m b e r m e a n s t h a n d i d t h e i r n o n s u p p l e m e n t e d c o u n t e r p a r t s . W h e n v i t a m i n E s u p p l e m e n t a t i o n w a s i s o l a t e d s t a t i s t i c a l l y a s a s e p a r a t e f a c t o r , t h e r e d u c t i o n i n T B A - n u m b e r m e a n d u e t o v i t a m i n

E s u p p l e m e n t a t i o n w a s s i g n i f i c a n t ( P < 0 . 0 5 ) . T a s t e p a n e l s c o r e s d i d n o t d i f f e r s i g n i f i c a n t l y d u e t o v i t a m i n E s u p p l e m e n t a t i o n o f d i e t s i n S t u d y 1.

S t u d y 2 T B A n u m b e r r e s u l t s ( T a b l e 5 ) a g a i n t e n d e d t o b e l o w e r w i t h i n c r e a s i n g U / S r a t i o s . V i t a m i n E s u p p l e m e n t a t i o n o f t h e f i n i s h e r d i e t s w i t h 1 1 . 2 I . U . v i t a m i n

E / k g o f d i e t a g a i n s h o w e d a p r o n o u n c e d

b e n e f i c i a l e f f e c t a s i n d i c a t e d b y T B A - n u m b e r m e a n s . H o w e v e r , t h e p a n e l w a s u n a b l e t o d e t e c t d i f f e r e n c e s b e t w e e n

p r o d u c t s f r o m s u p p l e m e n t e d a n d n o n s u p p l e m e n t e d d i e t s .

I n S t u d y 3 , t h e v e g e t a b l e o i l - C S O d i e t s y i e l d e d v a r i a b l e r e s u l t s b e t w e e n i n d i v i d u a l d i e t s f o r T B A n u m b e r s a n d o f f - f l a v o r

( T a b l e 6 ) . H o w e v e r , t h e v i t a m i n E s u p p l e m e n t a t i o n e f f e c t o n p r o d u c t T B A n u m b e r s w a s s t a t i s t i c a l l y s i g n i f i c a n t ( P

0 . 0 5 ) .S t u d y 4 r e s u l t s ( T a b l e 7 ) h i g h l i g h t t h e

b e n e f i c i a l e f f e c t s o f s u p p l e m e n t i n g d i e t s w i t h v i t a m i n E . I n a l l t h r e e d i e t s c o m

p a r e d , t h e v i t a m i n E - s u p p l e m e n t e d d i e t p r o v i d e d d r u m s t i c k s w h i c h p r o d u c e d l o w e r T B A n u m b e r s t h a n d i d i t s c o r r e s p o n d i n g n o n s u p p l e m e n t e d d i e t ( P ^

0 . 0 5 ) . A l t h o u g h t h e p a n e l r a n c i d f l a v o r s c o r e s d i d n o t d i f f e r s i g n i f i c a n t l y b e t w e e n t h e v a r i o u s t r e a t m e n t s , a t r e n d w a s n o t e d i n f a v o r o f v i t a m i n E s u p p l e m e n t a t i o n .

T h e p r o d u c t o f f - f l a v o r s c o r e w a s s i g n i f i c a n t l y ( P < 0 . 0 5 ) i m p r o v e d b y d i e t a r y v i t a m i n E s u p p l e m e n t a t i o n .

R e s u l t s i n E x p 2 r e i n f o r c e d r e s u l t s

f o u n d b y W e b b e t a l . ( 1 9 7 2 a , b ) a n d

W e b b e t a l . ( 1 9 7 3 ) . I n t h i s r e s e a r c h , a s i n t h e p r e v i o u s a r t i c l e s , v i t a m i n E s u p p l e m e n t a t i o n o f d i e t s c o n t a i n i n g v a r i o u s i n

g r e d i e n t s h a s i n m o s t i n s t a n c e s p r o v i d e d p r e c o o k e d f r o z e n s t o r e d p o u l t r y p a r t s w i t h i m p r o v e d s t a b i l i t y ( T B A v a l u e s ) a n d f l a v o r , e v e n w h e n r e l a t i v e l y l o w l e v e l s o f

v i t a m i n E a n d s h o r t f e e d i n g p e r i o d s w e r e u t i l i z e d .

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M. D. J U D G E , C. G. H A U G H . G. L . Z A C H A R IA H , C. E. P A R M E L E E a n d R. L . P Y L E

D e p a rtm e n ts o f A n im a l S ciences a n d A g r ic u ltu ra l E n g in e e rin g

P u rd u e A g r ic u ltu ra l E x p e r im e n t S ta tio n , W est L a fa y e tte , IN 4 7 9 0 7

SOYA ADDITIVES IN BEEF PATTIES

INTRODUCTIONT H E U T I L I Z A T I O N o f s o y a p r o d u c t s a s

g r o u n d m e a t e x t e n d e r s i s w i d e s p r e a d . Y e t , t e c h n o l o g i c a l i n f o r m a t i o n c o n c e r n i n g t h e i r p e r f o r m a n c e i n m e a t p a t t i e s i s v i r t u a l l y n o n e x i s t a n t i n s c i e n t i f i c l i t e r a t u r e . A n e x c e p t i o n i s t h e r e p o r t o f H u f f m a n a n d P o w e l l ( 1 9 7 0 ) w h i c h d e a l s w i t h t h e e f f e c t o f a s o y a p r o d u c t o n t h e t e n d e r n e s s o f g r o u n d b e e f p a t t i e s . T h e s e i n v e s t i g a t o r s o b s e r v e d t h a t p a t t i e s c o n t a i n i n g 2 % o f a t o a s t e d g r i t s o y a p r o d u c t

w e r e f o u n d t o b e m o r e a c c e p t a b l e b y a t a s t e p a n e l a n d h a v e l o w e r s h e a r f o r c e

s c o r e t h a n t h o s e w i t h o u t t h e e x t e n d e r .

T h e y a l s o r e p o r t e d t h a t p a t t i e s w i t h 3 5 % f a t w e r e m o r e t e n d e r t h a n t h o s e w i t h 1 5 o r 2 5 % f a t .

T h e r e a r e s e v e r a l c r i t e r i a o f q u a l i t y i n g r o u n d b e e f p a t t i e s i n c l u d i n g l o w n u m

b e r s o f b a c t e r i a , b r i g h t n e s s o f c o l o r a n d m i n i m a l c o o k i n g s h r i n k a g e . I n f r o z e n p a t

t i e s , t h e e a s e o f r e m o v a l o f t h e i n t e r l e a v

i n g p a p e r i s a l s o i m p o r t a n t . T h i s r e s e a r c h w a s c o n d u c t e d t o e v a l u a t e t h e e f f e c t s o f

t w o s o y a p r o d u c t s o n t h e s e i n d i c a t o r s o f q u a l i t y i n g r o u n d b e e f p a t t i e s .

EXPERIMENTALTWO C O M M E R C IA L L Y prepared soya products were used in this study. They w ere selected to represent typical (1 ) soya flour and (2 ) soya protein co n cen trate (SP C ). T he protein co n ten t o f the flour was 4 0 - 6 0 % and th at o f the SPC was not less than 70% . T he products w ere added to ground b e ef prepared from boneless chuck equivalent to U SD A U tility or Com m ercial grades, the fa t in which was adjusted to approxim ately 2 0 and 30% . T h e ground m eat m ixes were prepared by grinding (2 .5 cm plate), m ixing w ith ice and soya p rodu ct, and regrinding (3 mm plate). The protein co n ten t o f the tw o types o f m eat was roughly standardized by adding a relatively high level o f the soya additives to the b e e f contain ing 30% fat and a relatively low level to that contain ing 20% fat. Table 1 shows the com position o f the m ixtures used.

T he patties, form ed with a food portioning m achine, had a weight o f 57g , a diam eter o f 9 .2 cm and a thickness o f 75 m m . Single patties were considered the experim en tal units and a value was observed in each test for each patty . The patties were interleaved w ith w ax-coated com m ercially produced papers (tw o papers between each pair o f patties) and placed in stacks o f 8 patties each.

F or the bacteriological tests, each treatm en t cell consisted o f one stack (8 patties) and analysis o f variance was cond ucted for the e ffec ts

o f treatm en t (storage tim e) and additives. F or the co lo r, cooking shrinkage and paper release tests, each experim en tal treatm en t cell consisted o f five stacks (4 0 patties) and analysis o f variance was cond ucted w ithin each fat level for e ffec ts o f treatm en t (storage cond ition s), additives and stacks. T he variation due to stacks was the result, in part, o f the use o f a single but d ifferent type o f interleaving paper for each stack and was considered to be an estim ate o f the variability associated w ith paper to be found under com m ercial cond ition s. The same paper types were used w ithin each treatm en t group.

B a c t e r i a l c o u n t s

T otal num bers o f fresh-m eat spoilage organisms were estim ated by su bjecting rinses o f the patties to T ry p ton e G lucose E x tra c t Agar. A sample o f each p atty weighing lg was put di

rectly in a dilution blank and shaken prior to plating (A PH A , 1 9 6 7 ) . T he patties were sam pled im m ediately a fter preparation and at the end o f a 7-day storage period at 4 °C . The agar plates were incu bated for 2 days at 3 2 ° C.

L i g h t r e f l e c t a n c e

T he ob jectiv e o f the co lor evaluation in the study was to d etect any tend encies for the soya additives to change the apparent co lor o f the patties by “ diluting” the pigm ents present. Such an e ffe c t could be d etected by measures o f to ta l light reflectan ce at a wave length that is uniform ly absorbed by the pigm ents even though they exist in d ifferent chem ical states (isob estic po in t). An isobestic point for the fresh m eat pigments (reduced m yoglobin , oxy- m yoglobin and m etm yoglob in ) is 5 2 5 mp (Sn yd er, 1 9 6 5 ) . T h e patties were placed on clear glass and positioned on the reflectance

Table 1 —Ingredients o f ground beef patties

Fat content of meat Additives/100 lb meat (lb) Identification (%) Ice Soya

Control 20 SPCa-Low Flou rb -Low Control 30 SPC-High Flour-High

a Soya protein concentrate b Soya flour

Table 2—Effect of soya additives and storage tim e on bacterial numbers in beef patties

Storage tim ea (da)

Identification*3

Contro l0 SPC-Highi Flour-High SPC-Low Flour-Low

0 0.242d 0.172 0.336 0.732 1.4607 101 60 105 51 51

Analysis of variance

Source df M.S.

Storage time (T) 1 83 .10**Additives (A) 4 0 .37**T X A 4 0 .8 3 **Error 60 0.02

a 4°Cb See Table 1 for ingredients of meat mixtures c Combined control 20 and control 30 d Times 1 06 /g meat mixture ” P< 0.01

20 0 020 12 42C 12 43C 0 03C 18 630 18 6

Volume 3 9 ( 1974)—JO U RN A L OF FOOD S C /E /V C f-1 3 7


attachment of a Beckman Spectronic 20 spectrophotometer to determine percent reflectance of a circular area 2.5 cm in diameter at the center of each patty (40 patties per treatment cell).

Patties from each of the six groups described in Table 1 were assigned to one of three storage treatments which were believed to coincide with normal industry procedures. These were (1) storage for 24 hr at 4°C (unfrozen); (2) storage for 3 hr at 4°C followed by storage at -10°C for 5 days (delayed freeze); and (3) storage at -10°C for 5 days (immediate freeze). The freezing was accomplished by placing the stacks of 8 patties on freezer plates in a household freezer with an air temperature of — 10°C.

Prior to the reflectance measurements, the frozen patties were thawed by storage at 4°C for 24 hr.Cooking shrinkage

Shrinkage tests were conducted using a continuous gas-fired broiler with a standardized flame and conveyor speed. Heat was applied to both sides of the patties simultaneously. The patties were cooked to a moderate degree of doneness, i.e., the interior was light grey in color and the exterior showed some browning. A representative diameter for each patty was determined by averaging the maximum and minimum diameters of the 40 patties in each treatment cell measured immediately after cooking.

Table 3—Effect of soya additives and storage conditions on percent light reflectance® by beef patties

IdentificationbStorage

conditionsControl 20

(%)SPC-Low

(%)Flour-Low

(%)Control 30

<%)SPC-High

(%)Flour-High

(%)Unfrozen 12.5 13.8 13.4 13.6 14.9 11.6Delayed freeze 12.2 13.9 13.8 12.6 14.1 12.2Immediate freeze 12.5 12.8 13.5 14.4 13.8 11.7

Analysis of varianceSource df M.S. Source df M.S.

Storage conditions (C) 2 4.3 Storage conditions (C) 2 6.1Additives (A) 2 53.0** Additives (A) 2 192.2**Stacks (S) 4 37.4** Stacks (S) 4 31.5**C X A 4 6.4 C X A 4 23.3**C X S 8 2.4 C X S 8 4.0A X S 8 4.0 A X S 8 10.1C X A X S 16 7.7 C X A X S 16 7.5Error 315 6.28 Error 315 6.93

a 525 mMb See Table 1 for ingredients of meat mixtures **P < 0.01

Table 4—Effect of soya additives and storage conditions on cooking shrinkage in beef patties

Identification®Storage

conditionsControl 20

(cm)SPC-Low

(cm)Flour-Low

(cm)Control 30

(cm)SPC-High

(cm)Flour-High

(cm)Unfrozen 7.8b 8.1 8.1 7.2 7.9 8.1Delayed freeze 7.7 8.0 8.1 7.1 7.7 7.8Immediate freeze 7.6 8.0 8.1 7.2 7.8 7.9

Analysis of varianceSource df M.S. Source df M.S.Storage conditions (C) 2 0.19** Storage conditions (C) 2 1.43**Additives (A) 2 5.58** Additives (A) 2 19.39 * *Stacks (S) 4 0.32** Stacks (S) 4 0.25**C X A 4 0.10* C X A 4 0.05C X S 8 0.15** C X S 8 0.15**A X S 8 0.13** A X S 8 0.12**C X A X S 16 0.14** C X A X S 16 0.12**Error 315 0.039 Error 315 0.048

a See Table 1 for ingredients of meat mixtures b Average diameter after cooking

P < 0.05 **P < 0.01

The treatments used in the color evaluation phase of the study (unfrozen, delayed freeze and immediate freeze) were also used in the cooking shrinkage tests. The frozen patties were thawed by storage at 4°C for 24 hr prior to cooking.Paper release

A test was developed to simulate the removal of paper from frozen patties as it may occur prior to cooking. A special apparatus was built to hold the patty firmly on a flat plate with a spring-loaded slider. A clamp was used to grip the paper and was attached at a 20° angle from the patty to the tension load cell (lOOOg) of an Instron universal testing machine. The paper was removed at the rate of 50 cm per min and the maximum force required for its removal was recorded in grams. The frozen storage treatments described above (delayed freeze and immediate freeze) were utilized and the tests were performed immediately after removal of the patties from the freezer. The papers adhering to the bottom of the patties as placed in the stacks were used for the test (40 patties per treatment cell).

RESULTS & DISCUSSIONBacteriological quality

Tab le 2 sh o w s th e n u m b e r s o f b a c te r ia in the m e a t sam p le s as e s t im a te d f ro m p la te c o u n ts . T h e h igh ly s ig n i f ican t (P < 0 . 0 1) e f fe c t o f s to ra g e is obv io u s . S o m e in f lu e n ce o f add it ives was a lso f o u n d b u t o n ly a m o n g sam p le s t h a t w ere n o t s to re d . T he h igh ly s ig n if ican t (P < 0 .0 1 ) i n t e r ac t io n fo r s to rag e t im e a n d ad d it iv es re su l ted f r o m h ig h e r in i t ia l b ac te r ia l c o u n ts in th e m e a t sam p le s c o n ta in in g f lo u r as c o m p a r e d to th o se c o n ta in in g SPC. H o w ever, a f te r s to rag e fo r 7 days th e re were n o s ign if ican t d i f fe re n ce s a m o n g th e m e a t sam ples in bac te r ia l lo ad .

Light reflectanceT h e p e r c e n t l igh t r e f l e c ta n c e by the

p a t t ie s is sh o w n in T ab le 3 fo r t h e v a r ious m e a t m ix tu r e s a n d s to rag e c o n d i t io n s . Highly s ign if ican t (P < 0 . 0 1) e f fe c ts o f th e add it ives w ere o b se rv ed . In th e m e a t c o n ta in in g 20% fa t , b o t h so y a p r o d u c t s t e n d e d to increase l igh t r e f l e c t io n , b u t w i th 30% fa t o n ly th e SPC in c re ased l ight re f le c t io n as c o m p a r e d to th e c o n t r o l sam ples. In th e l a t t e r case, th e f lo u r t e n d ed to decrease l igh t re f l e c t io n . I t is l ike ly t h a t these e f fe c ts w ere c au sed by d i f fe r ences in p a r t ic le size o f th e ad d it iv e , so lu b i l i ty o f the ad d it iv e a n d / o r a d s o r p t io n o f m e a t ju ice s b y th e ad d i t iv e . C o n s e q u e n t ly, th e s ig n if ican t (P < 0 . 0 1) i n te r a c t io n b e tw e e n add it ives a n d s to ra g e c o n d i t io n s in the 30% fa t sam p le s ( th e w a te r in w h ich was p r o b a b ly r e d u c e d b y th e re la t ively h igh f a t c o n te n t ) p r o b a b ly re su l te d f ro m d i f f e re n t a m o u n t s o f t im e f o r s o lu b i l iz a t io n a n d a d s o r p t io n to ta k e p lace .

T h e c o n te n t io n t h a t th e re f l e c ta n c e m e a su re m e n ts m a d e in th is s t u d y w ere i n f lu en c ed by the d egree o f “ d i lu t i o n ” o f the m e a t p ig m e n ts is b o r n e o u t by th e c o m p a r is o n o f va lues o b se rv e d fo r 20% fa t sam p le s vs. th o se c o n ta in in g 30% fat.

S O Y A A D D I T I V E S I N B E E F P A T T I E S -1 3 9

Table 5—Effect of soya additives and storage conditions on paper release from beef patties

Identification3Storage Control 20 SRC-Low Flour-Low Control 30 SPC-High Flour-High

conditions (g ) (g) (g) (g) (g) (g)

Delayed freeze 375b 341 341 168 194 223Immediate freeze 338 294 308 169 186 238

Analysis of variance

Source df M.S. df M.S.Storage conditions (C) 1 68016 Storage conditions (C) 1 1253Additives (A) 2 26559 Additives (A) 2 78817**Stacks (S) 4 453504** Stacks (S) 4 133726**C X A 2 847 C X A 2 7554**C X S 4 53699 C X S 4 7285A X S 8 6624 A X S 8 29317*C X A X S 8 17344 C X A X S 8 15823Error 150 29056 Error 150 12152

3 See Table 1 for ingredients of meat mixtures b Release force

, P < 0.01 P < 0.05

T he h ig h er level o f fa t re su l te d in g rea te r a m o u n t s o f r e f le c te d light.

Cooking shrinkageT h e d iam e te rs o f th e c o o k e d p a t t ie s

i n d ic a te d th a t the ad d it iv es su b s ta n t ia l ly r e d u c e d (P < 0 .0 1 ) sh r inkage in b o t h 20% a n d 30% fa t sam p le s (T ab le 4) . In the c o n t r o l p a t t ie s w i th 20% fa t , sh r inkage was 16.3%. This m a y be c o m p a r e d to the sh r inkage o f p a t t ie s w i th lo w levels o f SPC an d f lo u r w h ich was 12.7% a n d 12.0% respec t ive ly . T h e average sh r in k ag e o f c o n t r o l p a t t ie s w i th 30 % fat was 22 .1% . T he h igh level o f SPC re d u c e d the sh r inkage to 15.2% a n d th e h igh level o f f lo u r re su l te d in 13 .8% sh r inkage .

A m easu rab le e f fe c t o f f reez in g on p a t ty sh r in k ag e was o b se rv ed . In all cases th e p a t t ie s c o o k e d a f te r 1 day o f s to rage in th e u n f r o z e n s t a te sh ra n k less t h a n th o se

w h ic h w ere f ro z e n a n d th a w e d pr io r to c o o k in g regard less o f the len g th o f s to r age p r io r t o f reezing. T h e average sh r in k age o f u n f r o z e n p a t t ie s was 13.0 and 15.9% re spec t ive ly fo r 20% a n d 30% fat sam ples . T h e average sh r in k ag e fo r the f ro ze n p a t t ie s was 13 .9% an d 17.6% re spec tive ly fo r the tw o k in d s o f m e a t s a m ples.Pap e r release

T h e d a ta on p a p e r re lease f ro m f ro zen p a t t ie s s h o w d is t in c t d i f fe re n ce s b e tw e e n th e t w o ty p es ( fa t levels) o f m e a t used (T ab le 5). Even w i t h o u t s ta t i s t ica l c o m p a r iso n , i t is o b v io u s t h a t the p apers were r e m o v e d f ro m th e p a t t i e s w i th 30% fat m u c h eas ie r t h a n f ro m th o se w i th 20% fa t. It is l ike ly t h a t the g re a te r m o is tu re c o n te n t o f the l a t t e r re su l te d in n u m e r o u s c o n ta c t s b e tw e e n p a p e r f ibers a n d ice

c rysta ls . C o n s e q u e n t ly i t is u n d e r s t a n d able t h a t th e e f fe c ts o f th e add it ives were dissim ila r in t h e tw o ty p e s o f m ea t . In p a t t ie s w i th 30% fa t , t h e so y a add it ives in c reased (P < 0 .0 1 ) th e fo rce r e q u i r e d to r em o v e th e p a p e r w h e reas th e re was no s ign if ican t e f fe c t o f ad d it iv es in the 20% fa t m ea t . It is poss ib le t h a t m a n y physical fa c to rs , su c h as degree o f s o lu b i l iz a t io n o f the add it ive o r c o n ta c t o f p a p e r a n d a d d i tive, w ere re sp o n s ib le fo r th e d if fe rences .

T h e e f fe c t o f s to ra g e c o n d i t i o n s on p a p e r re lease was n o t c lear c u t even t h o u g h th e d e la y e d freez in g t e n d e d to i n crease th e fo rce r e q u i r e d in sam p le s c o m p o se d o f 20 % fa t. T he m ig ra t io n o f free w a te r to th e m e a t -p a p e r in te r f a c e s d u r in g th e de lay p r io r to f reez in g is l ike ly to have o c c u r re d . In th e te s t using 30% fat m ea t , a s ign if ican t (P < 0 .0 1 ) i n te r a c t io n o f ad d it iv e a n d s to rag e c o n d i t i o n s was o b served . In th is i n s ta n c e th e de lay p r io r to f reez in g m a y have a l lo w e d t im e fo r w a te r a d s o r p t io n by th e f lo u r pa r t ic le s an d red u c t io n o f c o n ta c t b e tw e e n th e p a p e r a n d f lour .

In th e p a p e r re lease tes ts , a large p r o p o r t io n o f th e v a r ia t io n was assoc ia ted w i th s tack s . T h u s o n e co u ld e x p e c t to f in d large v a r ia t io n in p a p e r re lease u n d e r c o m m e rc ia l c o n d i t i o n s s ince th e p apers se lec ted w ere e x a m p le s o f p ap ers c u r r e n t ly in w id e sp rea d use.

REFERENCESAPHA. 1967. “ Standard Methods for the Ex

amination of Dairy Products,” 12th ed. American Public Health Association.

Huffman, D.L. and Powell, W.E. 1970. Fat content and soya level effect on tenderness of ground beef patties. Food Technol. 24: 1418.

Snyder, H.E. 1965. Analysis of pigments at the surface of fresh beef with reflectance spectrophotometry. J. Food Sci. 30: 457.

Ms received 6/28/73; revised 9/16/73; accepted 9/19/73._______________________________

Journal paper no. 5157 of the Purdue Agricultural Experiment Station, Lafayette, IN 47907.

G. R. S C H M ID T and S U N A R JO K E M A N '

Dept, o f Anim al Science, University o f Illinois a t Urbana-Champaign, Urbana IL 61 8 01

HOT BONING AND VACUUM PACKAGING OF EIGHT MAJOR BOVINE MUSCLES

INTRODUCTIONH O T B O N IN G M U SC L E S p re rigo r f o l lo w ed b y v a c u u m p a ck ag in g d u r in g chil l ing have several p o te n t i a l advan tages . By rem o v in g fa t a n d b o n e prerigor , re fr ige ra te d space fo r chil l ing is m in im iz e d . The subd iv is ion o f the w h o le carcass and rem o v a l o f the fa t p re r ig o r saves re fr ige ra t ion t im e an d sh ip p in g co s t can be re d u c e d ( K a s tn e r e t ah , 1 973) .

By a p p ly in g v a cu u m packaging , th e m e a t can be b o x e d and h a n d le d w i th o u t o v e rh ea d rails. T h e re is less risk o f la te r bac te r ia l c o n ta m in a t io n . H u m id i ty c o n t ro l in th e r o o m is n o t necessa ry an d li t t le w eigh t is lost d u e to e v a p o ra t io n (M inks and S tr inger , 19 7 2 ) . H ow ev e r , care is necessa ry so t h a t the plas tic bags are n o t d am ag ed . M eat he ld fo r t o o long in the p acks a cq u i res a c h eesy , s o u r a n d lac t ic acid sm ell ( J o s e p h , 1971) .

T h e ten d e rn e s s o f h e e f m usc les re m o v ed p re r igo r a n d s to re d a t 15°C fo r 2 4 —48 h r were c o m p a r e d w i th th e c o r re sp o n d in g c o n t r o l m usc les le f t on the c a r cass a n d chil led at 9 °C fo r 24 h r by S c h m id t a n d G i lb e r t ( 1 9 7 0 ) . T h e b iceps fem o ris (B F ) an d long iss im us dors i m u s cles s to re d at 15°C fo r 24 h r w ere o f e q u iv a len t ten d e rn e s s to th e i r co n tro ls , while th o se s to re d at 1 5°C fo r 48 h r were s ign if ican tly m o re t e n d e r . T h e se m im e m b r a n o s u s (SM ) m uscles sh o w ed no t r e a tm e n t e f fe c t , while the exc ised semi- t e n d in o su s (ST ) m usc les were to u g h e r th an th e i r c o n tro ls .

M arsh e t al. ( 1 9 7 2 ) exc ised p o rk m u s cles f ro m the h o t carcass a n d e x p o se d th e m to 0 C. C o m p le te exc is ion o f m u scles was c o m p a r e d to r o u t in e p rocess ing o f the e n t i re carcass. T he e f fe c t o f e x cis ion a n d cold e x p o su re o f seven muscles was s tu d ied . S ign if ican t t o u g h e n in g was o bse rved in long issim us , d a rk ST, t r iceps b rach i i a n d r e c tu s fem oris . T o u g h en in g , a l t h o u g h s ta t i s t ic a l ly n o n s ig n i f ic a n t , o c c u r re d in l ight ST and g lu teus m ed ius (G M ); h o w ev er , a lm o s t n o change in t e n derness was f o u n d in BF a n d psoas m ajo r (PM ) c o m p a r e d to the c o n tro l .

B on ing o f s t e e r carcasses a f te r being he ld as i n ta c t carcass fo r a p e r io d o f 2, 5 o r 8 h r p o s t m o r te m at 16°C was s tu d ied by K a s tn e r et al. ( 1 9 7 3 ) . T he c o r re sp o n d -

1 Present address: Gadiah Mada University, Bulaksumur Jogjakarta, Indonesia

ing sides were fa b r ic a te d in to m usc les and m usc le s y s te m s a f te r chill ing fo r 4 8 h r at 0 °C (c o ld -b o n e d ) . W hen h o t b o n in g was d o n e o n the in ta c t carcasses a f te r be ing he ld fo r 8 h r p o s t m o r t e m , s teaks were eq u a l o r su p e r io r t o th o se co ld -b o n e d . P e rc en t loss, sh ea r fo rce , co lo r value, c o o k in g loss, w a te r -b in d in g c a p a c i ty , p e r cen t m o is tu re and fa t w ere c o m p a r e d fo r b o th 5 and 8 h r h o ld in g pe r iods . T h e h o t b o n e d t r e a tm e n t had a sm alle r average p e rc e n t w e igh t loss th a n the co ld -b o n e d beef . T he 2-hr h o ld in g p e r io d was n o t s ign if ican tly d if fe re n t b e tw e e n the h o t - and c o ld -b o n e d . W arner B ra tz le r shear fo rce values fo r the h o t - b o n e d an d c o ld b o n e d s teak s w ere d i f f e re n t for 2-hr and5-hr h o ld in g pe r iods , b u t the d i f fe ren ces in sh e a r values at the 8-hr h o ld in g pe r iod were n o n s ig n i f ic a n t . T he d if fe ren ces in m ea t f lavor b e tw e e n th e h o t - a n d c o ld b o n e d sam ples were n o n s ig n i f ican t . T here were s ign if ican t d i f fe ren ces in raw m ea t co lo r b e tw e e n h o t - a n d c o ld -b o n e d s teak s fo r 2-hr h o ld in g pe r iod .

T he p u rp o se s o f th is s tu d y w ere : (1 ) to c o m b in e the b e n e f i ts o f h o t b o n in g an d v a c u u m p ackag ing in p r o d u c in g t e n der b e e f ; (2 ) t o d e te rm in e the q u a l i ty o f

b e ef a f te r s u b je c t io n to h o t b o n in g a n d v a c u u m pack ag in g ; a n d (3 ) to d e te r m in e changes in m usc le f iber d i a m e te r o f the b e e f p r o d u c e d by n o t b o n in g a n d v a c u u m packaging.

EXPERIMENTALA TOTAL of six Angus steers were slaughtered according to normal procedures after which each carcass was split into right and left sides. Right sides were hot-boned and vacuum-packaged as treatments, and left sides were conventionally cold-boned as controls. The left sides were placed in a 1°C room after each animal was killed. The hot boning of the right side was done about 1 hr after each animal was killed.

The boneless wholesale cuts fabricated were tib, clod arm inside chuck, top round, bottom round, sirloin tip, rump, tenderloin, sirloin and strip loin. Total lean trim, bone and fat were weighed from each side. The boneless wholesale cuts were weighed and kept at about 7°C for about 4 hr, after which they were placed in a 1°C room overnight. At 24 hr postmortem the cuts had reached an internal temperature of 3 to 7°C. At this time they were vacuum packaged and replaced in the cold room for 7 days. Cold boning of the left side was done after each side was chilled for 8 days at 1°C.

Muscle samples for taste panels, Warner Bratzler shear force tests and microscopic anal-

Table 1—Carcass traits of the six steers utilized

Mean ± S.E.Live weight, kg 481 ± 31Loin eye area, sq cm 82.5 ± 7.5Fat thickness, cm 1.57 ± 0.19Percent kidney and pelvic fat 3.37 ± 0.77USDA yield grade 3.23 ± 0.35USDA quality grade High choice

Table 2—Composition and yield of hot- and cold-boned sidesMeans ±S.E.

Cold-boned left side

Hot-boned right side

Hot carcass side weight, kg 157.7 ± 12.7 155.6 ± 12.1Percent retail yield 60.9 ± 1.8 63.7a± 2.2Percent fat 23.9 ± 2.0 20.7 ± 3.4Percent bone 12.1 + 0.7 12.9 + 0.8a Significant differences between hot- and cold-boned sides (P< 0.05)

UO-JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

_H O T B O N I N G O F B O V I N E M U S C L E S - 141

yses were taken from the boneless wholesale cuts of both hot-boned and cold-boned cuts at 8 days postmortem. Samples were cut 3 cm thick from the anterior longissimus dorsi (ALD) taken from the tenth rib of the boneless rib, posterior longissimus dorsi (PLD) from the fourth lumbar vertebrae of the boneless strip loin, SM from the geometric center of the boneless top round and GM from the geometric center of the boneless top sirloin. BF and ST were removed from the geometric center of the boneless bottom round and PM from the geometric center of the tenderloin.

Percent retail yield of the cold-boned and the hot-boned sides were calculated from the hot side weight. Loin eye area, percent kidney and pelvic fat, maturity, conformation, back fat and quality grade were measured on the control side at 24 hr postmortem. A 6-member trained taste panel was utilized to evaluate the flavor, juiciness, tenderness and overall acceptability of the meat on a 9-point hedonic scale.

At 9 days postmortem samples from the ALD, PLD, GM and PM from the control and treated sides were broiled to 67“C internal temperature in an electric oven. At 11 days postmortem, samples from the ST, SM, quadriceps femoris (QF) were roasted in plastic bags to 70°C internal temperature in a 164°C electric oven. Samples measuring approximately 3 x 2x 1 x cm were taken from both control and treated samples and subjected to taste pane! evaluation. Three cores 2.54 cm in diameter af each cooked sample were taken by hand and each core was measured twice at room temperature on the Warner Bratzler shear force instrument.

Two kinds of microscopic analyses were used to determine fiber diameter of hot-boned and cold-boned samples. 25 measurements were made from each sample of the control and each sample of the treated muscles. The first method to measure fiber diameter was described by Herring et al. (1965). Samples of each muscle

from both control and treated were frozen in liquid nitrogen and cross sections 8 microns thick were cut in a cryostat (Cryo-Cut Microtome, American Optical Corp.). 25 measurements were made randomly with a Zeiss R.A. 38-phase contrast microscope. The average of these 25 observations at a total magnification of I 00 was used as a fiber diameter estimation for that muscle.

Another microscopic analysis was made of a fiber suspension. Samples from both control or treated muscles were blended at about 7,000 rpm with a Lourdes blender for about 5 sec in physiological saline solution. These suspensions of fibers were examined directly with a Zeiss R.A. 38-phase contrast microscope. 25 observations were made for each muscle sample. These 25 measurements were used to estimate each sample fiber diameter at a total magnification of 100.

RESULTS & DISCUSSIONM E A N C A R C A S S T R A I T S fo r the six s tee rs u t i l iz e d a re given in T ab le 1. M ost ca t t le were in th e U S D A C ho ice q u a l i ty grade a n d h a d a b o u t average c u ta b i l i ty fo r t h a t q u a l i ty g rade. T h e average live w e ig h t w as 481 kg.

T h e p e rc e n t re ta i l y ie ld , in c lu d in g bone less ro a s ts , s te ak s a n d g ro u n d beef , was s ig n i f ican tly h ig h e r (P < 0 .0 5 ) fo r th e h o t - b o n e d side . M ore fa t was lef t o n the h o t - b o n e d ro a s ts , s ince i t was d if f icu l t to r em o v e a s im ila r a m o u n t o f fa t f ro m b o t h the h o t - a n d c o ld -b o n e d sides. H ow eve r , th e re was n o s ig n if ican t d i f fe re n ce in th e p e rc e n t fa t o r b o n e t r im b e tw e e n t r e a t m e n ts (T ab le 2).

T h e m ean s f o r p a la ta b i l i ty o f th e h o t - a n d c o ld -b o n e d s teak s a n d ro a s ts are given in T ab le 3. T h e h o t - b o n e d a n d c o ld b o n e d s te ak s a n d ro a s ts w ere n o t s t a t i s t i cally d i f f e re n t in sh e a r fo rce . T he ST, B F a n d SM ro a s ts were th e t o u g h e s t . These cu ts w ere c o o k e d in p las t ic bags w h ic h is a m o is t m e t h o d o f c o o k in g a n d t e n d e d to have a t o u g h e n in g e f fe c t . T h e t a s te panel did n o t sco re the s te a k s o r ro a s ts f ro m the h o t - b o n e d sides d i f f e re n t f ro m th o se f r o m th e c o ld -b o n e d s ides fo r f lavor , ju ic iness , t e n d e rn e s s o r overall a c c e p ta b i l i ty . H o ld in g th e c u ts a t 7 ° C fo r 4 h r b e fore p lac ing in the co ld r o o m a p p a re n t ly d e c reased s h o r t e n in g o r th e aging e f fe c t fo r 9 o r 11 days b e fo re t a s te p an e l ev a lu a t ion r e d u c e d the d if fe re n ce s o b se rv ed .

T h e m ea n s o f f ib e r d ia m e te r m ea su re d o n f ro ze n se c t io n s a n d h o m o g e n a te s are given in T ab le 4. H e rr in g e t al. ( 1 9 6 7 ) sh o w e d t h a t an in c rease in f iber d ia m e te r was cu rv il inea r ly re la te d w i th m usc le sh o r te n in g . T en d e rn e s s was sh o w n to be l inearly re la te d to f iber d ia m e te r . Tab le 3 sh o w s t h a t a l t h o u g h th e h o t b o n e d PM, GM a n d ST h ad s ign if ican tly (P < 0 .0 1 ) in c re a sed f iber d iam e te rs , t h e y w e re n o t t o u g h e r th a n th e i r c o ld -b o n e d c o u n te r parts . T he PL D also h a d s ign if ican tly ( P < 0 .0 5 ) in c re ased f ib e r d iam e te r . T h ere was very l i t t le e f fe c t o f th e m e th o d o f meas- u re in g m usc le f ib e r d iam e te r , c ry o s ta t or

Table 3—Palatability of hot- and cold-boned steaks and roasts

Steaks3 Roasts3ALD PLD pm GM ST SM BF QF

ShearbCold>< 6.4 5.8 5.8 6.6 9.7 7.5 9.1 6.5S.E. 1.4 0.7 1.4 1.2 1.9 1.2 1.3 1.9HotX 6.9 7.0 5.3 6.7 9.7 7.9 9.9 6.6S.E. 1.1 1.6 1.0 1.4 2.3 1.3 2.3 1.6

Flavor0ColdX 6.2 6.1 6.6 5.6 4.5 4.9 5.0 5.4S.E. 0.3 0.5 0.5 0.3 0.5 0.6 0.6 0.5HotX 6.1 6.1 6.8 6.0 4.7 5.0 5.1 5.3S.E. 0.3 0.4 0.6 0.3 0.5 0.6 0.3 0.7

Juiciness0Coldx" 6.5 6.6 6.6 6.3 4.0 4.1 5.1 5.6S.E. 0.4 0.6 0.8 0.5 0.8 0.2 1.2 1.1HotX 6.1 6.7 7.0 6.5 4.6 5.1 5.3 5.5S.E. 0.3 0 7 0.6 0.5 1.2 1.1 0.7 1.2

Tenderness0ColdX 5.9 5.9 7.2 4.8 3.6 4.2 3.2 5.2S.E. 0.7 1.0 0.8 0.4 1.0 0.6 0.6 0.7HotX 5.7 5.1 6.8 5.1 3.4 4.7 3.3 5.1S.E. 0.4 0.5 1.3 0.5 1.3 0.7 0.5 0.8

Overall acceptability0 ColdX 6.1 6.2 6.9 5.3 3.8 4.4 3.7 5.2S.E. 0.4 0.8 0.9 0.3 0.8 0.6 0.7 0.5HotX 5.9 5.7 6.8 5.5 3.7 4.7 3.8 5.2S.E. 0.3 0.4 0.9 0.5 1.2 0.7 0.6 1.0

a No significant differences between hot- and cold-boned steaks orroasts (P < 0.05)

13 Warner Bratzler shear in kg/2.54 cm diameter core c Scored on a 9-point scale with 9 being most desirable

*[42—JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

Table 4—Analysis of variance for fiber diameter measured on homogenates and frozen sections from hot- and cold-boned muscleFiber diameter(/±) ALD PLD PM GM ST SM BF QF

ControlHomogenized>< 72.2 73.5 51.3 68.2 69.6 75.5 72.4 64.2S.E. ±3.1 4.2 6.4 2.1 3.2 5.7 5.0 4.9Cryostatx: 67.8 69.5 52.9 67.3 68.0 70.6 70.8 65.1S.E. 3.8 4.9 6.1 4.7 4.7 4.0 2.3 5.5

TreatedHomogenizedX 73.8 76.0 61.1 78.2 76.1 76.0 72.6 70.6S.E. 2.4 5.3 5.9 8.5 2.7 3.0 4.9 5.8CryostatX 70.6 75.1 69.1 73.5 75.1 74.4 71.3 71.8S.E. 4.7 2.2 5.2 2.5 3.4 3.4 3.4 5.9

Fiber diameter ALD PLD PM GM ST SM BF QF

Cold x hotHomogenized X cryostat Cold-hot x

homogenlzed-cryostat

^Significant at the 5% level ^^Significant at the 1% level

h o m o g e n a te , o n the resu lts obse rved (T ab le 4 ) .

This w o r k agrees w i th p rev ious re p o r t s (S c h m id t a n d G i lb e r t , 19 7 0 ; K a s tn e r et al., 1 9 7 3 ) t h a t h o t b o n in g does n o t a f fec t f lavor o f beef . Since th e bone less w h o le sale cu ts were n o t e x p o se d to the cold t e m p e ra tu r e s o f less th a n 3 °C u n t i l a f te r 5 h r p o s t m o r t e m , o n e w o u ld n o t e x p e c t in c re a sed to u g h e n in g d u e to co ld s h o r t e n ing ( K a s tn e r e t al., 1 9 7 3 ; M arsh a n d L ee t ,1966) .

T h u s , h o t b o n in g o f b e e f can resu l t in a very a cc e p ta b le p r o d u c t . O n ly th e b o n e less re ta i l p r o d u c t n eed s to be chilled.

This w o u ld save co n s id e ra b le re fr ig e ra ted space a n d e n erg y w h ich is o f i m p o r t a n c e in b o t h dev e lo p in g an d d e v e lo p ed c o u n tries.

REFERENCESHerring, H.K., Cassens, R.G. and Briskey, E.J.

1965. Sarcomere length of free and restrained bovine muscle at low temperature as related to tenderness. J. Sci. Food Agr. 16: 379.

Herring, H.K., Cassens, R.G., Suess, G.G., Brun- gardt, V.H. and Briskey, E.J. 1967. Tenderness and associated characteristics of stretched and contracted bovine muscles. J. Food Sci. 34: 317.

Joseph, R.L. 1971. Production of tender beef. Food Mfg. Oct: 29.

Kastner, C.L., Henrickson, R.L. and Morrison, R.D. 1973. Characteristics of hot boned bovine muscle. J. Anim. Sci. 36: 484.

Marsh, B.B., Cassens, R.G., Kauffman, R.G. and Briskey, E.J. 1972. A research note. Hot boning and pork tenderness. J. Food Sci. 37: 179.

Marsh, B.B. and Leet, N.G. 1966. Studies in meat tenderness. 3. The effects of cold shortening on tenderness. J. Food Sci. 31: 450.

Minks, D. and Stringer, W.C. 1972. The influence of aging beef in vacuum. J. Food Sci. 37: 736.

Schmidt, G.R. and Gilbert, K.V. 1970. The effect of muscle excision before the onset of rigor mortis on the palatability of beef. J. Food Technol. (London) 5: 331.


1 31.67 98.13* 1736.55** 380.17** 278.94** 100.04 0.68 0.681 84.34* 14.52 3.46 44.78 10.38 9.25 13.09 13.09

1 2.25 14.52 3.79 19.87 0.33 0.64 0.17 0.17

J . H . H O D G E S , ' V . R . C A H I L L a n d H . W . O C K E R M A N

D e p t , o f A n i m a l S c i e n c e , T h e O h i o S t a t e U n i v e r s i t y , C o l u m b u s , O H 4 3 2 1 0

a n d T h e O h i o A g r i c u l t u r a l R e s e a r c h & D e v e l o p m e n t C e n t e r , W o o s t e r , O H 4 4 6 9 1

EFFECT OF VACUUM PACKAGING ON WEIGHT LOSS, MICROBIAL GROWTH AND PALATABILITY OF FRESH BEEF WHOLESALE CUTS

INTRODUCTIONT H E T R E N D to w a r d c en t ra l ize d p ro cess ing a n d large v o lu m e m erc h a n d is in g o f beef t h r o u g h s u p e r m a r k e t s has a c c e n tu a ted the n e ed to re d u c e th e sh r in k ag e o f w holesa le cu ts a n d to m a in ta in cu ts in a sa leable c o n d i t i o n t h r o u g h o u t t h e p r o c essing se q u e n c e . While w ho lesa le b e e f c u ts are n o r m a l ly v a c u u m p a c k a g ed a t the w a reh o u se o r w holesa le level o f d i s t r ib u t io n fo r s to rage a n d t r a n s p o r t a t i o n to re ta i l o u t le t s , re la t ive ly few s tu d ie s have been c o n d u c te d to observe th e e f fe c t iv e ness o f th is sy s te m . T h is lack o f c o n c lu sive in f o r m a t io n p r o m p te d th is s tu d y to observe t r e n d s in w e ig h t loss, bac te r ia l g ro w th a n d p a la tab i l i ty o f f resh b e ef w ho lesa le cu ts a f t e r s to rage u n d e r vacuum .

Ball e t al. ( 1 9 5 7 ) in an ex tens ive s tu d y o f 13 d i f fe re n t p a ck ag in g m ate r ia ls re p o r t e d shr inkage o f g ro u n d b e e f p ack ag ed in Cry-O-V ac e v ac u a te d packages ranged f ro m 0 .1% a t 1 day s to rage to 0 .7% a t 14 days s to rage . G ro u n d b e e f s to re d in m a te rials hav ing a re la t ive ly lo w o x y g e n a n d w a te r v ap o r p e rm e a b i l i ty gave th e h ighes t to ta l o rg a n o le p t i c q u a l i ty r a t in g an d flav o r sco re . H a l leck e t al. ( 1 9 5 8 ) r e p o r t e d t h a t b ac te r ia l g r o w th o n f resh g ro u n d m ea ts was s ign if ican tly c o r re la te d w i th gas and w a te r v a p o r p e rm e a b i l i ty o f the packag ing m ate r ia ls . T h e essen t ia l ly im p e rm e ab le pack ag in g f i lm s in c re a sed the lag phase a n d th e f ina l b a c te r ia l c o u n ts were n o t as h igh as w i th t h e m o re p e r m e able f i lms. T he p r e d o m i n a n t m ic r o o r gan ism s lo c a te d o n b e e f carcasses a t th e packag ing p lan t (S t r in g e r e t al., 19 6 9 ) were P s e u d o m o n a s f r a g i , P . g e n ic u la ta and M ic r o c o c c u s l u t e u s a n d a t the re ta i l s to re were P. f r a g i an d P. g e n ic u la ta .

Jay e e t al. ( 1 9 6 2 ) r e p o r t e d t h a t b a c te r io log ica l a n d o rg a n o le p t i c a cc ep ta b i l i ty o f g ro u n d b e e f was re ta in e d f o r a longer p e r io d (1 0 day s) w h e n p ack a g ed in gas im p e rm e a b le Saran as c o m p a r e d t o p a c k aging in c e l lo p h an e . G r o u n d b e e f p a c k aged in Saran a n d s to r e d a t —1.1 °C e x h ib i te d l i t t le change in t o ta l m ic ro b ia l

1 Present address: Stop & Shop Companies, Inc., P.O. Box 629, Marlborough, MA 01752

c o u n t t h r o u g h o u t 12 days o f s to rage (O rd a l , 1 9 6 2 ) . M a rr io t t e t al. ( 1 9 6 7 ) f o u n d t h a t th e use o f c e l lo p h an e and C ry-O -V ac h a d n o ap p rec ia b le e f fe c t on b a c te r ia l c o u n ts o f b e e f s teak s s to re d at —1.1 °C over a p e r io d o f 10 days. R e sea rch r e p o r t e d by B aran e t al. ( 1 9 7 0 ) i n d ica ted t h a t v a c u u m p ack ag ed h a m b u rg e r s to re d fo r 6 d ays re su l te d in a e ro b ic b ac terial g ro w th a t ta in in g a s t a t i o n a r y phase ; w h e reas , h a m b u r g e r p ack ag ed in a ir a n d s to re d fo r 21 days sh o w e d a c o n t in u e d d e v e lo p m e n t o f aero b es .

P ierson e t al. ( 1 9 7 0 ) r e p o r t e d t h a t the to ta l bac te r ia l c o u n t o f to p r o u n d s teaks in c reased m u c h m o re ra p id ly a n d was alw ays h ig h er w i th a e ro b ic pack ag in g as c o m p a r e d to a n a e ro b ic packag ing . Senso ry e v a lu a t io n in d ic a te d t h a t th e re was l i t t le d i f fe re n ce b e tw e e n f resh b e e f a t 0 days , u se d as a c o n t r o l , a n d b e e f p a c k aged an ae ro b ic a l ly fo r a p e r io d o f a t least 10 days. A e ro b ica l ly p a ck ag ed b eef s teaks were u n a c c e p ta b le a f te r 4 days o f s to rage .

E igh t days a p p ea rs t o be a reasonab le m a x im u m p e r io d fo r th e s to rage o f lam b loins in a v a cu u m package and unless a lo w t e m p e ra tu r e s to rag e is m a in ta in e d the s u b s e q u e n t re ta i l case life will be re d u c e d (R e ag a n e t al., 1971) . J e r e m ia h e t al.( 1 9 7 1 ) n o t e d t h a t l a m b cu ts aged in v a c u u m packages re su l te d in s ign if ican t a d v an tages in t e n d e rn e s s b u t overal l p a la tab i l i ty was r e d u c e d becau se o f the o ff-f lavors t h a t d ev e lo p ed d u r in g s to rage an d dur ing re ta i l d isp lay .

MATERIALS & METHODSPreparation and storage of samples

24 steers from a local feedlot were slaughtered at The Ohio State University Meat Laboratory to obtain 12 low grade (USDA low standard—low good) carcasses and 12 high grade (USDA average good-average choice) carcasses. Both sides of the carcasses were used to give a total of 48 sides. Each grade group was further divided into 6 carcasses (12 sides) aged 15 days at 2 ± 1°C after slaughter and 6 carcasses (12 sides) held 1 day postmortem (internal round temperature, 10 ± 5°C) before vacuum packaging. The rounds, short loins, ribs and arm chucks were designated for vacuum storage for 3, 7, 14 and 28 days using randomized incomplete blocks (Cochran and Cox, 1950) yielding three samples of each cut per subcell.

Beef quarters were placed in Kraft paper bags and transported in a refrigerated truck (3 ± 2°C) a distance of approximately 3 miles to a processing plant for cutting and vacuum packaging.

After wholesale cutting and initial sampling were performed, the rounds, short loins, ribs and arm chucks were placed into Cry-O-VacS-612 bags. The bone surface of the wholesale cuts were covered with Cry-O-Vac Boneguard to prevent bone punctures in the packaging film. A vacuum of 8 -1 0 in. (Tipper-Vac Model F Unit) was used to draw the air from the bag. The bag was sealed using Tipper Tie No. 450 clips. Shrinking of the film was accomplished by passing the package through a hot water tunnel (90 ± 2°C) for approximately 7 sec. The vacuum packaged beef primals were placed in corrugated cardboard boxes and transferred by refrigerated truck (3 ± 2°C) back to The Ohio State University Meat Laboratory for storage until final sampling. The primals were stored in a 3 ± 2°C cooler with a 90 ± 2% relative humidity.Measurement of weight loss

The wholesale cuts were weighed immediately after packaging. The net weight and the gross weight (packaging materials included) of each cut were recorded. After the designated storage period, the cuts were weighed with the film intact and again after removing the Cry-O- Vac bag, boneguard and accompanying juices. Weight loss due to evaporation was noted from the intact package weights. Total weight loss representing both evaporation and drip was calculated by subtracting the final wholesale cut weight from the initial cut weight.Bacteriological analysis

The bacterial flora were enumerated using standard plating techniques for aerobic and anaerobic bacteria. A swab technique recommended by the American Public Health Association (1960) was used to sample the surface of the semimembranosus muscle of the round, longissimus on the posterior end of the short loin, as well as on the posterior end of the rib and the triceps brachii (long head) of the arm chuck at the separation from the blade chuck. Initial sampling was accomplished immediately prior to packaging. Samples at 3, 7, 14 and 28 days of storage under vacuum were made by opening the vacuum package with a sterile scalpel.

A sterile cotton swab moistened with a 0.1% Bacto-peptone solution was used to swab the center of the exposed muscle defined by 12.3 sq cm sterile template.

The total bacterial counts were enumerated on Bacto Tryptone Glucose Extract agar (Difco). The anaerobic bacterial counts were enumerated by plating on Anaerobic agar

Volume 39 (I974)~J0URNAL OF FOOD SCIENCE-WZ


U r

1.0-O¿ 0.8-5bCD

Í 0.6-z

0I------------L.Wholesale Cuts

F i g . 1 - P e r c e n t a g e n e t w e i g h t l o s s o f v a c u u m

p a c k a g e d w h o l e s a l e b e e f c u t s .

(BBL) which contained methylene blue as an indicator of the reduced condition of the medium. The anaerobic dilution plates were placed in an evacuation-replacement jar and flushed three times with C 0 2 gas to remove the air present in the jar.

Organoleptic analysisSamples of the longissimus were evaluated

for tenderness, beef flavor, off-flavor and general acceptability after 0, 3, 7, 14 and 28 days storage under vacuum. One 1-in. steak was removed from the posterior end of each short loin (two samples per carcass) before packaging to be used for initial organoleptic evaluation. After the designated storage period two 1-in. steaks were removed from the posterior end of the short loin and broiled (4 in. from preheated coils) on a time constant basis (8 min first side, 7 min second side) under an electric broiler to achieve a medium degree of doneness (approximately 70°C). Following cooking, the longissimus of each steak was cut into uniform sections and duplicates were served to the six panel members for evaluation of juiciness, tenderness, natural flavor, off-flavor and general acceptability using a 10-point scale. A score of 10 was considered to be very juicy, most tender, possessed a very full natural beef flavor, exhibited no off-flavor and was highest in general acceptability.

R E SU L T S & D ISC U SSIO N

W eight loss d e te r m in a t io nW eight loss o f t h e p ackage (init ial

p ack a g ed w eigh t m in u s f inal packaged w e ig h t ) w as negligible regardless o f g rade, t im e p o s t m o r t e m p ackag ing , w ho le sa le c u t o r l e n g th o f s to rag e u n d e r v a c u u m . O n ly o c ca s io n a l w e ig h t losses o f less t h a n 0.1 p o u n d p e r w h o le sa le c u t o c c u r re d b u t th is d i f f e re n c e was t o o sm all t o b e m ea s

F i g . 2 — P e r c e n t a g e n e t w e i g h t l o s s o f h i g h ( a v e r

a g e g o o d - a v e r a g e c h o i c e ) a n d l o w ( l o w s t a n d

a r d - l o w g o o d ) g r a d e v a c u u m p a c k a g e d w h o l e

s a l e b e e f c u t s f o r v a r i o u s t i m e s d u r i n g t h e

s t o r a g e p e r i o d .

u re d w i th an y degree o f a cc u rac y . This w o u ld in d ic a te t h a t th e bags w e re p ra c t ica lly im p e rm e a b le t o m o is tu re loss, m ak in g e v a p o ra t io n losses negligible an d in d ica t in g t h a t p u rg e loss was t h e m ajo r sh r inkage fa c to r . F igu re 1 sh o w s th e p e r c en t n e t w e ig h t loss f ro m t h e m e a t ( e v a p o ra t io n a n d p u rg e ) o c cu r r in g u n d e r v a cu u m s to rag e (average fo r 3, 7, 14, 28 days s to rag e t im e ) fo r th e v a r io u s w h o le sale cu ts .

A n analys is o f va r ia n ce revea led th a t t h e n e t w e igh t loss w as s ign if ican t (P < 0 .0 1 ) f o r w h o le sa le c u ts a n d a c o m p a r i so n a m o n g th e m e a n s (S n e d e c o r , 1 9 5 9 ) in d ic a te d t h a t t h e sh o r t lo in h as signific an t ly less (P < 0 .0 5 ) sh r in k ag e t h a n th e a rm c h u c k an d th e ro u n d . Larger areas o f e x p o se d m usc le w e re p re sen t in t h e r o u n d and a rm c h u c k , th u s fac i l i ta t ing e x u d a t iv e f lu id loss.

P e rc en tag e n e t w e ig h t losses fo r all low a n d h igh grade c u ts a re p re se n te d in Figu re 2.

T h e analys is o f va r ian ce in d ic a te d th a t t h e n e t w e ig h t loss a p p r o a c h e d significan ce (P < 0 .1 0 ) fo r g rades w i th t h e h igh grades sh r in k in g less t h a n t h e lo w grades. T he h igh g rades w o u ld h ave a lo w e r m o is tu re c o n te n t a n d a m o re e x ten s iv e fat covering b o t h o f w h ic h sh o u ld d ecrease shr inkage . Th is agrees w i th r e p o r t s by S leeth e t al. ( 1 9 5 8 ) t h a t c h o ice b e e f q u a r te rs sh ra n k less t h a n c o m p a ra b le goo d q u a r te r s . T h e grade x s to rag e t im e i n t e r a c t io n f o r n e t w e igh t loss was n o t signific a n t even t h o u g h th e lo w grade sam ples h a d a g re a te r average n e t w e ig h t loss ear ly in t h e s to rag e p e r io d , w h e n c o m p a r e d to t h e h igh g rade , a n d less d i f fe re n ce s were

----- Aerobic. 15 Days Post-mortem PackagingAerobic. 1 Day Post-mortem Packaging

- - Anaerobic. 15 Days Post-mortem Packaging

F i g . 3 — L o g n u m b e r o f b a c t e r i a l o r g a n i s m s

p l o t t e d a g a i n s t s t o r a g e t i m e f o r a e r o b i c a n d

a n a e r o b i c b a c t e r i a l c o u n t s o f w h o l e s a l e b e e f

c u t s v a c u u m p a c k a g e d a t 1 a n d 1 5 d a y s p o s t

m o r t e m .

n o t e d l a te r in s to rage . All o f t h e o t h e r poss ib le in te r a c t io n s also p ro v e d to be n o n s ig n i f ican t .

T h e n e t w e ig h t loss c o m p a r i s o n b e tw e e n 1 a n d 15 d a y s p o s t m o r t e m p a c k aging was n o n s ig n i f ic a n t (P > 0 .1 0 ) . An overall m e a n p e rc e n t w e ig h t loss fo r all cu ts p ack a g ed a t b o t h 1 an d 1 5 d a y s p o s t m o r te m was 0.51 fo r 3 d a y s s to ra g e u n d e r v a c u u m , 0 .7 0 f o r 7 d a y s s to rag e , 0 .7 9 fo r 14 d a y s s to rag e an d 0 .9 3 f o r 2 8 d ays s to rage . T h ese w e ig h t losses w e re significan t (P < 0 .0 1 ) f o r s to rag e t im e a n d a g re a te r p e rc e n ta g e o f w e ig h t loss p e r d ay o c c u r re d d u r in g th e firs t 7 d a y s o f s to rag e .

B acter io log ica l changesF igure 3 sh o w s th e changes in a e ro b ic

as well as a n a e ro b ic b a c te r ia c o u n ts .T h e d a ta p r e se n te d r e p re se n t th e aver

ages o f c o u n t s fo r t h e r o u n d , s h o r t lo in , rib a n d a rm c h u c k a n d also averages b o t h th e h igh a n d lo w g ra d e g ro u p s . A n an a l ysis o f va r iance in d ic a te d t h a t n e i th e r o f these c lass if ica t ions p ro v e d to be significan t (P > 0 .0 5 ) .

A longer p o s t m o r t e m h o ld in g t im e (1 5 days vs. 1 d a y ) p r io r t o v a c u u m pack ag in g sign if ican tly (P < 0 . 0 5 ) in c re a sed b o t h a e ro b ic a n d a n a e ro b ic c o u n t s . A d d i t io n a l c o n ta m in a t io n and g r o w th d u r in g th e h o ld in g p e r io d is t h e p ro b a b le r e a so n fo r th e d e v e lo p m e n t o f in c re a sed n u m b e r s .

A e ro b ic m ic ro b ia l g r o w th in c re ased d u r in g th e 28 d a y s o f s to ra g e in v a c u u m . B aran et al. ( 1 9 7 0 ) r e p o r t s t h a t a e ro b ic bac te r ia l g r o w th r e ach es a s t a t i o n a r y phase a f t e r 6 d a y s o f v a c u u m ( in it ia l v a c u u m , 2 8 .5 in. o f Hg) p ack a g in g s to rag e b u t P ie rso n e t al. ( 1 9 7 0 ) f o u n d t h a t

VACUUM PACKAGING OF FRESH BEEF- 1 4 5

F i g . 4 — E f f e c t o f s t o r a g e t i m e a n d p o s t m o r t e m

p a c k a g i n g t i m e o n t h e t a s t e p a n e l t e n d e r n e s s

s c o r e s o f L o n g i s s i m u s d o r s i m u s c l e f r o m v a c u

u m p a c k a g e d b e e f s h o r t l o i n s .

F i g . 6 — E f f e c t o f s t o r a g e t i m e o n t h e o f f - f l a v o r

o f L o n g i s s i m u s d o r s i m u s c l e o f v a c u u m p a c k

a g e d b e e f s h o r t l o i n s .

aerobic bacteria counts increased fo r 15 days in “ anaerobically packaged” ( in it ia l vacuum, 15—20 in .) beef. The d iffe rence in vacuum achieved ( in it ia l vacuum in th is study 8—10 in .) o r the organism con tam inating the p roduct w ou ld p robab ly explain these differences. Research by C lark and Lentz (1969), Shaw and N ico l(1969), Baran et al. (1970) and Ledward et al. (1971 ) have ind ica ted tha t oxygen concen tra tion must be drastica lly reduced

------ Low Grade. 1 Day Post-mortem Packaging— Low Grade. 15 Days Post-mortem Packagirg

------ High Grade. 1 Day Post-mortem Packaging-------High Grade, 15 Days Post-mortem Packagirg

F i g . 5 — S t o r a g e t i m e a n d p a c k a g i n g t i m e p o s t

m o r t e m p l o t t e d a g a i n s t t a s t e p a n e l f l a v o r s c o p e s

o f L o n g i s s i m u s d o r s i m u s c l e f r o m v a c u u m p a c k

a g e d b e e f s h o r t l o i n s .

Low Grade. 1 Day Post-mortem Packaging - Low Grade, 15 Days Post-mortem Packaging

— High Grade, 1 Day Post-mortem Packagrg

F i g . 7 — E f f e c t o f s t o r a g e t i m e o n t h e g e n e r a l

a c c e p t a b i l i t y o f L o n g i s s i m u s d o r s i m u s c l e o f

v a c u u m p a c k a g e d b e e f s h o r t l o i n s .

to have a m ajor in fluence on aerobic bacteria l g row th . E ithe r the pa rtia l pressure o f oxygen in the vacuum packages or the residual air trapped around o r in the meat may have been high enough to m ain ta in aerobic bacteria l g row th over the 28-davs storage period. Partia l, but v isually undetectable, loss o f vacuum in the package may have caused add itiona l aerobic g row th during the la tte r part o f the storage period. Faculta tive anaerobes or

aerobes m ay also account fo r increase in numbers during storage.

Longer storage periods also increased anaerobic m ic rob ia l g row th . Anaerobic an d fa c u lta tiv e anaerobic m icrob ia l g row th may have been enhanced by the developm ent o f pa rtia l anaerobic cond itions in the package. The accum ula tion o f carbon d iox ide in the package due to m ic rob ia l m etabolism may have been suffic ie n t to support anaerobic g row th .

O rganoleptic analysisFigure 4 represents the e ffec t o f s tor

age tim e on the tenderness o f anaerobically packaged beef short lo ins.

Storage tim e greatly increased (P < 0 .01 ) tenderness in the short lo in packaged 1 day postm ortem w h ich agrees w ith research by Jeremiah et al. (19 71 ) on vacuum packaged lam b accep tab ility . No sign ificant (P > 0 .10) a lte ra tion in tenderness was noted during vacuum storage o f the carcass tissue held 15 days p r io r to vacuum packaging. Com parison o f 1 and 15-day postm ortem tim e yie lded a s ign ificant (P < 0 .0 1 ) increase in tenderness w h ich agrees w ith the w o rk o f Deatherage and Harsham (1947 ) w ho noted tha t ten derness increased fo r 7 days when beef was stored at 1 -2 °C . D ifferences in grades were also nonsign ifican t (P > 0.10). Com parison at the same age postm ortem is possible by com b in ing grades and com paring 15 day postm ortem packaging-zero day vacuum storage tim e w ith 1 day postm ortem packaging-14 day vacuum storage tim e and th is com parison approaches significance (P < 0.10). Muscle shortening may have resulted from cu ttin g the carcasses at 1 day postm ortem , thereby preventing op tim u m tenderiza tion . This w ou ld agree w ith the research o f G o ll et al. (1964), Love (1962) and R am sbottom and Strandine (1949) who reported tha t muscle ch illed in carcass fo rm was m ore tender than muscle tha t was boned and ch illed . Com parison at 29 days postm ortem is possible by com paring 15 day postm ortem packagin g -14 days vacuum storage tim e w ith 1 day postm ortem packaging-28 days vacuum storage tim e. This com parison yie lded a nonsign ifican t (P > 0 .10 ) d iffe rence suggesting tha t i f there were a muscle shortening effect the tenderiza tion d if ference becomes less pronounced as postm ortem tim e increases.

F lavor o f vacuum packaged beef short lo ins is presented in F igure 5.

Desirable beef flavo r in te ns ity in creased in the high grade short lo in packaged a fte r 1 5 days o f carcass aging. The reverse was true fo r the lo w grade short lo in as evidenced by a decline in flavor score a fte r 1 5 days o f aging before packaging. F lavor in te ns ity fluc tua ted around the in it ia l value fo r bo th the high and low grade short lo ins w h ich were packaged at 1 day postm ortem .


The de tection o f o ff- fla v o r by taste panel eva luation is illus tra ted in F igure 6.

The lo w grade short lo ins aged 1 5 days p r io r to packaging had a s ig n ifica n tly (P < 0 .0 1 ) m ore pronounced o ff- fla v o r than e ither the lo w grade held fo r 1 day or the high grade regardless o f prepackaging ho ld ing tim e. Packaging 1 day a fte r slaughter s ig n ifica n tly (P < 0 .05 ) reduced undesirable flavo r scores w h ich corresponds to low er bacteria l g ro w th in these trea tm ent groups. S ig n ifican tly (P < 0 .05) m ore evidence o f o ff- fla v o r existed as storage tim e increased over the to ta l28-day period w h ich agrees w ith Jeremiah et al. (19 71 ) who found increasing o ff- flavo r during storage. P lo ttin g o f these scores ind ica te tha t w ith the exception o f lo w grade-15 days postm ortem packaging samples o ff- fla v o r scores d id no t start to decline u n t il a fte r 7 days o f vacuum sto rage.

Mean taste panel scores fo r general accep tab ility are shown in F igure 7.

In it ia l general accep tab ility o f short lo ins packaged 1 day a fte r slaughter was s ign ifican tly increased (P < 0 .01) a fte r3 —7 days o f storage. A fte r 3 —7 days o f vacuum storage, general accep tab ility showed no s ign ificant (P > 0 .10) changes to 28 days o f storage. Over the to ta l s to rage period short lo ins packaged 1 day afte r slaughter tended to m a in ta in the ir des irab ility th rough 28 days o f vacuum storage. H igh grade short lo ins were rated s ig n ifica n tly (P < 0 .05) higher in general accep tab ility than lo w grade short lo ins. High grade short lo ins also were cons is ten tly rated higher at each storage tim e period when compared w ith lo w grade short lo ins tha t were packaged at the same tim e postm ortem . Carcass aging o f 15 days p r io r to packaging s ligh tly im proved (P < 0 .1 0 ) in general accep tab ility th rough 7 days o f storage a fte r w h ich tim e general accep tab ility scores declined (P < 0 .05). F o r the low grade short lo in th is de s ira b ility was s ig n ifica n tly (P < 0 .05) low er at 28 days storage than at 0 days storage.

These lo ins can be compared fo r general accep tab ility at the same age (15 days and 29 days) postm ortem . I f w ith in grade com p arisons are made the o n ly sign ifican t (P < 0 .05) d iffe rence encountered is tha t the lo w grade lo in , packaged

at 1 day postm ortem and stored fo r 14 days in vacuum is given a low er general accep tab ility score than the com parable lo w grade lo in , packaged at 15 days postm ortem and stored 1 day in vacuum. This lo w er accep tab ility score could p robab ly be a ttr ib u te d to its s ign ificant low er tenderness score.

In th is research general accep tab ility scores closely fo llow e d tenderness evaluations except fo r the lo w grade 15 day postm ortem packaging sample w h ich was given unfavorable o ff- fla v o r scores partic u la r ly a fte r 28 days o f storage.

C O N C L U SIO N S

FR O M T H E S T A N D P O IN T o f the packer o r beef processor, i t has been dem onstrated in th is study th a t vacuum packaging can provide a m ethod o f aging w ho lesale cuts w h ile reducing weight loss during storage. Bacterial g ro w th was enhanced by longer storage periods and w ould p robab ly render the p roduc t unacceptable to the average consumer some place between the 14 and 28 days storage tim e period. The general accep tab ility scores fo r the 15 days postm ortem samples were beginning to fa ll be low th e ir in it ia l values du ring th is same tim e period. Even the 1 5 day vacuum storage period should a llow ample tim e fo r norm al distr ib u tio n fro m slaughter to consum ption. Postm ortem ho ld ing o f the carcass p rio r to packaging tended to increase the m icrob ia l numbers as w e ll as the developm ent o f o ff-flavo rs p a rticu la rly in low grade carcasses du ring vacuum storage. However greater tenderness up to 15 days postm ortem was a ttr ib u te d to the ho ld ing process. This inverse re la tionsh ip between tenderness and flavo r indicates fu r th e r expe rim en ta tion is needed to determ ine the op tim u m tim e o f postm ortem aging before vacuum packaging.

R E F E R E N C E S

APHA. 1960. “ Standard Methods for the Examination of Dairy Products.” Publication Office, American Public Health Association, New York.

Ball, C.O., Clauss, W.E. and Stier, E.F. 1957. Factors affecting quality of prepackaged meat. 1. Physical and organoleptic tests. B. Loss of weight and study of texture. Food Technol. 11: 277.

Baran, W.L., Kraft, A.A. and Walker, H.W. 1970. Effects of carbon dioxide and vacuum packaging on color and bacterial count of

meat. J. Milk Food Technol. 33: 77.Clark, D.S. and Lentz, C.P. 1969. The effect of

carbon dioxide on the growth of slime producing bacteria on fresh beef. Cana. Inst. Food Technol. J. 2: 72.

Cochran, W.G. and Cox, G.M. 1950. “ Experimental Designs,” p. 259. John Wiley and Sons, Inc., New York.

Deatherage, F.E. and Harsham, A. 1947. Relation of tenderness of beef to aging time at 33—35° F. Food Res. 12: 164.

Goll, D.E., Henderson, D.W. and Kline, E.A.1964. Postmortem changes in physical and chemical properties of bovine muscle. J. Food Sci. 29: 590.

Halleck, F.E., Ball, C.O. and Stier, E.F. 1958. Factors affecting quality of prepackaged meat. 4. Microbiological studies. B. Effect of package characteristics and of atmospheric pressure in package upon bacterial flora of meat. Food Technol. 12: 301.

Jaye, M., Kittaka, R.S. and Ordal, Z.J. 1962. The effect of temperature and packaging material on the storage life and bacterial flora of ground beef. Food Technol. 16: 95.

Jeremiah, L.E., Reagan, J.O., Smith, G.C., Carpenter, Z.L. and Kammlah, H.W. 1971. Effects of vacuum packaging on the retail acceptability of lamb cuts. Research report PR-2926:53, Texas A&M Univ.

Ledward, D.A., Nicol, D.J. and Shaw, M.K. 1971. Microbiological and color changes during aging of beef. Food Technol. (Australia) 23: 30.

Love, R.M. 1962. Protein denaturation in frozen fish. 7. Effect of the onset and resolution of rigor mortis on denaturation. J. Sci. Food Agr. 10: 534.

Marriott, N.G., Naumann, H.D., Stringer, W.C. and Hedrick, H.B. 1967. Color stability of prepackaged fresh beef as influenced by pre-display environments. Food Technol. 21: 1518.

Ordal, Z.J. 1962. Anaerobic packaging of meats. In “ Proceedings 14th Research Conference,” p. 39. American Meat Institute Foundation, Chicago, 111.

Pierson, M.D., Collins-Thompson, D.L. and Ordal, Z.J. 1970. Microbiological, sensory and pigment changes of aerobically and anaerobically packaged beef. Food Technol. 24: 1171.

Ramsbottom, J.M. and Strandine, E.J. 1949. Initial physical and chemical changes in beef as related to tenderness. J. Anim. Sci. 8: 398.

Reagan, J.O., Jeremiah, L.E., Smith, G.C. and Carpenter, Z.L. 1971. Vacuum packaging of lambs. J. Food Sci. 36: 764.

Shaw, M.K. and Nichol, D.J. 1969. Effect of the gaseous environment on the growth on the meat of some food poisoning and food spoilage organisms. Proc. 15th European Meat Research Workers Meeting B-16:226.

Sleeth, R.B., Kelly, G.G. and Brady, D.E. 1958. Shrinkage and organoleptic characteristics of beef aged in controlled environment. Food Technol. 12: 86.

Snedecor, G.W. 1959. “ Statistical Methods,” 5th ed. The Iowa State College Press, Ames, Iowa.

Stringer, W.C., Bilskie, M.E. and Naumann,H.D. 1969. Microbial profiles of fresh beef. Food Technol. 23: 97.

Ms received 7/27/73; revised 10/9/73; accepted10/15/73.______________________

Journal Article No. 43—73, Ohio Agricultural Research & Development Center, Wooster.

Appreciation is expressed to Big Bear Stores,Inc., Columbus, Ohio, for packaging assistance.

D. R. H E L D M A N

A g r ic u ltu ra l E n g in e e rin g D e p t, a n d D e p t, o f F o o d S cience & H u m a n N u t r it io n

M ic h ig a n S ta te U n iv e rs ity , E ast La n s in g , M l 4 8 8 2 3

a n d G. A . H O H N E R

J o h n S tu a r t Research Labs, The Q u a ke r O ats Co., B a rr in g to n , IL 6 0 0 1 0

AN ANALYSIS OF ATM OSPHERIC FREEZE DRYING

INTRODUCTION

T H E U S E O F c o l d g a s w i t h l o w w a t e r v a p o r p r e s s u r e t o c a u s e s u b l i m a t i o n o f m o i s t u r e f r o m a f r o z e n f o o d a t o r n e a r a t m o s p h e r i c p r e s s u r e i s r e f e r r e d t o a s a t m o s p h e r i c f r e e z e d r y i n g . I t h a s g e n e r a l l y b e e n a s s u m e d t h a t t h e r a t e o f a t m o s p h e r i c f r e e z e

d r y i n g i s t o o s l o w t o b e e c o n o m i c a l l y f e a s i b l e . T h e r a t e - c o n - t r o l l i n g p a r a m e t e r i s m o l e c u l a r d i f f u s i o n o f w a t e r v a p o r w i t h i n t h e d r y p r o d u c t s t r u c t u r e a s c o m p a r e d t o v a c u u m f r e e z e d r y

i n g w h i c h i s g e n e r a l l y h e a t t r a n s f e r l i m i t e d . P o t e n t i a l a d v a n t a g e s o f t h e a t m o s p h e r i c p r o c e s s i n c l u d e t h e p o s s i b i l i t y o f a l o w e r i n i t i a l c o s t s i n c e e x p e n s i v e v a c u u m a s s o c i a t e d c o m p o n e n t s c o u l d b e e l i m i n a t e d . T h e p r o c e s s c o u l d b e d e s i g n e d i n t o a c o n t i n u o u s s y s t e m a n d s h o u l d p r o d u c e a h i g h e r q u a l i t y p r o d u c t b y e l i m i n a t i n g t h e h a r m f u l i n f l u e n c e o f e x c e s s i v e t e m p e r a t u r e i n t h e s e m i - d r y p o r t i o n o f t h e f o o d p r o d u c t .

T h e p o t e n t i a l f o r a t m o s p h e r i c f r e e z e d r y i n g w a s f i r s t d e m o n s t r a t e d b y M e r y m a n ( 1 9 5 9 ) . P r e l i m i n a r y s t u d i e s w i t h f o o d p r o d u c t s w e r e c o n d u c t e d b y L e w i n a n d M a t e l e s ( 1 9 6 2 )

a n d W o o d w a r d ( 1 9 6 1 , 1 9 6 3 ) . M a l e c k i e t a l . ( 1 9 6 9 ) h a v e a t t e m p t e d t o i n c r e a s e a t m o s p h e r e f r e e z e - d r y i n g r a t e s b y r e d u c

i n g p a r t i c l e s i z e i n a n i n v e s t i g a t i o n o f s p r a y f r e e z e d r y i n g o f

e g g a l b u m e n a n d a p p l e j u i c e . R e s u l t s o b t a i n e d b y D u n o y e r a n d L a r o u s s e ( 1 9 6 1 ) s u p p o r t t h e c o n c l u s i o n t h a t a t m o s p h e r i c f r e e z e - d r y i n g r a t e s o f s m a l l p a r t i c l e s c a n b e e q u i v a l e n t t o

v a c u u m f r e e z e - d r y i n g r a t e s . T h e e c o n o m i c a n a l y s i s o f W o o d w a r d ( 1 9 6 1 ) i n d i c a t e d t h a t e n e r g y c o s t s f o r m o i s t u r e r e m o v a l

c a n a l s o b e e q u i v a l e n t t o v a c u u m f r e e z e d r y i n g .T h e o b j e c t i v e s o f c u r r e n t i n v e s t i g a t i o n s a r e :

1. T o d e r i v e a m a t h e m a t i c a l m o d e l w h i c h d e s c r i b e s a t m o s

p h e r i c f r e e z e d r y i n g a n d c a n b e s o l v e d n u m e r i c a l l y ;2 . T o e v a l u a t e t h e a p p r o p r i a t e h e a t a n d m a s s t r a n s f e r p a r a m

e t e r s f o r t h e a t m o s p h e r i c f r e e z e - d r y i n g p r o c e s s b y u s e o f

t h e m a t h e m a t i c a l m o d e l ;3 . T o d e t e r m i n e t h e i n f l u e n c e o f v a r i o u s o p e r a t i n g v a r i a b l e s

o n t h e r a t e s o f a t m o s p h e r i c f r e e z e d r y i n g .

Fig. 1 -Schem atic diagram o f atmospheric freeze drying.

Theoretical considerationsA t m o s p h e r i c f r e e z e d r y i n g i s d e s c r i b e d b y a m a t h e m a t i c a l

m o d e l w i t h a p p r o p r i a t e a s s u m p t i o n s a n d s i m p l i f i c a t i o n s . T h e m a t h e m a t i c a l m o d e l i s t h e n t r a n s f o r m e d t o a f i n i t e - d i f f e r e n c e m o d e l w h i c h c a n b e s o l v e d b y n u m e r i c a l m e t h o d s .

The mathematical model. T h e s a m p l e u n d e r a t m o s p h e r i c f r e e z e d r y i n g c a n b e r e p r e s e n t e d s c h e m a t i c a l l y i n o n e d i m e n s i o n a s s h o w n i n F i g u r e 1 . A n i c e - v a p o r i n t e r f a c e ( f ) r e c e d e s t o w a r d t h e c e n t e r l i n e a s s u b l i m a t i o n p r o g r e s s e s , a n d t h e n e c e s s a r y h e a t o f s u b l i m a t i o n i s t r a n s p o r t e d f r o m t h e s u r f a c e ( s ) t o t h e i n t e r f a c e i n r e s p o n s e t o a t e m p e r a t u r e g r a d i e n t . S i m u l t a n e o u s l y w a t e r v a p o r i s t r a n s p o r t e d t h r o u g h t h e p o r o u s z o n e t o t h e s u r f a c e i n r e s p o n s e t o a p a r t i a l p r e s s u r e g r a d i e n t .

T h e e n e r g y a n d m a s s t r a n s f e r e q u a t i o n s f o r t h e p o r o u s z o n e w e r e d e r i v e d f r o m t h e a p p r o p r i a t e c o n s e r v a t i o n p r i n c i p l e a p

p l i e d t o a d i f f e r e n t i a l v o l u m e o f t h e p o r o u s z o n e , d V . C o n s i d e r i n g f i r s t t h e e n e r g y e q u a t i o n , w h i c h w a s w r i t t e n i n d i m e n s i o n l e s s f o r m a s :

P(Dpd f MCpW) 9 T

90

D„ 9 p v 9 t 3M- p w + p A H v —

9<p 90 90

( 1 )

L i k e w i s e t h e w a t e r v a p o r t r a n s p o r t e q u a t i o n w a s d e r i v e d

f r o m a m a s s b a l a n c e o n t h e s a m e d i f f e r e n t i a l v o l u m e e l e m e n t , a n d e x p r e s s e d i n d i m e n s i o n l e s s f o r m a s :

eMw 9PV 9 /b e 9PV\ 9M" R T 9 0 ~ = ~ 9 0 \ d “ 9 0 J ~ P W ’ i2)

I n g e n e r a l , t h e a m o u n t o f w a t e r a d s o r b e d o n a h y g r o s c o p i c s u r f a c e i s a f u n c t i o n o f b o t h t e m p e r a t u r e a n d v a p o r p r e s s u r e o f t h e s u r r o u n d i n g g a s . I n t h e t e m p e r a t u r e r a n g e w h e r e a t m o s p h e r i c f r e e z e d r y i n g c a n b e c o n d u c t e d , e x p e r i m e n t a l d a t a f o r

f r e e z e - d r i e d b e e f i n d i c a t e d t h a t t h e d e p e n d e n c e o f a d s o r b e d m o i s t u r e o n t e m p e r a t u r e i s n e g l i g i b l e w h e n c o m p a r e d t o d e p e n d e n c e o n r e l a t i v e h u m i d i t y . B e c a u s e o f t h e r e l a t i v e i n d e p e n d e n c e o f a d s o r b e d m o i s t u r e a n d t e m p e r a t u r e , t h e t i m e r a t e o f c h a n g e o f a d s o r b e d m o i s t u r e i n e q u a t i o n ( 2 ) w a s w r i t t e n

w h e r e u s e w a s m a d e o f t h e d e f i n i t i o n o f r e l a t i v e h u m i d i t y .

E q u a t i o n ( 2 ) w a s t h e n r e w r i t t e n t o g i v e :

V o lu m e 3 9 '1 9 7 4 1 -J O U R N A L OF F O O D SC IE N C E - 1 4 7

1 4 8 -J O U R N A L O F F O O D S C I E N C E -V o lu m e 3 9 (1 9 7 4 )

E q u a t i o n s ( 1 ) a n d ( 4 ) r e p r e s e n t t h e h e a t a n d m a s s t r a n s f e r e q u a t i o n s o f t h e m a t h e m a t i c a l m o d e l a n d m u s t b e s o l v e d s i m u l t a n e o u s l y t o y i e l d t e m p e r a t u r e a n d v a p o r p r e s s u r e a t a l l

p o i n t s a s a f u n c t i o n o f d i m e n s i o n l e s s t i m e . A p p r o p r i a t e b o u n d a r y c o n d i t i o n s m u s t b e s p e c i f i e d t o a l l o w s o l u t i o n . A t

t h e p r o d u c t s u r f a c e a f i n i t e p o r o u s z o n e i s a s s u m e d t o f o r m i n s t a n t a n e o u s l y a s h e a t a n d w a t e r v a p o r a r e e x c h a n g e d w i t h

t h e a i r s t r e a m b y c o n v e c t i o n . C o n v e c t i v e c o n d i t i o n s a t t h e

s a m p l e s u r f a c e g i v e t h e f o l l o w i n g b o u n d a r y c o n d i t i o n s :

B o u n d a r y c o n d i t i o n s a t t h e i c e - v a p o r i n t e r f a c e a r e m o r e c o m p l e x . V a p o r p r e s s u r e a t t h e i n t e r f a c e i s t h e s a t u r a t i o n v a p o r p r e s s u r e o f t h e i c e s u r f a c e a t t h e i n t e r f a c e t e m p e r a t u r e . T h e b o u n d a r y c o n d i t i o n f o r t h e e n e r g y e q u a t i o n i s d e r i v e d f r o m a n e n e r g y b a l a n c e o n t h e i n t e r f a c e ,

a h , | P (M „ - M ,) = - g _ f ♦ f „ (1 + M o ) C p c| i .

( 7 )

T h e r a t e o f v a p o r t r a n s f e r a w a y f r o m t h e i n t e r f a c e i s w r i t t e n a s

3 . C o n v e c t i v e s u r f a c e p a r a m e t e r s a r e a s s u m e d t o b e c o n s t a n t , t h a t i s , i n d e p e n d e n t o f w a t e r v a p o r c o n c e n t r a t i o n a t l o w c o n c e n t r a t i o n s i n v o l v e d a t t h e s a m p l e s u r f a c e .

4 . C e r t a i n p h y s i c a l p a r a m e t e r s i n c l u d i n g b u l k d e n s i t y o f t h e p o r o u s z o n e ( p ) , s p e c i f i c h e a t o f t h e p o r o u s z o n e ( C p ( j ) , s p e c i f i c h e a t o f w a t e r ( C p w ) , h e a t o f s u b l i m a t i o n ( A H S ) ,

h e a t o f v a p o r i z a t i o n ( A H V) , a n d s p e c i f i c h e a t o f t h e f r o z e n c o r e ( C p c ) a s s u m e d c o n s t a n t .

5 . T h e e n e r g y c o n t e n t o f t h e v a p o r i n t h e p o r e s i s n e g l e c t e d .

C o m p l e x i t y o f t h e s i m u l t a n e o u s s o l u t i o n o f e q u a t i o n s ( 1 )a n d ( 4 ) p r e c l u d e d c l o s e d f o r m i n t e g r a t i o n . I n s t e a d t h e m e t h o d s o f n u m e r i c a l a n a l y s i s w e r e u s e d , a n d t h e l a r g e a m o u n t o f

c o m p u t a t i o n n e c e s s a r y w a s d o n e o n a d i g i t a l c o m p u t e r ( C o n t r o l D a t a C o r p . M o d e l 3 6 0 0 ) .

T h e h e a t a n d m a s s t r a n s f e r e q u a t i o n s w e r e t r a n s f o r m e d i n t o

f i n i t e d i f f e r e n c e e q u a t i o n s u s i n g t h e p r o c e d u r e s o f C r a n k a n d

N i c h o l s o n ( 1 9 4 7 ) . A d e t a i l e d d i s c u s s i o n o f a c c u r a c y a n d

s t a b i l i t y c o n s i d e r a t i o n s i n t r o d u c e d d u r i n g s o l u t i o n o f t h e

e q u a t i o n s i s p r e s e n t e d b y H o h n e r ( 1 9 7 0 ) .

The mass transfer coefficientG u n n ( 1 9 6 7 ) a n d G u n n a n d K i n g ( 1 9 6 9 ) d e r i v e d a n e x p r e s

s i o n f o r v a p o r - p h a s e m a s s t r a n s p o r t i n p o r o u s m e d i a i n t h e

p r e s e n c e o f g r a d i e n t s o f t o t a l p r e s s u r e a n d c o n c e n t r a t i o n . T h e

e x p r e s s i o n i n c l u d e s e x p e r i m e n t a l l y d e t e r m i n e d c o n s t a n t s , w h i c h a r e f u n c t i o n s o f t h e p o r o u s p r o d u c t s t r u c t u r e . I n a t m o s p h e r i c f r e e z e d r y i n g , t h e t r a n s p o r t e d g a s i s w a t e r v a p o r w h i l e

t h e s t a g n a n t g a s i s a i r o r s o m e i n e r t g a s . S i n c e t h e m a x i m u m

t o t a l p r e s s u r e d i f f e r e n t i a l a c r o s s t h e p o r o u s z o n e i s a p p r o x i m a t e l y 4 . 5 m m H g ( a s c o m p a r e d t o a t o t a l p r e s s u r e o f o v e r

7 0 0 m m H g ) , t h e e f f e c t i v e m a s s t r a n s f e r c o e f f i c i e n t f o r a t m o s p h e r i c f r e e z e d r y i n g c a n b e w r i t t e n

d f

~7a P (Moe S / 9 P y \

D \ 3 0 / 0 = f ‘(8 ) D e

C 2 D M w

P R T(12)

F i n a l l y , s u b s t i t u t i o n o f ( 8 ) i n t o ( 7 ) l e a d s t o t h e a p p r o p r i a t e b o u n d a r y c o n d i t i o n f o r t h e e n e r g y e q u a t i o n a t t h e i c e - v a p o r i n t e r f a c e :

k s / ( T l \ D e s / 9 P V\

D \ ¥ / 0=f = AHs~ D \ W l = f9 T

f p ( l + M 0 ) C p c — - ( 9 )

A f t e r t h e i c e - v a p o r i n t e r f a c e r e a c h e s t h e c e n t e r l i n e a n d f r e e m o i s t u r e i s n o l o n g e r p r e s e n t i n t h e s a m p l e , t h e b o u n d a r y c o n d i t i o n s a t t h e c e n t e r l i n e a r e

T h e s t r u c t u r a l c o n s t a n t i n t h e e f f e c t i v e m a s s t r a n s f e r c o e f f i c i e n t , C 2 , . c a n b e v i e w e d a s a n a t t e n u a t i o n c o n s t a n t w h i c h a c c o u n t e d f o r t h e a m o u n t o f t h e f r e e - g a s v a l u e o f t h e m u t u a l d i f f u s i v i t y o f a i r a n d w a t e r v a p o r i s r e d u c e d d u e t o c o n s t r i c t i o n s o f t h e p o r o u s m e d i a . K r i s c h e r ( 1 9 5 9 ) h a s r e l a t e d t h i s c o n s t a n t t o t h e p o r o s i t y o f t h e p o r o u s z o n e b y a f a c t o r t o a c c o u n t f o r t h e t o r t u o s i t y o f t h e p a t h o f t h e w a t e r v a p o r m o l e c u l e t h r o u g h t h e d r i e d p o r t i o n o f t h e p r o d u c t .

EXPERIMENTAL

(ST \

3 0 J<j>= o0 , a t 0 = 0 . ( 1 0 )

T h e a m o u n t o f s p e c i f i c h e a t l i b e r a t e d d u e t o c h a n g e i n i c e c o r e t e m p e r a t u r e w i t h t i m e h a s a n e g l i g i b l e e f f e c t u p o n t h e v e l o c i t y o f t h e i c e - v a p o r i n t e r f a c e , b u t c h a n g e s i n i c e c o r e t e m p e r a t u r e h a v e a s i g n i f i c a n t e f f e c t u p o n t h e v a p o r p r e s s u r e a t t h e i n t e r f a c e a n d t h u s u p o n t h e r a t e o f m a s s t r a n s f e r . T h e r e

f o r e , t h e r e m a i n i n g b o u n d a r y c o n d i t i o n f o r t h e m a s s t r a n s f e r e q u a t i o n w a s w r i t t e n

P v = F ( T C) = s a t u r a t i o n c o n d i t i o n , 4> - f. ( 1 1 )

V a r i o u s a d d i t i o n a l a s s u m p t i o n s a n d s i m p l i f i c a t i o n s n o t s p e c i f i c a l l y m e n t i o n e d d u r i n g d e r i v a t i o n o f t h e m o d e l w e r e m a d e . T h e s e a r e a s f o l l o w s :

1 . I n a m a c r o s c o p i c s e n s e t h e p r o d u c t i s a s s u m e d t o b e o f u n i f o r m a n d c o n s t a n t p o r o s i t y ; t h u s s h r i n k a g e i s a s s u m e d t o b e z e r o .

2 . A l l t r a n s p o r t i s a s s u m e d t o b e i n o n e d i m e n s i o n .

P r e p a r a t io n a n d a s s ig n m e n t o f s a m p le s

E x perim en ta l sam ples w ere prepared from th e lo in eye m uscle o f beef, longissim us dorsi. A section o f the m uscle , grade USDA C hoice, was roasted a t 1 6 3 °C un til the tem p era tu re at the cen ter o f mass reached 74°C. Average com position o f 10 sam ples o f th e co oked b eef was 9.8% fat (e ther ex trac t) w ith a m oistu re co n ten t o f 150% , dry basis. T he cooked m eat was then frozen a t - 2 9 ° C and la te r c u t in to app rox im ate ly 1-cm cubes w ith an electric band saw. Special e f fo r t was m ade w hen cu ttin g the cubes to align the n a tu ra l fibers o f th e m eat w ith the planes o f th e cube. The cubes w ere w rapped in foil and sto red a t - 2 9 ° C in a sealed con ta iner un til needed fo r a tes t. C ubes in w hich the fibers o f the m eat p ro jec ted a t an ob lique angle to the faces o f the cube were discarded. F rom the rem aining p o p u la tio n , cubes w ere assigned random ly to a particu lar set o f tes t conditions.

T e s t c o n d i t i o n s

Prelim inary tests cond u c ted in the cu rren t research in d ica ted significan t p ro d u c t shrinkage in b ee f dried w ith air tem p era tu res above 0°C . C alculation o f th e am o u n t o f unfrozen w ater in b ee f from ap p a ren t specific h ea t da ta ind icates a large change o f un fro zen w ater per u n it change in p ro d u c t tem p era tu re in th e range from - 1 0 ° C to 0°C (R iedel, 1957). These considerations and th e fact th a t su b lim atio n is exceedingly slow a t tem p era tu res below -1 0 ° C led to se lection o f tw o levels o f air tem p era tu re , - 2 .8 ° C and -8 .2 ° C . F o r purposes o f adequate ly testing the p roposed m athem atica l m odel, d a ta w ere ob ta in ed at tw o levels o f to ta l pressure, 0 .97 a tm o sp h e re and 0 .58 a tm osphere .

A N A L Y S IS O F A T M O S P H E R IC F R E E Z E D R Y IN G - 1 4 9

E x p e r i m e n t a l t e s t s w e r e c o n d u c t e d w i t h p r o d u c t f i b e r s p a r a l l e l a n d p e r p e n d i c u l a r t o t h e d i r e c t i o n o f h e a t a n d m a s s t r a n s f e r . T h r e e t r i a l s o f e a c h e x p e r i m e n t a l s i t u a t i o n w e r e c o n d u c t e d .

Experimental apparatusT h e a t m o s p h e r i c f r e e z e - d r y i n g r a t e s w e r e m e a s u r e d in a n a p p a r a t u s

d e s c r i b e d in d e t a i l b y H o h n e r ( 1 9 7 0 ) . T h e s y s t e m w a s d e s i g n e d t o p r o v i d e a c o n t i n u o u s h i s t o r y o f s a m p l e w e i g h t a n d t e m p e r a t u r e s a t t h e s u r f a c e a n d c e n t e r o f t h e c y l i n d r i c a l l y - s h a p e d b e e f s a m p l e s . T h e s a m p l e h o l d e r s w e r e d e s i g n e d s o t h a t h e a t a n d m a s s t r a n s f e r o c c u r r e d o n l y in t h e a x i a l d i r e c t i o n . T h e t e m p e r a t u r e o f t h e a i r , w h i c h p a s s e d t h r o u g h s i li ca ge l d u r i n g e a c h c y c l e , w a s c o n t r o l l e d b y p a s s i n g t h r o u g h a h e a t e x c h a n g e r s u b m e r g e d in a c o n t r o l l e d t e m p e r a t u r e w a t e r b a t h a n d w a s m o n i t o r e d b y a t h e r m o c o u p l e a n d r e c o r d i n g p o t e n t i o m e t e r .


Numerical solutionThe mathematical model assumed adsorbed moisture in the

porous zone was in equilibrium with the air-water vapor mixture in the pores. The saturated pressure of water vapor as a function of temperature was also required in the numerical solution. This expression was obtained by fitting a third degree polynomial to saturated vapor pressure date over the temperature range from -30°C to 0°C. Maximum absolute deviation of the polynomial was within 3% of the data over the temperature range of interest.

Various physical parameters which were assumed constant in the model also required evaluation. The constant numerical values used in the solution and the source of information are listed in Table 1.

The ice core temperature and air temperature were monitored during tests performed to collect parameter estimation data. For atmospheric freeze drying it was found that the surface temperature remained nearly constant within 0.5°C of the air temperature. Therefore, surface temperature was entered in the computer program as a constant value.

Solution of finite-difference equations to accurately approximate the partial differential equations from which they were written required careful selection of the incremental step size in the independent variables. There are two independent variables in the proposed model: dimensionless time and dimensionless distance. Size of the increment in dimensionless distance, A <p, was selected to be 0.1 by repeatedly solving the model while varying the size of the increment.

The maximum time step, A d , compatible with stability of the convective mass transfer boundary condition was com

puted and found to be approximately 40 sec, real time. Computation time for one step in real time on the CDC-3600 computer varied depending on the number of nodes in the numerical solution at any given time; however, average simulation speed for solving the entire model was approximately 60 hr/min of computer time.

Trial solutions of the numerical model gave indication that the model was at least qualitatively correct. Figure 2 shows typical vapor pressure and temperature profiles computed from the proposed model for one-dimensional atmospheric freeze drying of beef. For the elapsed time shown in Figure 2, three-fourths of the free moisture had been removed. The position of the ice-vapor interface is indicated by a dashed line. The most outstanding characteristic of the computed profiles is their almost perfect linearity. These computed results strongly support the pseudo steady-state assumption used by previous investigators (Sandall et al., 1967; Dyer and Sunderland, 1968). Clearly, movement of the ice front was so slow that the time derivatives of dependent variables in the porous zone were insignificant compared to the space derivatives.

Linearity of the profiles increased the accuracy with which the derivatives of vapor pressure and temperature were computed at the interface. The velocity of the interface and the rate of drying are dependent on calculation of these derivatives.

Figure 3 illustrates a solution of the proposed model in terms of dimensionless mean moisture content versus dimensionless time. The model solution has been converged to a typical set of atmospheric freeze-drying data obtained from a one-dimensional sample. In addition, Figure 3 illustrates a solution which neglected the same components of the heat and mass transfer equations as does the pseudo steady-state model. The most important component neglected was the adsorbed moisture. Both solutions included the same values for all constants and transport parameters of the model. The proposed model, at least qualitatively, predicted the extended time required to remove the absorbed moisture after the ice front reached the center line. Previous investigators (Sandall et al.,1967) have noted that models which neglected the adsorbed moisture were significantly in error after 75—90% of the original moisture had been removed. This observation is supported by results of the proposed model.

Results shown in Figure 3 indicated pseudo steady-state models are in error chiefly due to neglecting the adsorbed moisture in the porous zone. Clearly the assumption of linear

Table 1—Numerical values of physical constants used in the mathematical model

Density Value Source

Bulk density of dry 0.46g/cm3 Mean value of experimentalproduct, p measurements

Specific heat o f dry 138 cal/g-° C Computed from specific heatproduct, Cpcj of frozen beef at —40° C

(Short and Staph, 1951)Specific heat o f ice

core, Cpc 1.15 cal/g-°C Riedel (1957)Porosity o f dry

product, e 0.76 Harper (1962)Mutual d iffus iv ity , air

and water vapor,1 atm, 0°C, D 0.22cm2 /sec Perry (1967)

Heat o f sublimation, AHS 676 cal/g Threlkeld (1962)Initia l moisture

content, M0 approx 1.5 db Measured fo r each testHalf-thickness of

sample, s approx 0.45 cm Measured fo r each test

1 5 0 -J O U R N A L O F F O O D S C I E N C E -V o lu m e 3 9 (1 9 7 4 )

dependent variable profiles is acceptable. For purposes of parameter estimation, the more complete description of atmospheric freeze drying as included in the proposed model is preferred.

Eighteen estimates each of three parameters are presented in Table 2. The three parameters of the mathematical model are adjusted in repeated computer solutions of the model to obtain the best fit to each set of experimental data. In this way the numerical values are obtained for all parameters from each experiment. The parameters, k and C2, were estimated near the values expected. Harper (1962) reported the value of k in freeze-dried beef for pressures above approximately 0.5 atm in the absence of a counter-flow of water vapor to be approximately 1.5 x 1CT4 cal/cm-sec/°C. The esimates of k shown in Table 2 are near to this value. Sandall et al. (1967) evaluated C2, the structural constant in the effective mass transfer coefficient, in the breast meat of turkey and found values between 0.44 and 0.66 for transport parallel to fibers and approximately 0.27 for transport perpendicular to the fiber orientation. Similar to slightly higher values are presented in Table 2 for precooked beef.

An analysis of variance of all three parameters was conducted. The variance of the experimental results was analyzed for significance due to air temperature, system pressure and orientation of the fibers. Interactions between these factors were assumed negligible. Testing for significant differences was done by use of the F-test at the 90% level of significance.

Fig. 2 —Com puted profiles o f vapor pressure and temperature for typ ical conditions o f atmospheric freeze drying.

The value of hD was not expected to vary with temperature or pressure over the small range of these variables that was considered. Since hD is not a function of product properties it was not expected to be a function of orientation of the fibers. Statistical analysis of the dependence of hD on temperature, pressure and orientation confirmed that no significant differences exist for any of these factors at the 90% confidence level. The mean value of all estimates of hD was approximately 0.0095 g/sec-cm2 -atm.

The internal heat and mass transfer parameters, being functions of the product under consideration, are of great interest. The porosity of freeze-dried beef has been reported by Harper(1962) to be approximately 0.76. Since it was expected that the tortuosity factor was greater for transport perpendicular to the fiber orientation of the meat than parallel to the fibers, C2

was expected to be less in those tests conducted with water vapor transport perpendicular to the fibers of the meat. The structural constant was not expected to be a function of any operating variable.

The estimates of C2 shown in Table 2 were tested for significant differences due to temperature, pressure level and orientation of the fibers. Only differences in C2 due to orientation of the fibers were significant when tested at the 90% confidence level. Differences due to fiber orientation were also significant at the 95% confidence level. The mean for estimate of C2 for vapor diffusion parallel to the fibers was 0.81 and 0.62 for diffusion perpendicular to the fibers.

Estimates of the effective thermal conductivity, k, present an interesting comparison to results obtained by Harper(1962). Using steady-state methods on freeze-dried beef with no water vapor flux, Harper found the mean value of k to be1.5 x 1 0' 4 cal/cm-sec-°C. An overall mean value of 1.0 x 10“4

cal/cm-sec-°C was found in current research. These estimates were made in the presence of a counterflow of water vapor and by parameter estimation from transient experiments. The estimated effective thermal conductivity was especially sensitive to variations in structure and composition of the meat

Table 2—Summary of parameter estimates and variance of the residuals

Test no. hD c 2 k o 2

A ir Temp -8 .2 ° C Pressure = 0.97 atm Orientation = parallel1 0.0084 0.56 0.46 X 10’4 1.97 X 10 '5

17 0.0091 0.75 0.44 9.782 0.0079 0.86 0.47 5.72

A ir Temp -8 .2 ° C Pressure = 0.97 atm Orientation = perpendicular7 0.0087 0.64 2.77 X 10‘4 1.05 X 10 '58 0.0117 0.80 0.95 29.90

18 0.0086 0.51 1.59 13.80A ir Temp -2 .8 ° C Pressure = 0.97 atm O rientation = parallel3 0.0126 0.89 0.53 X 10-4 21.82 X 10 s4 0.0116 0.95 1.35 1.97

15 0.0056 0.99 0.51 16.4416 0.0092 0.61 0.31 5.36A ir Temp -2 .8 ° C Pressure = 0.97 atm Orientation = perpendicular5 0.0072 0.62 0.96 X 10-4 9.24 X 10‘ 56 0.0111 0.64 0.99 17.26

A ir Temp -2 .8 ° C Pressure = 0.58 atm O rientation = parallel9 0.0087 0.74 1.21 X 10'4 8.52 X 10 '5

10 0.0087 0.99 1.00 12.3612 0.0095 0.74 1.02 7.54A ir Temp -2 .8 ° C Pressure = 0.58 atm Orientationi = perpendicular11 0.0133 0.42 1.03 X 10'4 14.13 X 10 's13 0.0091 0.72 1.75 8.7614 0.0107 0.62 1.09 11.40

A N A L Y S IS O F A T M O S P H E R IC F R E E Z E D R Y IN G - 151

sample as is evident from the results of Table 2. 95% confidence limits were computed for the estimated value of k. Using the t-test (Snedecor, 1956) these limits were found to be 0.7 x 10' 4 to 1.3 x 10' 4 cal/cm-sec-°C. Variations in k due to system pressure, air temperature and orientation were all insignificant at the 90% confidence level.

Evaluation of k and C2 allowed certain observations to be made concerning details of the mechanisms, of atmospheric freeze drying. Heat transfer through the porous zone of the product has been assumed to be by conduction and convection through the gas-filled pores. The mean of the current estimates of k was approximately two-thirds of the magnitude which Harper (1962) found. While variability of the estimates of k was large, it is noteworthy that the 95% confidence limits on the mean of the current estimates did not include the mean value Harper obtained. Furthermore, the mean estimated value of k was between the values Harper (1962) found for atmospheric pressure and vacuum conditions. These points tend to support the above concept of the mechanism of heat transfer with some additional insight. Apparently, the counterflow of water vapor throughout the drying process substantially reduced the contribution of the pores to transfer of heat in the opposite direction. Thus the effective value of thermal conductivity measured under dynamic conditions at atmospheric pressure was found to be near the value obtained under static conditions in a vacuum.

Implications of the above results are that the effective transfer of heat through the porous zone during the drying process is substantially less than measured under steady-state conditions. Such findings are important to optimization of the freeze-drying process.

Results of statistical analysis of the mass transfer parameter, C2, confirmed the hypothesis that the mechanism of mass transfer was water vapor diffusion through stagnant air in the pores of the dried layer. Further insight into this process was gained from the magnitude of the estimated values of C2

both parallel and perpendicular to the fiber orientation. The mean value of C2 parallel to the fibers was determined to be 0.81 compared to a value of one under free-gas conditions. In

other words, the mean free path of the water vapor molecule led to contact with the porous solid only often enough to reduce the effective value of the free-gas mass diffusivity by 19%. Similarly transport perpendicular to the fibers was reduced by 38%. From the viewpoint of a water vapor molecule at freeze-drying temperatures, freeze-dried beef is a highly porous medium. Sandall et al. (1967) estimated C2 to be between 0.44 and 0.66 parallel to the fibers and 0.27 perpendicular to the fibers of turkey meat. This indicates the structure of turkey meat is less porous than that of beef.

Simulation of atmospheric freeze drying in three dimensions

In all tests discussed previously, the transport of heat and water vapor in the sample has been limited to one dimension. Such tests were used for parameter estimation and analysis of the mechanisms of atmospheric freeze drying. Obviously, practical application of the process occurs in samples where heat and water vapor are transported in three dimensions. Complexity of the mathematical model would defy even numerical solution if it were derived initially in three space coordinates. However, the one-dimensional model was transformed into a reasonably accurate approximation of atmospheric freeze drying in cubical samples with transport of heat and water vapor from all six surfaces. Mean values of all parameters determined in the previous section were used, and the anisotropic effect induced by the fiber structure was disregarded.

Geometrically the three-dimensional model was visualized as a pyramid with height one-half the length of the base. The apex of the pyramid was located at the center of the cubical sample with the base of the pyramid on the sample surface. All transport of heat and water vapor was assumed to move perpendicular to the sample surface. Actually, of course, flow of heat and water vapor were not perpendicular to the surface of the cube except along a line from the center perpendicular to the surface. Nevertheless, considering the sample variability and the effect of this variability on the product-dependent constants of the model, the three-dimensional model was considered sufficiently accurate for process analysis work.

Fig. 3-C om parison o f predicted drying curves o f the proposed m odel and pseudo steady statemodeI in one-dimensional samples.

1 5 2 -J O U R N A L OF F O O D S C IE N C E -V o lu m e 3 9 (1 9 7 4 )

Results of the three-dimensional solution using the mean values of parameters are compared to experimental results of atmospheric freeze drying of cubes of precooked beef in Figure 4. Both sets of experimental results shown in Figure 4 were obtained at —2.8°C air temperature and 0.97 atm total pressure. One sample had a half-thickness of 0.7 cm and the other 0.5 cm. These results confirm that the approximations included in the three-dimensional model were reasonably accurate until the dimensionless mean moisture content dropped

below 0.1. At low moisture contents the three-dimensional model predicted excessively long drying times.

At low moisture content the ice core in the three-dimensional model was assumed to be reduced to a small cube in the center of the sample. Water vapor was assumed to flow outward only along a path with cross-sectional area equal to the area of the ice core in the center of the sample. Clearly, near the end of the process, this assumption neglected a substantial amount of the effective transport area of the sample. In subse-

Fig. 4 —C o m p a ris o n o f s o lu t io n s o f th e th re e -d im e n s io n a l m o d e l to e x p e r im e n ta l re su lts in c u b ic a l sam ples.

Fig. 5 -P red ic ted effect o f air temperature on atmospheric freeze drying rates in 1-cm cubes o fprecooked beef.


quent discussion where the three-dimensional model will be used for analysis of the atmospheric freeze-drying process, prediction of the model will be disregarded below M = 0 . 1 .Analysis of atmospheric freeze drying in cubical samples

The power and economy of a proven computer simulation for analysis of the effect of operating variables upon a physical process quickly becomes apparent when the speed and flexibility of the model solution are compared to acquiring the same information from experimental tests. The approximate three-dimensional model discussed above was used to investigate the effect of air temperature, system pressure, sample size and magnitude of the surface mass transfer coefficient on the rate of atmospheric freeze drying in cubical samples of cooked beef.

The practical operating range of all variables was investigated by changing the variables one at a time while holding all others at a standard condition. The standard condition was the following:

Air temp, Ta System pressure, P Sample half-thickness, s Surface mass transfer coef., h[> Structural constant, C2

Thermal conductivity, k Initial moisture content, M0

= —3.0°C = 0.97 atm = 0.5 cm= 0.0095 gm/cm2 -sec-atm = 0.725= 0.0001 cal/cm-sec-°C = 1.5 gm-H2 0/gm-dry solid

Other product dependent constants were evaluated as shown in Table 1.

In Figure 5 the effect of air temperature on the rate of atmospheric freeze drying of 1 cm cubes of cooked beef is shown. Air temperature was investigated at —3.0°C, —8.0°C and — 13.0°C. This modest range in air temperature caused a greater change in the predicted drying time than the changes investigated in any other variable. The predicted increase in drying rate with air temperature was approximately of the order witnessed by Woodward (1961) and Lewin and Mateles(1962), As has previously been noted, the practical upper limit of air temperature is approximately —3.0°C due to depression

of the freezing point caused by dissolved solutes. Results of Figure 5 demonstrate that any practical application of atmospheric freeze drying must be carefully designed to operate as near the maximum allowable temperature as possible.

The large dependence of drying rate on air temperature was caused by two factors. First, higher air temperature resulted in a higher ice core temperature which in turn caused a higher saturated vapor pressure at the ice-vapor interface. The higher vapor pressure at the interface represented an increase in the mass transfer potential and caused more rapid vapor transport across the porous zone. The second and minor cause was an increase in the vapor diffusivity due to increase in temperature.

The effect of reducing system pressure on the rate of freeze drying in 1 cm cubes of precooked beef is shown in Figure 6 . Little discussion is required concerning these results since it has been previously established that the maximum rate of freeze drying occurs in the range of 8—25 Hg, far below atmospheric conditions. The justification of atmospheric freeze drying centers about elimination of equipment related to providing and maintaining a vacuum condition. However, process design for atmospheric freeze drying should take full advantage of the increased drying rate due to reduced system pressure by arranging equipment to minimize the total pressure in the drying chamber.

The effect of sample size on the rate of atmospheric freeze drying in beef cubes at the standard_conditions listed above is shown in Figure 7. Time to dry to M = 0.1 decreases rapidly with decrease ir. the dimension of the cubical samples. For freeze drying with a very large surface mass transfer coefficient (so that the surface vapor concentration approximates the free-stream vapor concentration) the time to dry to any given dimensionless moisture content should vary with the ratio of the square of the sample size. Results shown in Figure 7 were computed with h^ = 0.0095 g/cm2 -sec-atm which was not large enough to fulfill the above criterion. Nevertheless sample size was shown to greatly affect the drying rate.

Effect of the surface mass transfer coefficient on the rate of atmospheric freeze drying is best illustrated by definition of a

Fig. 6 —Predicted effect o f system pressure on atmospheric freeze drying rate in 1-cm cubes o fprecooked beef.

1 5 4 - J O U R N A L OF F O O D S C I E N C E -V o lu m e 3 9 (1 9 7 4 )

ratio of external to internal mass transfer coefficients analogous to the heat transfer Biot number,

H = hDs/De = h DsRTP/C2 DMw (14)

The drying time and, therefore, the drying rate are independent of when it is greater than approximately 100. For values of H greater than 100 the rate of mass transfer is effectively controlled by the internal mass transfer mechanism.

Economic analysis of the atmospheric freeze-drying process is beyond the scope of this research; however, some observations can be made from the results presented concerning its practical usefulness. Ooviously the rate of atmospheric freeze drying is slow even in relatively small samples. The most promising area of the operating variable space is where hD is high and sample size is small. This vicinity was of interest to Malecki et al. (1969) but other problems concerning fluidization of frozen particles in the fluidized bed hampered the investigation. Perhaps other configurations of equipment which could investigate this domain of the operating variable space would meet with more success.

Economic viability of the process must be based on low capital investment for equipment and a continuous process. In both of these areas great improvement is possible over conventional freeze drying

CONCLUSIONS

THE MOISTURE content history of a sample during atmospheric freeze drying can be described by a mathematical model of simultaneous heat and mass transfer.

Nonlinear estimation can be used to evaluate appropriate heat and mass transfer parameters for atmospheric freeze drying of beef.

Transport of heat in beef during atmospheric freeze drying is primarily by conduction through the matrix of the products solids.

Reduction of particle size and an increase in surface mass transfer coefficient appear to be the most effective ways to increase rate of atmospheric freeze drying.

The effect of particle size and surface mass transfer coefficient on atmospheric freeze-drying rate can be described most effectively through the use of a dimensionless number which describes the ratio of external to internal mass transfer in the sample.

NOMENCLATURE

C 2 C o n s t a n t , d i m e n s i o n l e s s

C p c S p e c i f i c h e a t o f i c e , c o n s t a n t p r e s s u r e , c a l o r i e / g - ° C

C p(i S p e c i f i c h e a t o f d r y p r o d u c t , c o n s t a n t p r e s s u r e , c a l o r i e / g - ° C

C p w S p e c i f i c h e a t o f w a t e r , c o n s t a n t p r e s s u r e , c a l o r i e / g - ° C

D M u t u a l f r e e - g a s d i f f u s i v i t y , a i r a n d w a t e r v a p o r , c m 2 / s e c

D D P , c m 2 - a t m / s e c

D e E f f e c t i v e w a t e r v a p o r t r a n s f e r c o e f f i c i e n t in t h e p o r o u s z o n e , d e f i n e d b y e q u a t i o n ( 1 2 ) , g / s e c - c m - a t m

e P o r o s i t y o f t h e p o r o u s z o n e , d i m e n s i o n l e s s

f P o s i t i o n o f i c e - v a p o r i n t e r f a c e , d i m e n s i o n l e s s

F ( . ) F u n c t i o n o f

H D e f i n e d b y e q u a t i o n ( 1 3 )

h S u r f a c e h e a t t r a n s f e r c o e f f i c i e n t , c a l o r i e / c m 2 - s e c - ° C

h D S u r f a c e m a s s t r a n s f e r c o e f f i c i e n t , g / c m 2 - s e c - a t m

k E f f e c t i v e t h e r m a l c o n d u c t i v i t y , p o r o u s z o n e , c a I / c m - s e c - ° C

k c E f f e c t i v e t h e r m a l c o n d u c t i v i t y , f r o z e n c o r e , c a l / c m - s e c - ° C

M M o i s t u r e c o n t e n t , d r y b a s i s

M D i m e n s i o n l e s s m e a n m o i s t u r e c o n t e n t

M a M o l e c u l a r w e i g h t , a i r , g / g - m o l e

M w M o l e c u l a r w e i g h t , w a t e r , g / g - m o l e

M f A d s o r b e d m o i s t u r e c o n t e n t in e q u i l i b r i u m w i t h s a t u r a t e d w a t e r v a p o r , d r y b a s i s

M 0 I n i t i a l m o i s t u r e c o n t e n t , d r y b a s i s

rh M as s f l u x r a t e , g / c m 2 -sec

P T o t a l p r e s s u r e , a t m o s p h e r e

P a P a r t i a l p r e s s u r e o f a i r , a t m o s p h e r e

F:g. 7—Predicted effect o f sample size on atmospheric freeze drying rate in cubes o f precookedbeef.


P s a t S a t u r a t e d v a p o r p r e s s u r e , a t m o s p h e r e

P v P a r t i a l p r e s s u r e o f w a t e r v a p o r , a t m o s p h e r e

P va P a r t i a l p r e s s u r e o f w a t e r v a p o r in t h e a i r s t r e a m

R U n iv e r s a l g a s c o n s t a n t , c r r .3 - a t m / m o l e - ° C

r R e l a t i v e h u m i d i t y , d i m e n s i o n l e s s

s H a l f - t h i c k n e s s o f t h e s a m p l e , c m

T T e m p e r a t u r e i n t h e p o r o u s z o n e , ° C

T a T e m p e r a t u r e i n t h e a i r s t r e a m , ° C

T c T e m p e r a t u r e o f t h e f r o z e n c o r e , ° C

t T i m e , s ec

x D i s t a n c e , c m

A H S H e a t o f s u b l i m a t i o n , c a l / g

A H V H e a t o f v a p o r i z a t i o n , c a l / g

0 D t / s 2 , d i m e n s i o n l e s s t i m e

p B u l k d e n s i t y o f p o r o u s z o n e , g / c m 3

0 x / s , d i m e n s i o n l e s s d i s t a n c e

REFERENCES

Crank, J. and Nicholson, P. 1947. A Practical Method for Numerical Evaluation of Solutions of Partial Differential Equations of the Heat Conduction Type. Proceedings of Cambridge Phil. Soc. 43(17): 50-67.

Dunoyer, J.M. and Larousse, J. 1961. Experience Nouvelles sur la Lyophilisation. Trans, of Eighth Vacuum Symposium and Second International Congress 2: 1059.

Dyer, D.F. and Sunderland, J.E. 1968. Transfer mechanisms in sublimation dehydration. J. Heat Transfer, ASME Trans. 90C: 379.

Gunn, R.D. 1967. Mass transport in porous media as applied to freeze drying. Ph.D. dissertation, University of California, Berkeley.

Gunn, R.D. and King, C.J. 1969. Mass transport in porous materials under combined gradients of composition and pressure. A.I.Ch.E. Journal. 15(4): 507.

Harper, J.C. 1962. Transport properties of gases in porous media at reduced pressures with reference to freeze drying. A.I.Ch.E. Journal. 8(3): 298.

Hohner, G.A. 1970. An analysis of heat and mass transfer in atmospheric freeze drying. Ph.D. dissertation, Michigan State University, East Lansing.

Krischer, O. 195S. Die Wissenschaftlichen Grundlagen der Trockung- stecknik. Springer, Berlin.

Lentz, C.P. 1961. Thermal conductivity of meats, fats, gelatin gels and ice. Food Technol. 15: 243.

Lewin, L.M. and Mateles, R.F. 1962. Freeze drying w ithout vacuum, a preliminary investigation. Food Technol. 16(1): 94.

Malecki, G.J., Shinde, P., Morgan, A.I. Jr. and Farkas, D.F. 1969. Atmospheric fluidized-bed freeze drying of apple juice and other liquid foods. Food Technol. 24(5): 93.

Mery man, H.T. 1959. Sublimation freeze drying w ithout vacuum. Science 130: 628.

Perry, J.H. 1967. “ Chemical Engineers’ H andbook,” 3rd ed. McGraw- Hill Book Co., Inc., New York.

Riedel, L. 1957. Kalorimetrische Untersuchen über das Gefrieren von Fleisch. Kältetechnik 9(2): 38.

Sandall, O.C., King, C.J. and Wilke, C.R. 1967. The relationship between transport properties and rates o f freeze drying of poultry meat. A.I.Ch.E. Journal. 13(3): 428.

Short, E.B. and Staph, H.E. 1951. The energy content of foods. Ice & Refrig. 121(5): 23.

Smith, G.D. 1965. “ Numerical Solution of Partial D ifferential Equations.” Oxford University Press, New York & London.

Snedecor, G.W. 1956. “ Statistical M ethods.” Iowa State University Press, Ames, Iowa.

Threlkeld, J.L. 1932. “ Thermal Environmental Engineering.” Prentice- Hall, Inc., Englewood Cliffs, N.J.

Woodward, H.T. 1961. Study of vapor removal systems in dehydration of food products having piece of block conform ation. QM Contract Report (DA 19-129-QM-l 597).

Woodard, H.T. 1963. Freeze drying w ithout vacuum. Food Engr. 35(6): 96.

Ms received 5/4 /73; revised 8 /17/73; accepted 8/20/73.Journal Article No. 6359 of the Michigan Agriculture Experim ent

Station.Presented a t the International Symposium on Heat & Mass Trans

fer Problems in Food Engineering, Oct. 27, 1972, Wageningen, TheNetherlands.

M. PELEGInstituto Venezolano de Investigaciones Tecnológicas e Industriales

IN V E S T I, Apartado 7668, Carmelitas, Caracas, Venezuela

DETERMINATION OF FRESH PAPAYA'S TEXTURE BY PENETRATION TESTS

IN T R O D U C T IO N

A L A R G E N U M B E R c f va r ie ties o f p a p a ya f ru i t a re g ro w n in m a n y t ro p ic a l c o u n t r ies; h o w e v e r , d u e to the f r u i t ’s h igh ra te o f d e te r io r a t io n , i t is c o n s u m e d m ain ly as a f resh f ru i t in c o u n t r i e s o f i ts origin. In r e c e n t y e a rs , h o w e v e r , large-scale e x p o r t o p e ra t io n s have been car r ied o u t m ain ly f ro m H aw aii to th e U.S. m a in lan d . As the use o f p o s t-h a rv es t te c h n iq u e s a n d air t r a n s p o r t is sp read in g , i t m ig h t be assu m e d t h a t th is t ro p ic a l f ru i t will be m o re available in N o r t h A m e r ic an a n d E u r o pean m a rk e ts . I t m ig h t be also e x p e c te d th a t the c o n s u m p t i o n o f p ro cessed p a p a ya p r o d u c t s su c h as p u rees a n d th e i r der iva tives will also e x p a n d in the fu tu re .

O n e o f t h e basic ch a ra c te r i s t ic s o f p a p a y a f ru i t q u a l i ty is i ts t e x tu r e . M a tu r i ty o f p a p a y a is a c c o m p a n ie d b y the so f te n in g o f th e flesh. T h e degree o f s o f t ness o b v io u s ly c o n t r ib u t e s t o th e o rg a n o lep t ic c h a ra c te r i s t ic s o f t h e f resh f ru i t b u t i t m ig h t also p lay a m a jo r ro le in p ro cess ing o p e ra t io n s . T h e v iscosi ty , h e a t t r a n s fer a n d u n i f o r m i t y o f th e p ro cessed p a p a ya p r o d u c t s will necessa r i ly be d e p e n d e n t u p o n th e in i t ia l t e x tu r e o f the raw fru i t a n d th e re fo re t e x t u r e s t a n d a r d s a n d class i f ica t io n w o u ld be essen tia l .

P o s t-h a rv es t changes in p a p a y a f ru i ts have b e en th o r o u g h ly in v es t ig a ted a n d su rv ey ed (U niv . o f Hawaii , 19 7 0 ; Aka- m ine a n d G o o , 196 9 , 19 7 1 ) . T e x tu r e aspec ts o f p a p a y a r ip en in g w ere s tu d i e d b y B ru k n e r a n d K in ch ( 1 9 6 8 ) . A p p ly in g f o r c e - d e f o rm a t io n devices t o w h o le in ta c t f ru i ts t h e y f o u n d c o r re la t io n s b e tw e e n w h o le f ru i t s t r e n g th , c o lo r d e v e lo p m e n t a n d f lesh t e x tu r e . A l th o u g h s o m e te x tu r e m e a su re m e n ts o f p a p a y a us ing a p e n e t r o m e t e r are r e p o r t e d ( T h o m p s o n a n d Lee, 1 9 7 1 ) , th e re is l i t t le d e ta i led i n f o r m a t io n on th e m e c h a n i sm o f th e tes ts them se lves . T h e m a n y m e th o d s o f t e x tu re d e te r m i n a t i o n o f f ru i ts have b e e n th o r o u g h ly d iscussed in t h e l i t e ra tu re (F in n e y , 19 6 9 ) . O ne o f the m o s t s im ple and c o n v e n ie n t w ays o f f ru i t t e x tu r e d e te r m in a t io n is the use o f th e h a n d y “ F r u i t Pressure T e s te r ” de sc r ib e d by Magness a n d T a y lo r ( 1 9 2 5 ) . T h e physical a sp ec ts o f th is m e t h o d were s tu d ie d by several w o rk e r s , ( B o u rn e e t al., 1966 ; B o u rn e , 1 9 6 5 ) , b u t th e a p p l i c a t io n s re

p o r t e d did n o t in c lu d e the p a p a y a fru i t . In th is w o r k w e have e v a lu a te d th e p e n e t r a t io n t e s t as a t e c h n iq u e fo r ev a lu a t in g the t e x t u r e o f f resh p a p a y a fru i t .

E X PE R IM E N T A L

MaterialsLocal Venezuelan papaya fruits (also called

lechosa) of the elongated hermaphrodite type were collected in commercial plantations. Fruit

size was between 3 -6 kg and their initial maturity state when yellow bands were apparent on the peel. Fruits were washed and hot-water treated for 20 min at 49° C as described by Akamine (1969). After treatment, fruits were allowed to ripen at room temperature varying in the range of 20-24°C . Fruits were selected for analysis in various degrees of ripeness. Tests were performed on 4 -5 cm thick slices freshly cut from the fruit (Fig. 1). When the effect of peel was studied, whole intact fruits were tested.

Table 1—Penetration of papaya using different plungers Coefficient

Penetration Plungers Yield force of Texture Yield point TypeTSS speed diam avg variance strength3 distance of

(“ Brixl (cm/min) (in.) (g) (%» (g/cm2) (mm) curve

8.4 20 3/16 83 27 466 2.0 ST5/16 246 16 497 2.5 ST7/16 527 18 543 2.9 FL9/16 908 26 566 3.0 FL

11/16 1516 16 633 3.6 FL(541)

100 3/16 140 19 787 2.0 ST5/16 336 19 679 2.0 ST7/16 676 27 697 2.0 FL9/16 1058 25 660 3.0 FL

11/16 1778 17 742 4.5 FL(712)

8.0 20 3/16 126 18 798 2.3 ST5/16 316 19 638 2.7 ST7/16 633 11 653 2.9 FL9/16 980 22 611 3.4 FL

11/16 1360 16 568 3.5 FL(636)

100 3/16 153 19 859 1.3 ST5/16 380 21 768 1.0 ST7/16 844 26 870 1.5 FL9/16 1398 12 872 2.4 FL

11/16 1668 12 696 2.3 FL(813)

Not 20 3/16 3275 7.5 18400 3.8 FLripe 5/16 8485 7.3 17140 7.0 FL"yehow" 7/16 16530 11 17040 8.4 FLzone 9/16 24500 7.4 15280

(16965)9.9 FL

Not 20 3/16 4275 2.5 24020 7.0 FLripe 5/16 12400 14 25050 7.0 FL"white" 7/16 21500 0 22160 9.5 FLzone 9/16 30660 7.3 19310

(22590)10.7 FL

a Numbers in parentheses represent average results of sets of tests at the same penetrationspeed.

1 5 6-JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

FRESH PAPAYA TEXTURE DETERMINATION- 1 5 7

Test procedureAn Instron Universal Testing Machine Model

TM was used in a compression arrangement. Cylindrical metal plungers were mounted on the moving arm of the machine (Fig. 1). The plungers employed had diameters of 3/16, 5/16, 7/16, 9/16 and 11/16 in. Each was about 10 cm long and the penetrating end had a spherical shape similar to the Fruit Pressure Tester described by Magness and Taylor (1925). Fruits were penetrated at rates of

20, 50 and 100 cm/min to a distance o f at least 15 mm and the plungers withdrawn by an automatic command at the end o f each test. Force- distance curves were plotted directly by the recorder of the Instron Machine. Between three and eight penetrations were performed for each set of tests. The lower number represents generally the two bigger plungers for which the available fruit surface was technically limited. In the case of definitely unripe fruits, where two colored zones could be easily distinguished,

measurements were separately treated according to their location. Any two measurements taken were at least 15 mm apart to avoid the influence of the former test. No measurements were taken at a distance closer to about 10 mm of either the peel or the internal space of the fruit. Total soluble solids content (TSS) was determined in each fruit tested. Peeled slices were disintegrated in a domestic Osterizer and the TSS o f the puree was determined by a re- fractometer. Results are given in °Brix.


T y p es o f fo rce -d is tan c e curves

T he fo rce -d is tan c e curves o b ta in e d sh o w e d a l in e a r z o n e w i th a d i s t in c t y ie ld p o in t . T h e y ie ld p o in t o c c u r r e d a t a distan c e o f 1 .0—5.3 m m in r ipe f ru i ts an d2 . 4 —10.7 m m in u n r ip e f ru i ts (T ab les 1 a n d 2). T h is d is tan ce (Fig . 2 ) r e p re s e n te d the d is tan ce w h ic h th e c ro ssh e ad m o v ed u n t i l t h e y ie ld p o in t o c c u r re d a n d i t c o n ta in e d the c o m b in e d d e f o rm a t io n s o f the so lid mass o f f lesh a r o u n d th e p e n e t r a t i o n zone a n d th e d e f o r m a t io n o f th e flesh w i th in th e p e n e t r a t e d z o n e . T h e sh a p e o f the curve b e y o n d th e y ie ld p o in t varied a cc o rd in g to th e d i a m e te r o f th e p lu n g e r e m p lo y e d (F ig . 2 ) . T h e sm a lle r p lu n g ers (i .e . , 3 / 1 6 a n d 5 /1 6 in . ) p r o d u c e d a s t e a d y o r s lo w ly d ec l in in g fo rce c o n t i n u a t io n while th e bigger p lu n g ers p r o d u c e d w ide f l u c tu a t io n s in th e r e c o r d e d fo rce . T h e l a t t e r e f fe c t m ig h t be d u e to th e p r o d u c t i o n o f c o m p a c t e d t issue mass d u r ing th e m o v e m e n t o f the p lu n g e r w h ic h was f r a c tu re d in se q u e n c e w i th excessive force .

T h e d is tan c e t o t h e y ie ld p o in t sh o w e d t h a t th e s t a n d a r d p e n e t r a t i o n l ine m a rk e d on th e Magness a n d T a y lo r a p p a r a tu s o f7 .9 m m e x c e e d e d th e a c tu a l y ie ld p o in t o f t h e p a p a y a f ru i t f lesh e spec ia l ly w h e n r ipe f ru i ts w e re e x a m in e d .

E f fec t o f p lu n g e r d ia m e te r a n d p e n e t r a t i o n sp e e d

Yie ld fo rce s o b ta in e d w i th th e d if fe r e n t p lu n g ers a t v a r io u s p e n e t r a t i o n sp eed s are given in T ab les 1 a n d 2. T h e m ag n i tu d e o f th e p e n e t r a t i o n fo rce was n o t u n i fo rm a n d th e c o e f f ic ie n ts o f va r iance ( i.e ., s t a n d a r d d e v ia t io n d iv ided by th e average fo rce ) w ere g enera l ly in t h e range o f5 - 3 0 % a n d r e a c h e d a m a x im u m value o f 38%. This e f fe c t is p r o b a b ly c au sed by th e n o n u n i f o r m t e x t u r e o f p a p a y a a n d the u n e v en so f t e n in g o f th e f ru i t . D e m o n s t r a t io n o f th is e f fe c t can be seen in Tab le 1. A c o m p le te ly u n r ip e f ru i t has tw o d if fe re n t z o n es o f d i s t in c t ly d i f f e re n t t e x tu ra l s t r e n g th . H o w ev e r , in r ipe f ru i ts th e co lo r is a p p a r e n t ly m u c h m o re u n i f o r m a n d th e re fo re i t was m u c h h a r d e r t o lo ca te th e d i f f e re n t zones .

R esu l ts in T ab les 1 a n d 2 s h o w th a t th e average y ie ld fo rce o b ta in e d was p ro p o r t io n a l t o t h e c ross-sec t iona l area o f th e p lu n g er . S u p p o r t f o r th is re la t io n sh ip is given in T ab le 3, w here m o re d a ta are

Table 2—Effect of penetration speed on yield force of papaya

TSS(°Brix)

Plungersdiam(in.)

Penetrationspeed

(cm/min)

Yield force avg(g )

Coefficientof

variance(%)

Texture strength (g/cm! )

Yield point distance

(mm)

6.6 5/16 20 506 7 1022 2.4(Completely 50 591 12 1194 2.4

unripe) 100 787 9.5 1590 3.57/16 20 890 9.5 918 2.3

50 1100 13 1134 2.5100 1442 13 1487 4.0

7.8 5/16 20 496 7.3 1002 2.750 597 6.4 1206 2.7

100 706 10 1426 2.87/16 20 917 5.8 945 2.9

50 1093 5.6 1127 3.0100 1282 6.8 1322 4.0

8.0 5/16 20 300 24 606 2.350 340 38 687 3.2

100 504 37 1018 2.87/16 20 632 10 652 2.3

50 731 39 754 2.2100 911 28 939 3.1

8.8 5/16 20 548 23 1107 2.650 650 25 1313 3.1

100 565 15 1141 3.07/16 20 998 21 1029 2.8

50 960 12 990 2.7100 1692 22 1744 5.3

11.7 5/16 20 658 24 1329 2.650 720 9.8 1455 2.5

100 793 19 1602 4.17/16 20 1274 33 1313 3.1

50 1560 34 1608 2.9100 1586 21 1634 4.4

Table 3—Ratios between yield forces obtained by penetration tests of papaya with different plungers

Diam of plungers (in.) 3/16-5/16 5/16-7/16 7/16-9/16 9/16-11/16

Ratio between perimeters length (calc) 0.600 0.714 0.777 0.818

Ratio between cross-sectional areas (calc) 0.360 0.510 0.605 0.670

Exp ratio between yield forces 0.375a 0.542a 0.659a 0.710b

Coefficient of variance <%) 19 16.5 12.3 13.5

a Results of 19 sets of tests b Results of 13 sets of tests


r e p re s e n te d . T h e e x is te n c e o f th is re la t io n s h ip suggests t h a t the m a jo r e f fe c t o f th e p lu n g e r was c o m p re s s io n o f the f ru i t tissues. T h e sl igh tly h ig h e r th a n t h e o r e t i cal re su l ts (T ab le 3) are p r o b a b ly d u e to

the m in o r c o n t r i b u t io n o f sh e a r e f fec ts o c cu r r in g a lo n g th e p e r im e te r o f the p lu n g er . T h e fac t t h a t th e d o m in a n t e f fec t was c o m p re s s io n m ig h t have ju s t i f ie d the c a lcu la t io n o f y ie ld s tresses as force

d iv ided by c ross -sec t iona l a rea , t h u s e x pressing the t e x tu r e o f p a p a y a in t e rm s o f s t r e n g th u n i ts ( g / c m 2 ) ( T h o m p s o n a n d Lee, 19 7 1 ) . P e n e t r a t i o n resu l ts fo r app les (B o u rn e , 1 9 6 6 ) s h o w e d th a t th e c o n t r i b u t io n o f sh e a r e f fe c ts was a b o u t 25% o f th e to ta l fo rce . In th e p a p a y a f ru i t , i f sh e a r h a d a c o n s id e ra b le ro le t o p lay , i t was e x p e c te d t h a t d ev ia t io n s f ro m th e th e o re t i c a l c o m p re s s io n r a t io (T a b le 3) w o u ld be g re a te r fo r the sm a l le r p lu n g ers , a n d w o u ld decrease w i th th e in c rease in p lu n g er d ia m e te r as a re su l t o f th e d e creasing r a t io b e tw e e n p e r im e te r a n d cross-sec t iona l area . T h is e f fe c t , h o w e v e r , was n o t o b se rv ed , s u p p o r t i n g th e suggest ion t h a t th e d o m in a n t com p re ss iv e fa ilu re m e c h a n i sm is a c h a ra c te r i s t i c o f the p a p a y a t e x t u r e s t ru c tu re .

T h e inc rease o f p e n e t r a t i o n sp e e d in the range o f 2 0 —100 c m /m in h a d th e t e n d e n cy t o increase th e m a g n i tu d e o f the y ie ld fo rce o b ta in e d , (T ab les 1 a n d 2). T he e x p la n a t io n o f th is can be s p e c u la te d on th e basis t h a t th e p re ssu re e x e r t e d p r o d u c ed t issue c o m p a c t i o n a n d e x t r a c t e d liquid . As th e ra te o f p e n e t r a t i o n in c reased th e re was less t im e availab le fo r the c o m p a c t io n to rea r ran g e i ts f o rm a n d fo r th e l iq u id to f lo w th r o u g h th e n e ig h b o r in g t issues th u s p r o d u c in g m o re resistan c e to the p lu n g e r ad v an ce . I t seem s advisable t h a t w h e n the p e n e t r a t i o n t e c h n iq u e is a p p l ie d as a s t a n d a r d t e x t u r e ev a lu a t io n t e s t f o r p a p a y a , th e sp e e d o f p e n e t r a t i o n sh o u ld be h e ld c o n s t a n t in o rd e r t o m in im ize d e v ia t io n s c au sed by the sp eed e f fec ts .

Yield p o i n t d is tan c e was f o u n d to have a s light t e n d e n c y to in c rease w i th b o t h the p e n e t r a t i o n sp e e d a n d the d i a m e te r o f the p lu n g er . H o w ev e r , th e changes were very sm all a n d s e e m e d to be o f m in o r s ignificance.

Texture and TSSNo. c o r re la t io n was f o u n d b e tw e e n t e x

tu re s t r e n g th an d to ta l so lu b le so l ids c o n ten t . R esu l ts in T ab les 2 a n d 4 d e m o n s t ra te t h a t f ru i ts h av ing h ig h e r TSS c o n te n t c o u ld have to u g h e r t e x t u r e th a n th a t o f f ru i ts h av ing lo w e r T SS c o n te n t . I t has also b e en o b se rv ed t h a t t e x t u r e c o l lapse o f p a p a y a f ru i ts c o u ld o c c u r p r io r to th e full d e v e lo p m e n t o f t h e TSS i .e ., at8 - 9 ° B r i x level. Th is p h e n o m e n o n suggests t h a t th e s o f te n in g r a te o f the p a p a y a f ru i t can be i n d e p e n d e n t o f th e TSS d e v e lo p m e n t ra te , p r o b a b ly due to genet ica l a n d a g r icu ltu ra l fac to rs .

Effect of the peelR esu l ts o f p e n e t r a t i o n tes ts t h r o u g h

the peel are given in T ab le 4. T h e t y p e o f fo rce -d is tan ce curve sh o w e d a c lear y ie ld p o in t a t a d is tan c e o f 4 . 6 - 7 . 8 m m fo llo w e d b y a fo rce dec rease t o a level close to th e va lues o f th e y ie ld fo rc e o f the f ru i t flesh. T h e y ie ld p o i n t d is tan c e was f o u n d to be b igger in c o m p a r i s o n to those

Fig. 1 —Assembly for penetration tests o f papaya slices.

3 0 0 0

2000

Fig. 2 —Typer o f force-distance curves obtained in penetration tests o f papaya. S T curves obtained with 3 /1 6 and 5 /1 6 in. plungers; F L —fluctuations in force during penetration with 7 /16, 9 /1 6 and 1 1 /1 6 in. plungers; WP—curves obtained when fruits were penetrated through the peel.

FRESH PAPAYA TEXTURE DETERMINATION- 1 5 9

Table 4—Effect of peel on the penetration test of papaya3

TSS(°Brix)

Fruit with peel on Lower point after yield

Yieldforce(g)

Coefficientof

variance(%)

Yield point distance

(mm)Force

(g)

Coefficientof

variance(%)

Yieldforce

withoutpeel(g)

6.6 6772 12 7.8 744 35 5917.8 2173 15 4.6 534 9.7 5978.8 2897 12 5.9 528 32 650

11.7 4608 22 5.6 694 22 7208.7 2890 19 6.1 647 19 519

a All tests were carried out using a 5/16 in. plunger at a penetrationspeed of 50 cm/min.

o b ta in e d in r ipe f ru i t slices (Fig . 2). T he bigger d is tan ce m ig h t be e x p la in e d by the a d d i t io n a l c o n t r i b u t io n o f b o t h the peel d e f o rm a t io n a n d th e w h o le f ru i t s t ru c tu re d e f o rm a t io n . T h e f ru i t s t r u c tu r e d e f o r m a t io n was a p p a r e n t ly b igger th a n b o t h the peel an d th e f ru i t f lesh d e f o rm a t io n s a n d it m ig h t be c o n s id e re d as th e m ain f a c to r w h ich a f fec ts th e r e c o r d e d y ie ld p o in t d is tance . I t seem s t h a t the m a jo r c o n t r i b u t io n to the y ie ld fo rce was th e peel s t re n g th . N o c o r re la t io n was f o u n d betw een e i t h e r the y ie ld fo rce an d th e to ta l so lub le so l ids c o n te n t o r w i th the results o b ta in e d f o r th e f ru i t flesh. T h e m e c h an ism o f s t r e n g th d e v e lo p m e n t o f the peel an d i ts re la t io n s h ip to o t h e r changes in the f ru i t co u ld n o t be c o n c lu d e d a n d n eed s f u r t h e r s tu d y .

C O N C L U SIO N S

T H E P E N E T R A T I O N T E S T fo r th e evalu a t io n o f t e x tu r e o f p a p a y a has th e a d van tages o f be in g s im ple , e n ab l in g m a n y rep l ica t io n s a n d r e q u ir in g a re la tive ly sm all p o r t io n o f the f ru i t o n ly . Being a

de s t ru c t iv e t e s t i t has an o b v io u s d isad van tage a l t h o u g h in the case o f big p a p a ya f ru i ts the r e m a in in g pa r ts can still be u t i l ized fo r process ing . R esu l ts o f this s tu d y sh o w e d t h a t th e d o m in a n t e f fec t o f the p e n e t r a t i o n te s t was o f a c o m p re ss io n n a tu r e so th a t the t e x tu r e o f the f ru i t f lesh c o u ld be e v a lu a te d in s t r e n g th u n i ts ; t h a t is, in t e rm s o f fo rce p e r u n i t area te s te d . T h e c o e f f ic ie n t o f va r iance o f the resu lts was f o u n d to be in th e range5 —30%, re ac h in g a m a x im u m o f 38%. F o rc e v a r ia t io n s w i th in th e sam e se t o f tes ts were m a in ly d u e to th e n o n u n i f o r m n a tu r e o f the t e x tu r e . Y ield p o in ts o f m a tu re an d m a tu r in g f ru i ts o c cu r re d w i th in a d is tan c e s h o r t e r th a n the 7.9 m m line m a rk e d o n th e s t a n d a r d F ru i t P ressu re T es te r . As n o h ig h er fo rces were genera lly r e c o r d e d in th is range, i t seem s th a t th e te s t p r o c e d u r e suggested by Magness a n d T a y lo r ( 1 9 2 5 ) was a p p r o p r ia te fo r th e p a p a y a f ru i t . H ow ev e r , e x p e r im e n ta l resu lts sh o w e d t h a t the sp eed o f p e n e t r a t i o n a f fe c te d th e m a g n i tu d e o f the y ie ld fo rces a n d th e re fo re a p r o c e d u re t h a t in c lu d es a c o n s t a n t sp eed o f p enerra - t io n seem s advisable.

T h e use o f d i f f e re n t d ia m e te r p lungers seem s to be i n te rc h a n g e a b le f ro m b o th a sp ec ts o f s t r e n g th m a g n i tu d e an d results d ispe rs ion . H o w ev e r , w h e n it is desired to m ea su re b o t h s t r e n g th d i s t r ib u t io n and zone lo c a t io n s w i th in the sam e f ru i t , the use o f p lu n g ers w i th sm all d iam e te rs ( 3 / 1 6 a n d 5 /1 6 in . ) is m o re advisable.

N o c o r re la t io n s w ere f o u n d b e tw e e n t e x tu r e a n d to ta l so lu b le so l ids c o n te n t or w i th peel s t r e n g th , in d ic a t in g th e c o m p lex n a tu r e o f the p a p a y a r ip en in g p ro c ess an d leaving m u c h sp ace fo r f u r th e r in ves t iga t ion .

R E F E R E N C E S

Akamine, E.K. and Goo, T. 1969. Effects of controlled atmosphere storage of fresh papayas with special reference to shelf life extension of fumigated fruits. Res. Bull. No. 144. Hawaii Agricultural Experiment Station, University of Hawaii.Akamine, E.K. and Goo, T. 1971. Relationship between surface color development and total soluble solids in papaya. Hort. Sci. 6(6): 567.Bourne, M.C. 1965. Studies on punch testing of apples. Food Technol. 19(3): 113.Bourne, M.C., Moyer, J.C. and Hand, D.B. 1966. Measurement of food texture by universal testing machine. Food Technol. 20(4): 170.Brukner, P.F. and Kinch, D.M. 1968. Force- deformation ratio as an index of papaya maturation. Trans, of the ASAE. 11: 437. Finney, E.E. Jr. 1969. Objective measurements for texture in foods. J. Texture Stud. 1: 19. Magness, J.R. and Taylor, G.F. 1925. An improved type of pressure tester for the determination of fruit maturity. Circular No. 350. U.S. Dept, of Agriculture.Thompson, A.K. and Lee, G.R. 1971. Factors affecting the storage behaviour of papaya fruit. J. Hort. Sci. 46: 511.University of Hawaii. 1970. Papayas in Hawaii. Circular No. 436. Cooperative Extension Service, University of Hawaii.Ms received 6/13/73; revised 8/21/73; accepted8/24/73.____________________This study was financed by the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICIT), (National Venezuelan Council of Scientific & Technological Research).The author expresses his thanks to Mr. Gomez Brito for his technical aid, and to Mrs. Helga and Mr. Peter Bastiansen for the fruit supply.

M. A. R A O ' and L. N. O T O Y A P A L O M IN O Faculdade de Tecnologia de Alimentos, State University o f Campiñas, Caixa Postai 1170, Campiñas, S.P., Brazil

and L. W. B E R N H A R D TInstituto de Tecnologia de Alimentos, Caixa Psotal 1170, Campiñas, S.P., Brazil

FLOW PROPERTIES OF TROPICAL FRUIT PUREES

IN T R O D U C T IO N

T H E S T U D Y o f the viscosi ty o f f ru i t purees has rece ived inc reas ing a t t e n t io n du r ing the past few years . H o ld sw o r th( 1 9 7 1 ) s u m m a r iz e d the resu lts o f a n u m ber o f in v es t ig a t io n s a n d p o in te d o u t the n o n - N e w to n ia n n a tu r e o f m o s t o f the f ru i t pu rees s tu d ied . E x c e p t fo r a re ce n t ly r e p o r t e d s tu d y ( A n o n y m o u s , 19 7 2 ) , it a p p ea rs t h a t v iscosi ty d a ta on t rop ica l f ru i t p u rees have n o t been available. T he p re sen t s t u d y was u n d e r ta k e n to fill this need . A tu b e v is c o m e te r was c h o se n fo r the s im p lic i ty o f c o n s t r u c t io n an d o p e r a t io n , an d th e re liab i l i ty o f the results . C h a rm ( 1 9 6 0 ) a n d Saravacos ( 1 9 6 8 ) e m p lo y e d tu b e v i sco m e te rs , an d th e l a t te r also d iscussed several ty p es o f v isco m e te rs used fo r s tu d y in g the f low p ro p e r t ie s o f fo o d s tu f fs . T h e f low p ro p e r t i e s o f the p u rees o f b a n a n a , m a n g e , guava a n d p a p a y a were d e te r m in e d w i th a capil la ry visc o m e te r t h a t was des igned to e l im in a te c o r re c t io n s fo r e n t r a n c e an d e x i t e ffec ts .

W ith the tu b e v i sc o m e te r the shea r stress a t th e wall T w and the p seu d o shear ra te 32Q/7tD 3 can be ca lcu la ted f ro m th e e x p e r im e n ta l variables . T he t ru e shea r ra te a t the wall can be o b ta in e d by u s i n g t h e W e is s e n b e rg -R a b in o w i tc h - M o o n e y r e la t io n sh ip , E q (1):

d V z 3 32 Q T w d (3 2 Q /7 rD 3 )

dr ) w “ 4*<íñD3) + T d ( T w )

(1)A n a l t e rn a te an d m o re p o p u la r version o f Eq (1 ) , based on a p o w e r - ty p e r e la t i o n ship b e tw e e n th e sh e a r s tress a n d the p se u d o sh e a r ra te is

d V 7(--I— ) dr v

3n ' + 1 32 Q

4 n ' ^ D 3~)(2)

T h ere are several c o n d i t io n s t h a t m u s t be m e t b e fo re the e q u a t io n s can be e m p lo y ed c o r re c t ly and these are discussed later. 1

1 Present address: Dept, of Food Science, New York State Agricultural Experiment Sta- tion, Geneva, NY 14456


ApparatusThe viscometer employed consisted essen

tially of a 7.5 liter stainless steel reservoir for the fruit puree sample and a straight horizontal tube with pressure taps. Nitrogen under controlled pressure was used to force the fruit puree through the tubes. Commercial stainless steel tubes having average diameters of 0.454 cm and 0.812 cm were used. The pressure difference AP over a fixed length L of a tube was measured with a U-tube differential manometer with mercury as the manometric fluid. This length was 63.5 cm for the small tube and 11 5.5 cm for the large tube.

The distance Le between the tube entrance and the upstream pressure tap was 74.0 cm for the small tube and 1 23.5 cm for the large tube. These distances are more than the 100 tube diameters recommended by Bogue (1959) for the realization of fully developed laminar flow. The distance Lx between the downstream pressure tap and the exit of the tube was 36.0 cm for the small tube and 38.5 cm for the large tube. The pressure tap tubes, having an internal diameter of 0.4 cm, were soldered to the capillary tubes and care was taken to eliminate protrusions that would distort the velocity profiles. The average diameter of each tube was calculated from the weight of mercury required to fill 30 cm lengths of the tubes. Both the tubes were employed in studies with the banana puree, but only the larger tube was employed for the other purees.Procedure

The first operation of filling the connecting tubes to the manometer and its legs with the fruit puree was accomplished by forcing the puree under pressure. After ensuring that the reservoir was nearly full with the sample and that no air was trapped in the capillary tube or the connecting tubes to the manometer, the lid of the reservoir was closed air-tight, the open end of the capillary tube was plugged with a stopper and the reservoir was pressurized. About 2 min later the puree was allowed to flow. After the difference in the levels of the mercury in the manometer legs became constant, it was noted; and duplicate samples of the puree were collected for known intervals of time for subsequent weighing. Densities of the purees were determined with a pycnometer for use in the calculation of the volumetric flow rate Q and the difference in pressure AP. For each puree between 7 and 14 values of AP and the corresponding values of Q were obtained. All measurements were made at the prevailing ambient temperature as indicated in Table 1. Materials

The purees were made front fruits grown in

the state of Sao Paulo. The mango puree was prepared by disintegration, followed by fiber removal by means of a screen with 0.020 in. openings, after which it was deaerated, heatec in a votator with boiling water (97.5°C) and subsequently cooled and frozen. The frozen puree was stored at -30°C for 9 months. Before use the puree was thawed at room temperature and 2,000 ppm of sodium meta bi-sulfite was added. In the preparation of the guava puree, the steps after fiber removal were not employed and after the addition of sodium nrcta bi-sulfite (2,000 ppm.) the puree was stored in plastic containers at room temperature for 9 months before the viscosity tests. The banana puree was made in essentially the same manner as the mango puree, but the deaerated product was pasteurized at 105°C and canned. The canned puree was stored for 9 months at 16°C before use. In the preparation of the papaya puree, after the disintegration step, sodium meta bi-sulfite (2,000 ppm.) was added and the puree was stored in plastic containers at room temperature for 1 month before it was employed in the viscosity experiments.


IN O R D E R T O use e i t h e r E q (1 ) o r (2 ) , the v iscosi ty o f th e f lu id m u s t be i n d e p e n d e n t o f t im e , a n d the f low c o n d i t io n s m u s t be i so th e rm a l , l a m in a r a n d s te a d y w i th n o slip a t th e wall. In a d d i t io n the shea r s tress at the wall m u s t be d e te r m in ed u n d e r c o n d i t io n s c o r re s p o n d in g to a fu l ly d ev e lo p ed v e lo c i ty p rof i le . T h e last r e q u i r e m e n t , w h ic h has b e en n e g lec ted in so m e s tu d ies , can be a c c o m plished e i th e r by m e a su r in g th e p ressure d ro p a f te r fu lly d e v e lo p ed f low has been e s tab l ished o r by su i tab ly c o r re c t in g the m ea su re d pressure d ro p . T h e l a t t e r is u sua l ly necessa ry in the case o f h igh ly viscous f lu ids . T he c o r re c t io n m e th o d s involve th e use o f several tu b es , e ac h w i th a d i f f e re n t (L /D ) r a t io ( B r o d k e y , 1967) . T h e re is n o re liable m e t h o d to c o r re c t the pressure d ro p d a ta w i th a single t u b e in w h ich b o t h an e n t r a n c e f low reg ion a n d a fu lly d ev e lo p ed f lo w reg ion are p re sen t .

T he tu b e s e m p lo y e d in the p re se n t s tu d y were des igned to m easu re th e p re s sure d r o p per u n i t len g th a f te r fu l ly dev e lo ped f low c o n d i t io n s w ere e s tab l i sh e d . An e n t r a n c e len g th L e g re a te r th a n 150 tu b e d ia m e te r s was p ro v id e d fo r each tu b e . F u r th e r , th e re were n o p ipe o r tu b e f i t t ings a t the e n t r a n c e o f the tu b e to i m pede the d e v e lo p m e n t o f the f low . It was

1 GO-JOUR NAL OF FOOD SCIENCE-Volume 39 (1974)

FLOW PROPERTIES OF TROPICAL FRUIT PUREES-ÌGÌ

assum ed t h a t th e p u rees e m p l o y e d p o sse ssed t im e . i n d e p e n d e n t rheo lo g ica l p ro p e r t ie s a n d t h a t th e i r v e lo c i ty a t the tu b e wall was zero . T h e o t h e r r e q u i r e m e n ts t h a t the f lo w c o n d i t io n s be l am inar , s te a d y a n d i so th e rm a l d u r in g a run w ere sa t is f ied . F igu re 1 is th e cap il la ry f low d iag ram fo r all the p u rees , w h e re in th e shea r s tress D A P /4 L is p l o t t e d against the p se u d o sh e a r ra te 32Q/77D3 . T he n o n - N e w to n ia n n a tu r e o f th e p u rees is c learly e v id e n t an d in p a r t icu la r all the pu rees are sh e a r- th in n in g o r p se u d o p la s t i c flu ids (s lope o f th e lines is less th a n u n i ty ) . In a d d i t i o n all th e pu rees , over the range o f sh e a r ra te s e m p lo y e d , o b e y the p o w e r - ty p e re la t io n .

DAP , , , 3 2 Q , " ’T T k ^ (3)

T he p r im es on th e c o n s t a n t s k ' a n d n ' are used to i n d ic a te t h a t t h e y are der ived f ro m a cap i l la ry f low d iag ram a n d to dist ingu ish th e m f ro m the c o n s ta n t s in the m o re p o p u la r p o w e r law

D A P d V z n(4)

T he c o n s i s t e n t re su l ts o b t a in e d w i th b o t h the tu b e s fo r th e b a n a n a pu ree is an in d i ca t io n t h a t fu l ly d e v e lo p ed f lo w c o n d i t ions w ere ach ieved in b o t h th e tu b es . D ue to th is reason o n ly the large tu b e was e m p lo y e d fo r the o t h e r purees .

T h e m a g n i tu d e s o f k a n d n fo r each p u re e are s u m m a r i z e d in T ab le 1 and b e

Table 1—Rheological parameters and other data of the fruit purees

Fruit ° Brix Temp °C rV, dynes s11

k —— — cmBanana 17.7 22.0 ± 1 0.283 123.30Guava 10.3 23.4 ± 1 0.494 43.63Mango 9.3 24.2 ± 1 0.334 23.56Papaya 7.3 26.0 ± 1 0.528 10.12

cause o f the s t ra ig h t line r e la t io n sh ip s in F igure 1, th e m a g n i tu d e s o f th e p o w e r law c o n s t a n t s can be easily c a lcu la te d , n = n ’ a n d k ’ = k [ ( 3 n + l ) / 4 n ] n . F r o m th e va lues o f n ' we n o te t h a t th e p u rees in the o rd e r o f in c reas in g p se u d o p la s t i c i ty are p a p ay a , guava, m a n g o a n d b a n a n a re sp ec tively.

T h e p ressu re d i f fe re n ce was m ea su re d w i th an a cc u ra c y o f ± 0 .2 cm o f m e rc u ry a n d th e v o lu m e t r ic f lo w ra te was m easu re d w i th an a cc u ra c y o f ±0.1 c m 3 . T he c o n s e q u e n t u n c e r t a i n t y in th e m ag n i tu d e o f th e sh e a r s tress was a b o u t ± 4 d y n e s / c m 2 a n d t h a t o f the sh e a r ra te was ± 2 .4s"1 . T h e s c a t te r in th e d a ta p o in ts fo r th e p u rees o f guava a n d m a n g o is a t t r i b u t e d to th e u n c e r ta in t ie s in th e m ag n i tu d e s o f th e sh e a r s tress an d the shear ra te . T h e v a r ia t io n o f ± 1 ° C in th e a m b ie n t t e m p e ra tu r e d u r in g th e course o f the e x p e r im e n t s w i th e a c h pu ree was n o t co n s id e re d to be a so u rc e o f s ign if ican t e rro rs . This is becau se o f th e w e a k in f lu ence o f t e m p e r a t u r e o n the v iscosi ty o f

f ru i t p u rees (Saravacos , 1970) . F o r the sam e reaso n th e p re se n t resu l ts co u ld be use fu l a t o t h e r t e m p e ra tu r e s also.

It is c o n c lu d e d t h a t t h e t u b e visc o m e t e r e m p lo y e d in th e p re sen t s tu d y p ro v ed to be s a t i s f a c to ry fo r s tu d y in g the f low p ro p e r t i e s o f t h e p u rees o f b a n an a , m an g o , guava a n d p a p a y a . T h e purees were f o u n d to be p se u d o p la s t i c n on- N e w to n ia n f lu ids a n d o b e y e d th e p o w e r law rh eo lo g ica l e q u a t io n over th e range o f sh e a r ra tes e m p lo y e d . S tu d ie s w i th a B ro o k f ie ld v i s c o m e te r on th e p u rees m ad e f ro m t ro p ic a l f ru i ts o f C en tra l A m e r ic a ( A n o n y m o u s , 1 9 7 2 ) sh o w e d th a t t h e y were a lso p se u d o p la s t i c in n a tu re . H o w ev e r , in c o n t r a s t w i th o u r f in d ings th e p u rees in in c reas in g o rd e r o f p se u d o p la s t i c i ty w e re b a n a n a , guava, m an g o a n d p a p a y a , re sp ec t iv e ly . T h e d if fe re n t re su l ts n o t e d in t h e t w o s tu d ies is a t t r i b u t e d t o the d i f f e re n t g eograph ica l origins o f t h e f ru i ts .

N O M E N C L A T U R E

D = Inside diameter of tube, cm k = Fluid consistency index, dyne sn/cm2 L = Tube length, cm n = Flow behavior index, dimensionless AP = Pressure difference, dyne/cm2 Q = Volumetric flow rate, cm3 /s r = Radial coordinateVz = Axial velocity, cm/s Superscripts

= Quantity based on capillary flow diagram

Subscriptsa = Apparent property e = At the entrancew = At the wallx = At the exitGreek letters M = Viscosity, poise Tw = Shear stress, dyne/cm2

R E F E R E N C E S

Anonymous. 1972. Informe anual 71/72. Conservación y processamiento de frutas tropicales. Instituto Centroamericano de Investigación y Tecnologia Industrial. Guatemala City, Guatemala.Bogue, D.C. 1959. Entrance effects and prediction of turbulence in non-Newtonian flow. Ind. Eng. Chem. 51: 874.Brodkey, R.S. 1967. “The Phenomena of Fluid Motions,” p. 365. Addison-Wesley, Reading, Mass.Charm, S.E. 1960. Viscometry of non-Newtonian food materials. Food Res. 25: 351. Holdsworth, S.D. 1971. Applicability of rheological models to the interpretation of flow and processing behavior of fluid food products. J. Texture Studies 2: 393.Saravacos, G.D. 1968̂ Tube viscometry of fruit purees and juices. Rood Technoi. 22. 1585. Saravacos, G.D. 1970. Effect of temperature on viscosity of fruit juices and purees. J. Food Sci. 35: 122.Ms received 6/28/7 3; revised 8/29/73; accepted 9/6/73._____________________This work was supported by the Organization of American States. Janet S. Rao helped in the preparation of the manuscript. Presented at the 33rd Annual Meeting of the Institute of Food Technologists, Miami Beach.

S H E R M A N L E O N A R D and F R A N K W IN T E R

Dept, o f Food Science & Technology, University o f California, Davis, CA 9 5 61 6

PILOT APPLICATION OF FREEZE-HEAT PEELING OF TOMATOES

INTRODUCTIONC O N T IN U O U S S Y S T E M S fo r peel ing f ru i ts a n d v egetab les have been in ex isten c e fo r m a n y d eca d es an d are in m a n y w ays re sp o n s ib le f o r the tec h n o lo g ic a l d e v e lo p m e n t a n d e c o n o m ic success o f the m o d e r n d ay f ru i t a n d v egetab le p reservat io n in d u s t ry .

T h ese m ec h an ic a l , abrasive a n d c h e m ical peel ing sy s te m s o r c o m b in a t io n s t h e r e o f invo lved th e lu x u r io u s use o f w a ter . A t th e t im e these p rocesses w ere d e v e lo p e d , w a te r was available in a b u n d a n t q u a n t i t ie s and w as te d isposa l w i t h o u t g o v e rn m e n t re g u la t io n o r c o n c e rn fo r p o l lu t io n was c o n v e n ie n t and ch eap . T o d a y w a te r su p p ly [i t is e s t im a te d t h a t U.S. in d u s t ry a lo n e c o n su m e s 32 bill ion gallons o f w a te r daily (B a rn s te a d , 1 9 7 1 )] and p o l lu t io n c o n t r o l have rese t th e p a r a m e ters f o r f ru i t an d vegetab le peeling. A d d i t io n a l ly , in th e last few years th e d e v e lo p m e n t o f a so p h i s t i c a te d te c h n o lo g y has given us m a n y n e w o p t io n s t o be c o n s id e red in f ru i t a n d v egetab le peeling.

T he largest so u rc e o f w aste m ate r ia ls is peels a n d t r im f ro m fru i ts an d vegetab les ( E n v i r o n m e n ta l P r o t e c t io n A g en cy R e p o r t , 1 9 7 1 ) to g e th e r w i th ch em ica ls a n d d e te rg e n t s d i lu te d in w a te r to aid in th e i r rem ova l. If these chem ica ls , n a m e ly so d i u m h y d r o x id e , a n d d e te rg e n t s can be e l im in a te d , i t m igh t be poss ib le to c o n s id e r salvaging peels w h ic h m ay be f u r th e r re f in e d a n d p ro c essed , o r poss ib le d e h y d ra te d , fo r o t h e r r e m a n u f a c tu r in g p u r poses.

A m o n g th e p ro cessed f ru i ts a n d vegetab les , t o m a t o e s c o m p r ise the largest t o n nage o f raw m a te r ia l , o f w h ich pee led t o m a t o e s are a s ign if ican t p a r t . C a li fo rn ia a lo n e p rocesses over 4 -1 /2 m il l ion to n s o f t o m a to e s a n n u a l ly (C a li fo rn ia C ro p R e p o r t in g Service, 1 9 7 2 ) o f w h ic h a p p r o x i m a te ly 25% are p ee led . I f the w a te r used in pee l ing can be e l im in a te d o r r e d u ce d by 95% , th e in d u s t r y co u ld save over tw o bill ion gallons o f w a te r p e r y e a r (O r lo b , 1965 ; S p ich er e t al., 1 9 6 7 ) in C a li fo rn ia a lone . B ecause o f th e large q u a n t i t ie s o f w a te r and the B .O .D . invo lved , so m e c o m m e rc ia l fa c to r ie s t o d a y are o v e r ta x in g m u n ic ip a l waste sy s te m s (N C A , 19 7 0 ) a n d are th u s faced w i th h eav y in v e s tm e n t in p r e t r e a t m e n t faci li t ies o r s h u t d o w n (P o r te r -C o lo g n e A c t a n d Pub l ic Law9 2 - 5 0 0 ) .

E arly re sea rch ers in te re s te d in s t u d y ing t o m a t o peel ing m e th o d s were c o n c e rn ed m a in ly w i th im p r o v e m e n t o f q u a l i ty a n d r e d u c t io n o f losses. C agnoni ( 1 9 5 5 ) r e p o r t e d a p rocess o f chil l ing fo r 20 o r 3 0 sec in a p r e c o o le d b r ine fo llo w e d by s u b s e q u e n t th a w in g in h o t w a te r o r h o t gas t h a t w o u ld lo o sen the sk ins fo r easy rem o v a l . His p r im a ry p u rp o se was to s t u d y the p h e n o m e n o n involved w h e r e b y th is p ro c e d u re w o u ld rem o v e th e peels. B ro w n e t al. ( 1 9 7 0 ) e x te n d e d th is re sea rc h u t i l iz in g m o d e r n ra p id c r y o genic m e t h o d s o f f reez in g n o t available to C agnon i. T h e y w ere , h o w e v e r , c o n c e rn e d m ain ly w i th the q u a l i ty e v a lu a t io n o f the final p r o d u c t . N e i th e r C ag n o n i n o r B row n et al. a t t e m p t e d to o b t a i n c o m m e rc ia l i n f o r m a t io n o n m e c h an ic a l ly h a rv es ted f ield ru n to m a to e s . F o r th e i r e x p e r im e n tal w o r k th e y used ca re fu l ly se lec ted , full-ripe t o m a t o e s a n d all re sea rch was c o n d u c te d e x p e r im e n ta l ly in c lu d in g the h a n d s t r ip p in g o f the lo o se n e d peels. T h e i r re sea rch , h o w e v e r , was i m p o r t a n t a n d in s t r u m e n ta l in s e t t in g the p a r a m e te rs fo r th is in v es t ig a t io n .

T h e ob jec t ive s o f e x p e r im e n ta l w o rk a t th e U n ivers i ty o f C a li fo rn ia (Davis) were t o dev e lo p th e o p t im u m p a ra m e te r s fo r f reeze peel ing , to d e v e lo p i n f o r m a t io n c o n c e rn in g w a te r usage a n d th e c o m p a r a tive q u a l i ty o f c o m m e rc ia l ly lye-pee led a n d f reeze-pee led to m a t o e s a n d to d e te r m in e in a c o n t in u o u s e x p e r im e n ta l m o d e l the y ie ld fa c to rs a n d peel loss as a f fe c te d by d i f f e re n t qua li t ie s o f to m a to e s .


Raw material selectionMechanically harvested commercial toma

toes were used. The principal variety was U.C. 145-7879. Small lots of other varieties were used, as indicated later in the report, for comparative purposes.

These tomatoes were classed as follows:A -G ood. Well colored whole tomatoes

larger that 1 -1 /2 in. diam and perfect for peeling. They are firm, the skin unbroken, free from scars and defects and permit only a green or yellow area centered on the stem end of the tomato no larger than a nickel.

B-G ood; less than 1 in. cracks. The description of this tomato is exactly the same as “Good” except that these tomatoes have skin breaks or cracks totaling less than 1 in. in length.

C -G ood; greater than 1 in. cracks. This classification of tomato is the same as “Good” except that these tomatoes have skin breaks or cracks totaling greater than 1 in. in length but no visible locuies.

D-Visible locuies. This classification is the same as “Good-greater than 1 in. cracks” but in addition may have been crushed to the point of misshaping the fruit and have visible seed cavities (with visible seeds).Freezing

The surface freezing of the tomatoes was effected by the use of a modified pilot scale freezant system where freezing times could be controlled using both “Freezant” (Du Pout’s trade name for food grade Freon 1 2, dichloro- difluoromethane) immersion and spray, or spray alone, for a period of from 2 to 30 sec. Heating

The temperature of the water in the scalding system was controlled with an outside heat exchanger with temperature control and a good water recirculation system. The time of scalding was positively controlled with a paddle wheel which determined the residence time of the tomatoes in the scalding water. This paddle wheel could be speeded up or slowed down to give the desired treatment.Skin cutting

A series of razor blades, extending 1/16 in., were embedded in polyurethane forming a chute which could be placed immediately after the freezer or following blanching in order to cut the tomatoes before they entered the scrubbing operation.Scrubbing

The Magnuson scrubber (Magnuson Engineers Inc.) used in this experimental work is composed of a large variable speed auger that drives the tomatoes through the scrubbing device. The scrubbing device itself is composed of a series of shafts fitted with soft rubber abrasive discs (6 in. diam). These discs can spin at variable speeds. The shafts themselves are fitted onto a cage forming a tunnel of spinning discs. As the auger drives the tomatoes through the scrubbing device, each tomato has ample opportunity to make contact with the spinning discs and thus have the skin removed and thrown against the catching shell where the skins are conveyed to the discharge opening.Canning

The scrubbing device discharged tomatoes directly onto the canning table where ail peeled tomatoes were canned directly into 2 -1 /2 cans.

In those cases where there was excessive skin or tiny flags remaining on the stem or blossom end of the tomato, they were removed either by hand or in a mechanical grape cap stemmer.

The canned tomatoes were covered with

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FREEZE-HEAT PEELING OF TOMATOES—^63

tomato juice, including 1.0% sodium chloride and 0.025% calcium chloride and sterilized for 31 min at 100°C in a rotary atmospheric cooker.

The “Freezant” residual in the tomato samples was determined by the vapor phase chromatography method proposed by Du Pont (Hoffman, 1972).


IN 1971 p re l im in a ry s tu d ie s were ru n in w h ich very sm all q u a n t i t ie s o f to m a to e s were d ip p e d b y h a n d in to “ F r e e z a n t ” and th e n im m e d ia te ly i n to very h o t (8 8 ° C ) o r bo i ling w a te r . T hese p re l im in a ry runs clearly sh o w e d t h a t re s id en ce t im e in the f reez ing s e c t io n , as well as the h e a t in g sec t io n , was crit ica l in o rd e r to e f fec t ive ly loosen th e sk ins w i th o u t f u r th e r dam age to the t o m a t o . T o o long a t im e in the “ F r e e z a n t ” re su l te d in t o o g reat a f reez ing e f fec t a n d th u s a h ig h er p e rce n ta g e o f peel ing loss. In a s im ila r fa sh ion it was fo u n d t h a t t o o long a t im e in th e sca ld ing sec t ion w o u ld cause th e su rface o f the pee led t o m a t o to s lough o f f in the peel ing o p e ra t io n . I t was f o u n d t h a t f ro m 2 —10 sec were all t h a t w ere re q u ir e d in the f reez ing sec t ion to freeze su ff ic ie n t ly t h ro u g h th e sk ins. T h e o p t im u m te m p e r a tu re re q u ire d in th e sc a ld e r to loosen the skins f ro m th e t o m a to e s was fo u n d to be 88°C . While these h a n d d ip p in g p ro c e dures lo o se n ed th e sk ins, th e y d id n o t faci l i ta te th e sk in rem ova l . W ith a very small c u t (1 in. o r less) f ro m a ra zo r blade i t was f o u n d t h a t the sk ins c o u ld be s l ipped f ro m th e t o m a t o w i th a lm o s t n o dam age to th e c h a ra c te r o r t e x tu r e o f the fru i t . W ith p re se lec ted peel ing f ru i t , n o t sh o w in g defec ts , a 2-sec dip in the “ F r e e z a n t ” a n d 5 sec in 8 8 ° C w a te r gave a peel ing loss o f less th a n 5% a n d since the t o m a to e s w ere sc ru b b e d by h a n d , a p e rfec t peel ing e ffic iency .

In 1972 the first c o n t in u o u s m o d e l , as de sc r ibed u n d e r E x p e r im e n ta l , was u t i lized w i th a s t a n d a r d 5-sec “ F r e e z a n t ” t r e a tm e n t fo l lo w ed by a c u t t in g c h u te and a h e a t in g sy s te m p ro v id in g 15 sec at 88°C . T he peels w ere th e n r e m o v e d an d s e p a ra ted by th e M a g n u sc ru b b e r rolls set at 22 (fas t varidrive se t t in g ) a n d the auger set a t 20 ( fas t varidrive se t t in g ) . T h e re sults are su m m a r iz e d in T ab le 1. A series o f varie ties w ere c o m p a r e d to U.C. 1 4 5 -7 8 7 9 , w h ic h h ad 9 0 .7 % o f th e f ru i t p e r fec t ly pee led w i th an 8 .2% peeling loss (T ab le 1). T h e figures u sed fo r U.C. 145 are a m ea n o f 104 lo ts a n d sh o w a s t a n d ard d ev ia t io n o f 3.5 in th e p e rc e n t pee led and 0.5 in the p e rc e n t peel ing loss. T he eleven var ie ties c o m p a r e d w i th U.C. 1 4 5 -7879 w ere o n e -sam p le lo ts c o m p o s e d o f 3 0 - 1 0 0 lb o f f ru i t w i th o u t rep lica tions. These sam p le s d o n o t l en d t h e m selves t o s ta tis t ica l analysis b u t d o t e n d to sh o w the d if fe rences in th e p e e lab i l i ty o f to m a to e s o f d i f fe re n t varie ties . S o m e o f

Table 1—Comparison of several varieties of preselected, firm, ripe tomatoes, peeled by liquid freezant spray and hot water

VarietyPercentpeeled

Percent peel loss

U.C. 145-78793 90.7 ± 3.5 8.2 ± 0.5105 Cal J 72.0 13.036b 99.0 8.726 95.3 9.541 98.8 11.145 82.9 4.833 97.3 14.687 79.2 9.263 95.8 IM.A.C66 96.0 N .A .C37 76.0 8.347 82.0 8.2

a Main commercial variety now mechanically harvested and canned in California 15 Varieties selected from breeder's trials. Numbers are arbitrarily assigned so as not to reflect breeder's selection. c Data not available. These varieties were marked "not peel" by the plant breeders. Peeling was not the problem in the freeze peeling system but because these tomatoes were elongated varieties, they tended to get mangled in the pilot system which was built based on round tomato character s- tics.

these varie te is , p a r t icu la r ly the e lo n g a ted type (V ar ie t ie s 63 a n d 6 6 ) , were rep o r te d ly n o n p e e l in g varie ties , as ju d g ed by the p lan t b reed e rs , because th e y did n o t lend them se lves t o success fu ll lye peel ing by c o n v e n t io n a l p ro c e d u re s . It

sh o u ld be n o t e d , h o w e v e r , t h a t all these varie ties were p ee led success fu lly w ith so m e d if fe re n ce s in th e p e rc e n t peeled a n d in th e p e r c e n t peel ing loss, an d the resu lts were c o n s id e ra b ly b e t t e r th a n fo r c o n v e n t io n a l lye pee l ing p ro c ed u re s .

T h e pee l ing e f f ic ie n c y , t h a t is th e pe rcen t o f t o m a t o e s p ee led a n d go ing d i r e c t ly t h r o u g h the p rocess i n t o the can , varied f ro m 7 2 - 9 9 % d e p e n d in g u p o n v a r i e t y . P ee l in g losses w ere f ro m4 . 8 - 1 4 . 6 % . It sh o u ld be n o t e d th a t all these n e w e r va r ie ties w ere b re d fo r m e chan ica l h a rv es t in w h ic h f i rm flesh a n d to u g h skin are a d v a n tag e o u s . Varie t ie s su c h as 105 Cal J can be p ee led in a c o n v e n t io n a l lye p e e le r w i th e x t e n d e d t im e, h igher t e m p e r a t u r e lye t r e a tm e n t an d in c reased so d iu m h y d r o x id e c o n c e n t r a t io n . W hen th is is d o n e , h o w e v e r , pee l ing losses are excessive . T h ese sam e var ie ties , as sh o w n in T ab le 1, w h e n freeze-pee led , have a 13% peeling loss, less t h a n h a lf c o n v e n t io n a l pee l ing loss e n c o u n t e r e d by in d u s t ry . As a re su l t , th e f inal grade o f th e f ree z an t -p ee led p r o d u c t m ak es “ fan c y ” r a th e r t h a n “ e x t r a s t a n d a r d ” as w o u ld be th e case w h e n lye pee l ing th e sam e to m a to e s w h e re the v igo rous a c t io n re m oves th e o u t e r layers o f h igh ly c o lo red t o m a t o flesh.

In o rd e r to c o m p a r e th e peel ing c h ar acte r is t ics o f d a m a g e d raw m ate r ia l in the f reeze-pee ling sy s te m a n d c o n v e n t io n a l lye peel ing , a series o f 3 0 - 5 0 lb lo ts were ru n on th e f o u r c lass if ica t ions l is ted u n der R aw M aterial S e lec t io n . T h e results are p re se n te d in T ab le 2 in te rm s o f com -

Table 2—Comparison of peeling loss and efficiency between conventionally lye-peeled and freezant-peeled tomatoes

Conventionally Freezantlye peeled peeledb

TomatoesLotsruna

Peelingloss

Percentpeeled

Peelingloss

Percentpeeled

A —G oo d /no damage 42 15.23 ± 1.64 95 7.8 93.1B— < 1 in. crack 9 25.41 ± 5 .36 95 8.7 93 .8C— > 1 in. crack 23 28.20 ± 5.39 95 11.8 93.5D—V is ib le locules 3 51.15 + 7.53 N .A .C 19.0 88.5

a Size of lots ranged from 30—50 lb b No replications c Data not available

Table 3—Comparison of the percent original tomato retention between lye-peeled and freezant-peeled processes (U.C. 145-7879 tomatoes)

TomatoesNo. of cans

Conventional lye peeled

Freezantpeeleda

A —G ood/no damage 258 97.1 ± 6.9 94.9 ± 1.6B— < 1 in. crack 148 90.8 ± 2.9 94 .5 ± 1.1C— > 1 in. crack 191 91 .2 ± 2.7 93 .6 ± 1.7D —V is ib le locules 19 91 .9 ± 3.0 94 .6 ± 0.6

a Lots of five cans each

ÎM-JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

Table 4—Influence of exposure time in Freezant on percent peeled and peeling loss of U.C. 145-7879 tomatoesa

Time Percent peeled Percent peel losses

5 sec 90.7 ± 3.5 8.2 ± 0.510 sec 95.4 ± 1.2 12.4 ± 0.620 sec 94.1 ± 1.2 16.1 ± 0.430 sec 97.3 ± 3.0 21.6 ± 0.5Hand dipped and hand peeled 2 sec 100.0 4.8 ± 1.0

a The data presented are on tw o lots weigh ing 3 0 —50 lb fro m the same load, using variable in-Freezant residence tim es and keeping o th e r variables o f the standard program constant. Tem perature was m a inta ined at 8 8 °C and heat d ip was fo r 1 5 sec.

Table 5—Influence of blanching water temperature on peeling losses and peeling efficiency of U.C. 145-7879 tomatoes

PercentTemperature No. of peel and trim

(°C) lotsa Percent peeled losses

77 3 93.3 i 1.9 11.3 ± 0.282 2 90.1 ± 2.5 8.7 ± 1.088 3 94 .2 ± 2.4 11.3 ± 2.3

100 3 96.2 ± 1.6 13.6 + 2.8

a Lot size varied from 30—50 lb

Table 6—Influence of scrubber variables on the peeling loss and efficiency of U.C. 145-7879 tomatoes3

Roll setting

Auger setting13

Slow Medium Fast

Slow 96 .8 (14.2) 95.3 (8.9) 88 .4 (8.0)Medium 94.1 (17.0) 90 .0 (17.4) 88.8 (8.2)Fast 93.3 (17.8) 90.1 (9.7) 92 .6 (8.7)

a All figures represent one lot each of from 30—50 lb.13 First figure represents peeling efficiency {percent of tomatoes peeled); figure in parenthesis represents percent loss in weight.

p le te peel r e m o v a l e f fe c te d a n d peel ing losses. I t can be seen th a n “ F r e e z a n t ” pee l ing loss increases f ro m 7.8% fo r the c o n t r o l sam p le t o 19% f o r th e visible loeu les w h e reas lye-pee i ing losses increase f ro m 15 .23 ± 1.6 t o 5 1 .1 5 ± 7 .53% . This is e x c e p t io n a l since visible loeu les are lost w h e n s u b m i t t e d to a c o n v e n t io n a l lye peel ing p ro c e d u re . T he v igorous lye t r e a t m e n t a n d s u b s e q u e n t th ree -s tage wash ing sy s tem d e s t ro y su c h to m a to e s c o m p le te ly, r e n d e r in g th e m useless fo r cann ing . F o r th e u n d a m a g e d f ru i t th e lye peeler has a 95% e ff ic ien cy w h e reas the “ F re e z a n t ” p ee led to m a to e s have 93 .1% y ie ld o f the f ru i t fo r c an n in g in th is co m p a ra t iv e test.

U s ing th e sy s te m d esc r ib ed in the e x p e r im e n ta l p ro c e d u re s , sam ples were d ra w n to t race the presence o r absen ce o f “ F r e e z a n t ” t h r o u g h eac h s tep o f the p rocess t o the f in ished p r o d u c t . These sam ples were d ra w n as fo l low s: (1 ) Im m e d ia te ly a f te r a 10-sec “ F r e e z a n t ” t r e a t m e n t ; (2 ) I m m e d ia t e ly a f te r 15 sec at 8 2 °C h e a t t r e a tm e n t ; (3 ) I m m e d ia te ly a f te r peel ing in th e M agnuson sc ru b b e r ; and (4 ) T h e c an n e d s te r il ized f in ished p r o d u c t .

T he “ F r e e z a n t ” residuals sh o w n b e lo w w ere d e te r m in e d by D u P o n t f ro m e x p e r i m en ta l sam p le s p rocessed in Davis, Calif.

Sample N Avg Range1 3 126 9 8 - 163 ppm2 3 39 30-50 ppm3 3 21 12-30 ppm4 25 5.56 ± 1.80 < 1 -9 ppm

Comparative evaluationC o m p ara t iv e e v a lu a t io n o f processes

o f t e n o v e r lo o k s the r e la t io n sh ip o f d ra in ed w e igh t , at e q u i l ib r iu m , to the orig inal pee led t o m a t o fill w e igh t. T ab le 3 in d ic a te s a s l igh tly h ig h er p e rc e n t r e te n t io n in lye peel ing o f go o d f ru i t th a n w i th “ F r e e z a n t ” peeling, w h e reas fo r to m a to e s hav ing less th a n 1 in. cracks, m o re t h a n 1 in. c racks a n d visible loeu les , the p e rce n t re ta in e d is h ig h er w i th “ F r e e z a n t ” p ee l ing.

T h e in f lu e n ce o f res idence t im e in the “ F r e e z a n t ” can be seen in Tab le 4. T he p r e c e n t e f f ic ien cy in peel ing in c reased f ro m 9 0 .7 ± 3.5 to 9 7 .7 ± 3 .0, as the res idence t im e in the “ F r e e z a n t ” in c reased f ro m 5 sec to 30 sec. A t the sam e t im e , h o w e v e r , the p e rc e n t peel ing loss in creases t r e m e n d o u s ly w i th th e increase in res idence t im e in the “ F r e e z a n t , ” going f ro m 8 .2 ± 0.5 to 2 1 .6 6 ± 0 .5. In each case the d a ta re p re se n te d h e re are f rom lo ts w eigh ing f ro m 30 — 50 lb each.

In o rd e r to d e te r m in e the in f lu e n ce o f b lan c h in g w a te r t e m p e ra tu r e on peel ing loss a n d peel ing e f f ic ien cy , series o f tests w ere ru n u t il iz ing U.C. 1 4 5 - 7 8 7 9 t o m a to es a n d t e m p e ra tu r e s o f 7 7 ° C th ro u g h 100°C . T ab le 5 in d ic a te s a slight increase in pee l ing e f f ic ie n c y w i th an increase in

t e m p e ra tu r e a n d a t the sam e t im e , peel loss ten d s to increase w i th the increase in the t e m p e ra tu r e o f the sca ld ing w a te r over 82°C .

D a ta i l lu s t ra t in g the in f lu e n c e o f the s c ru b b in g variables o n peel ing loss and e f f ic iency are p re se n te d in T ab le 6. A fast auger sp e e d , w h ich d e te rm in e s res idence t im e in the sc ru b b e r , in all cases red u ces the p e rce n ta g e o f peel ing losses. This seem s reaso n ab le since the f ru i t has less res idence t im e in the sc ru b b in g device. T he e f f ic ien cy o f the s c ru b b in g a c t io n , h o w e v e r , te n d s to be in c reased at the s low es t possible au g er speed .

T h e t re n d s w i th the r o ta t io n a l speed o f th e so f t r u b b e r abrasive rolls are n o t as c lear-cu t . While it w o u ld a p p e a r f ro m T ab le 6 th a t th e m o s t logical se t t in g w o u ld be a m e d iu m au g er sp eed an d a s low rp m fo r the r o t a t i o n o f the so f t abrasive discs to give 9 5 .3 % peeling e f f i

c iency a n d 8 .9% pee l ing loss, f u r th e r w o r k m u s t be d o n e to ver ify this .

Water usageA t all t im es t h r o u g h o u t these tes ts w a

ter usage was cr it ica lly e v a lu a te d . T h e w a te r used in th e s c r u b b e r h a d a tw o f o ld p u rp o se : first , to lu b r ic a te th e e q u ip m e n t ; a n d se c o n d , t o fac i l i ta te b o t h the s e p a ra t io n o f sk in f ro m th e t o m a t o and its d ischarge f ro m the sc ru b b e r . W ith the rolls se t fast ( 2 2 ) a n d the auger se t fast(2 0 ) , i t was poss ib le to o b ta in 9 0 .7 % e f f i c iency a n d a 9 1 .8 % re co v e ry o f p ee led to m a to e s fo r cann ing . T h e 10 gal o f w a te r used per to n o f f ru i t p ee led in th is f reeze-peeling sy s te m was neglig ib le c o m pared to the w a te r usage in a s t a n d a rd c o m m e rc ia l p ee le r w h ic h uses f ro m 2 , 4 0 0 —2 ,6 0 0 gal pe r t o n o f f ru i t peeled .

T h e m a r k e t value o f the pee led t o m a toes p r o d u c e d by th e “ F r e e z a n t ” peel ing

FREEZE-H EAT PEELING OF TOMATOES - 1 6 5

Table 7—Weight and wholeness of canned Freezant-peeled tomatoes compared to USDA Standards for 401 X 411 cans

Freezant-peeled tomatoesUSDA Grade A

Min requirements

Net weight 29.56 ± 0.52 26.8Drained weight Fresh tomato

20.72 ± 0.72 19.6

fill weight 21.90 ± 0.55 NoneHeadspace (gross)3 12.82 ± 2.86 20.0pHSoluble solids

4.19+ 0.05 None

(° B) 5.70 ± 0.60 NonePeel residue (in.2) 0.65 ± 0.45 0.9Wholeness 90% 80%a Headspace reported in thirty-seconds of an inch

Table 8—Peel residue (in.2 per 401 X 411 can) lye-peeled and Freezant-peeled tomatoes3

in conventionally

Tomato Conventionally Freezantclassification lye peeled peeled

Good (no damage) 0.56 ± 0.82 0.62 ± 0.51<1 in. cracks 1.71 ± 1.38 0.57 ± 0.28>1 in. cracks b 0.55 ± 0.30Visible locules b 1.08 ± 0.40a Average USDA Grade A (Fancy) tolerance for 401 X 411 can lots is

0.9 in.b No information available

sy s te m is bes t e v a lu a te d in t e r m s o f U S D A G rades . S o m e o f the m o s t i m p o r t a n t c o n s id e ra t io n s are t a b u la te d in Tab le 7 a n d 8. T h ese tab les w ere c o m p i le d f ro m a p p r o x im a te ly 38 lo ts o f c o m m e rc ia l ly ha rves ted t o m a t o e s w h ic h w ere “ Freez- a n t ” pee led an d p a ck e d in to 401 x 411 cans d u r in g S e p te m b e r , 1972 . T h e results o f th e 6 - m o n th c u to u t s ( 1 8 7 cans) are in c lu d ed (T ab les 7 a n d 8). T h e U S D A G rad e A ( F a n c y ) m in im u m r e q u i r e m e n ts are ta b u la te d w h e n a p p licab le a n d ca lcu la ted fo r 401 x 411 can lo ts .

T h e d ra in e d w e igh t an d p e rc e n t w h o le ness o f “ F r e e z a n t ” p ee led f ru i t c o m p a re favorab le w i th the r e q u i r e m e n t s o f U.S. G rad e A F a n c y sh o w n in T ab le 7.

T h e a m o u n t o f res idua l peel (T ab le 8) in “ F r e e z a n t ” p ee led t o m a t o e s is c o m p a red to the p e r c e n t re s idua l pee l in a lye-peeling sy s te m .

S y s te m advan tagesT h e d isadvan tages o f th e c o n v e n t io n a l

lye peeler , h igh peel ing losses (2 6 % ) and the use o f large v o lu m e s o f w a te r (2 ,6 0 0 g a l / to n o f f ru i t p ee led ) can be a llevia ted in t h e “ F r e e z a n t ” pee l ing sy s te m de sc r ibed in th is pap er .

T h e h o t w a te r pee l ing s y s te m , w hich p re ce d ed th e m o d e r n - d a y lye peeling

s y s te m , u se d less w a te r t h a n th e lye p ee le r b u t t e n d e d to d e s t ro y th e q u a l i ty o f th e f ru i t b e cau se o f the h igh res idence t im e in h o t w a te r ( 3 —4 m in ) . Th is h o t w a te r sy s te m also h a d p ro h ib i t iv e ly high l a b o r cos ts fo r peel r em o v a l a n d w o u ld be u n a b le to peel s o m e o f the n e w f i rm , to u g h -sk in n ed var ie ties d ev e lo p ed fo r m ech an ica l ha rves t .

“ F r e e z a n t ” p ee l ing will peel th e n e w m ec h an ic a l h a rv es te r var ie ties in sp i te o f th e to u g h sk in . T h e “ F r e e z a n t ” peel ing sy s te m also has a m u c h lo w e r peel ing loss an d , th e re fo re , te n d s to g rea t ly im p ro v e th e case y ie ld per to n o f f ru i ts p u rc h ase d .

A n o t h e r ad v an tag e o f t h e “ F r e e z a n t ” peel ing sy s te m is th e p o te n t i a l t o re co v e r sk ins fo r d e h y d r a t io n o r p rocess ing fo r f u r t h e r m a n u f a c tu r in g use. I t app ea rs th e re are c o m p a n ie s lo o k in g fo r a n a tu ra l f o r m o f ce l lu lose w h ic h can be in c o r p o r a te d i n to ce r ta in d ie t packs .

F r o m a p o l lu t io n s t a n d p o in t , the m ark e d r e d u c t io n in w a te r c o n s u m p t io n o f t h e “ F r e e z a n t ” pee l ing sy s te m gives m a n y adv an tag es over the c o n v e n t io n a l lye pee l ing s y s te m in w h ic h u p to 2 ,6 0 0 gal o f w a te r p e r t o n o f f ru i t p ee led m ay be n e e d e d as c o m p a r e d to 10 gal in the “ F r e e z a n t ” pee l ing sy s te m .

Im p ro v e d q u a l i ty , w h ic h can be re f lec ted in im p r o v e d grade y ie ld p e r to n , has a h igh p o te n t i a l w i th th e “ F r e e z a n t ” peel ing s y s te m . In c e r ta in o f th e n e w varie ties d e v e lo p ed f o r m e c h an ic a l han d lin g , the lye-pee led p r o d u c t y ie lds an e x t r a s t a n d a r d q u a l i ty in t e rm s o f U S D A co lo r scores w h e rea s t h e “ F r e e z a n t ” peeled sy s te m , w i th i ts ease o f sk in s e p a ra t io n a n d re m o v a l , re ta in s a h ig h e r p e rce n ta g e o f th e f lesh c o n ta in in g ly c o p e n e (B ro w n e t a l . , 1 9 7 0 ) on th e very su rface o f the t o m a t o a n d th u s a U S D A G ra d e A ( F a n cy ) c o lo r sco re is ach ieved .

Because o f th e gen tle a c t io n o f the “ F r e e z a n t ” p ee l ing s y s te m , a n d th e a b sence o f h o t lye a n d th e s u b s e q u e n t n e cessi ty o f a 3-stage ag i ta t in g w ash so a k s y s te m , i t b e c o m e s poss ib le t o u t i l ize n o t o n ly p e r fe c t t o m a t o e s f o r pee l ing b u t also t o m a to e s w i th less t h a n 1 in . c racks , m o re th a n 1 in. c racks a n d , i f de s i red , t o m a to e s w i th visible locu les . T h u s , w i th the m u c h w id e r h o r i z o n o f r a w m a te r ia l s e lec t io n f o r “ F r e e z a n t ’’ peel ing , i t b e c o m e s poss ible t o c o n s id e r “ F r e e z a n t ” pee l ing fo r d iced w h o le t o m a to e s , s l iced w h o le t o m a toes , w e d g ed w h o le t o m a to e s , o r sa lad p ack s o f w h o le p ee led t o m a t o e s as well as w h o le t o m a to e s .

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HIGH-LYSINE CORN FRACTIONS AND THEIR CHARACTERISTICS

INTRODUCTION

G E N E T IC IS T S H A V E f o u n d o p a q u e -2 a n d f lo u ry -2 m u t a n t genes t h a t s u b s ta n tia l ly inc rease th e to ta l lysine a n d t r y p t o p h a n c o n te n t o f c o rn e n d o s p e rm (M ertz e t al., 19 6 4 ; Ne lson e t al., 1 9 6 5 ) . High- lysine (o p a q u e -2 ) c o m c o n ta in s h igher levels o f lysine a n d t r y p to p h a n th a n o r d in a ry c o rn ( F r o s t a n d R o b in so n , 1971) . T h e g o o d q u a l i ty o f h igh-lys ine co rn p r o tein has b e e n c o n f i r m e d b y h u m a n fe e d ing e x p e r im e n t s (Bressan i , 19 6 6 ; C lark , 19 6 8 ; Y o u n g e t al., 19 7 1 ) . R e p o r te d ly the n u t r i t iv e va lue o f o p a q u e -2 c o rn p r o tein is c o m p a ra b le t o t h a t o f m o s t p r o te ins o f a n im a l orig in . High-lysine co rn o ffers an o p p o r t u n i t y t o im p ro v e th e p r o te in q u a l i ty o f d ie ts in c lu d in g o rd in a ry c o m .

O rd in a ry c o rn c o n ta in s h o r n y e n d o sp e rm , w h e rea s high-lys ine c o rn has n o n e . N o f lak ing grits w ere r e co v e red f ro m h igh-lys ine c o rn in a mil l ing s tu d y re p o r t e d by B rek k e ( 1 9 7 1 ) . High-lysine c o m derived f ro m the o p a q u e -2 m u ta n t s t ra in c u r re n t ly p r o d u c e d is m o re f lo u ry , is s o f t e r t h a n n o rm a l co rn an d has a larger germ . Poss ib ly th ese d i f fe ren ces m ig h t pose p r o b le m s in a d a p t in g to so m e fo o d uses. Th is c o n c e rn p r o m p te d us to c o m pare t h e chara c te r i s t ic s o f o r d in a ry d e n t c o rn w i th th o se o f h igh-lys ine c o rn a f te r d ry mil ling. T es ts i n c lu d e d c o lo r m e a su re m e n ts , a m y lo g ra p h c h arac te r is t ic s , s t o r age s ta b i l i ty , e x t r u s io n c o o k in g , f e rm e n t a t i o n a n d bak ing .

MATERIALS & METHODSCorn and its dry m illing

H i g h - l y s i n e ( o p a q u e - 2 ) F u n k 2 5 4 1 0 , a t h r e e - w a y c r o s s , a n d y e l l o w d e n t S c h w e n k 2 1 1 , a s in g l e c r o s s , w e r e t h e h y b r i d c o r n s s e l e c t e d . G r i t s , l o w - f a t m e a l , l o w - f a t f l o u r a n d d e g e r m e r f i n e s f r a c t i o n s w e r e p r e p a r e d a c c o r d i n g t o t h e d r y - m i l l i n g p r o c e d u r e d e s c r i b e d b y B r e k k e e t a l . ( 1 9 7 1 ) .

Chemical and physical testsA v a i l a b l e l y s i n e w a s m e a s u r e d b y a m e t h o d

b a s e d o n r e a c t i o n w i t h d i n i t r o f l u o r o b e n z e n e ( R a o e t a l . , 1 9 6 3 ) . M o i s t u r e a n a l y s e s w e r e m a d e o n a B r a b e n d e r m o i s t u r e t e s t e r ( 1 2 0 ° C , 6 0 m i n ) . O t h e r c h e m i c a l a n a l y s e s w e r e p e r f o r m e d b y s t a n d a r d p r o c e d u r e s ( A A C C , 1 9 6 2 ) . C o l o r w a s m e a s u r e d w i t h a H u n t e r M o d e l D - 2 5 c o l o r d i f f e r e n c e m e t e r . G e l a t i n i z a t i o n p r o p e r t i e s o f t h e v a r i o u s f r a c t i o n s a t 9 % s o l i d s w e r e c h a r a c t e r i z e d i n a B r a b e n d e r - V i s c o - A m y l o g r a p h . M e t h o d s f o r d e t e r m i n i n g w a t e r a b s o r p t i o n i n d e x a n d w a t e r s o l u b i l i t y i n d e x h a v e b e e n d e s c r i b e d b y A n d e r s o n e t a l . ( 1 9 6 9 ) .

Storage stabilityT h e h i g h - l y s i n e a n d d e n t c o r n f r a c t i o n s w e r e

p a c k a g e d in g la s s c o n t a i n e r s w i t h f o i l - l i n e d s c r e w c l o s u r e s . S u c h c o n t a i n e r s m a i n t a i n e d m o i s t u r e a t c o n s t a n t l e v e l s i n s a m p l e s s t o r e d a t 3 7 ° a n d 4 9 ° C a n d a t a p p r o x i m a t e l y 2 5 ° C r o o m t e m p e r a t u r e . C o n t r o l s w e r e h e l d a t - 1 8 ° C . W i t h d r a w a l s f r o m s t o r a g e w e r e m a d e a t 4 9 ° C a f t e r 0 , 2 8 a n d 5 6 d a y s ; a t 3 7 ° C a f t e r 9 0 a n d 1 8 2 d a y s ; a n d a t 2 5 ° C a f t e r 1 8 2 a n d 3 6 5 d a y s .

T h e s a m p l e s w e r e t e s t e d f o r c h a n g e s in a v a i l a b l e l y s i n e , f a t a c i d i t y a n d f l a v o r .

T h e f r a c t i o n s w e r e m a d e i n t o c o o k e d g r u e l s ( 1 0 % s o l i d s ) f o r f l a v o r e v a l u a t i o n b y a n e x p e r i

e n c e d 1 6 - m e m b e r t a s t e p a n e l d u r i n g t h e 3 6 5

d a y s o f s t o r a g e . T h e s a m e f r a c t i o n s o f b o t h

h ig h - l y s i n e a n d y e l l o w d e n t c o r n s t o r e d f o r d i f f e r e n t t i m e s a t — 1 8 ° C a n d a t t h e p a r t i c u l a r e l e v a t e d t e m p e r a t u r e w e r e t a s t e d i n e a c h t r i a l , m a k i n g f o u r s a m p l e s c o m p a r e d a t a t i m e . H ig h -

l y s i n e f l o u r w a s a l s o t e s t e d a g a i n s t d e g e r m e r

f i n e s s t o r e d a t - 1 8 ° C a n d a t e l e v a t e d t e m p e r a t u r e s . S a m p l e s w e r e r a t e d f o r f l a v o r a c c o r d i n g t o t h e 1 0 - p o i n t s c o r e s h e e t d e s c r i b e d b y B o o k -

w a i t e r e t a l . ( 1 9 7 1 a ) . A s c o r e o f 1 0 r e p r e s e n t s “ e x c e l l e n t ” ; 9 , “ v e r y g o o d ” ; 8 , “ g o o d ” ; e t c . S c o r e s o f 6 a n d a b o v e w e r e c o n s i d e r e d a c c e p t a b l e . S a m p l e s w i t h o b j e c t i o n a b l e f l a v o r s w e r e

Table 1—Analyses2 of high-lysine and yellow dent corn and their fractions

Cornvarieties

andfractions

Particle size range (U.S. std

mesh)Yield(%)

Moisture(%)

Protein13(%)

Fat(%)

Ash(%)

Crudefiber(%)

High-lysine Whole kernels 11.6 10.3 5.7 1.6 3.1Grits (1st, 2nd, 3rd break) -16+25 12 11.2 6.7 0.3 0.3 0.3

Low-fat meal -25 +50 24 11.3 7.2 0.4 0.3 0.6Low-fat flour -50 14 11.3 5.8 0.4 0.3 0.3Degermer fines -50 10 11.3 6.6 0.4 0.4 0.5

Yellow dent Whole kernels 13.0 11.6 4.8 1.5 2.2Grits (1st, 2nd, 3rd break) -16+25 35 11.2 10.6 0.6 0.3 0.3Low-fat meal -25 +50 17 10.8 8.1 1.1 0.3 0.3Low-fat flour -50 1 11.2 7.0 2.4 0.4 0.5Degermer fines -50 1 9.0 10.4 5.4 1.2 2.1

a Dry matter basis k Percent nitrogen X 6.25

Table 2—Hunter color values for high-lysine and yellow dent corn fractions

Corn fractionsL

(lightness)a

(+red, —green)b

(+yellow, —blue)

High-lysineGrits (1st, 2nd,3rd break) 85.1 1.8 25.3

Low-fat meal 86.5 0.6 23.5Low-fat flour 89.2 -0.8 20.5Degermer fines 87.9 -0.8 20.1

Yellow dentGrits (1st, 2nd,

3rd break) 72.3 9.6 34.1Low-fat meal 82.3 2.4 30.3Low-fat flour 85.6 -1.1 24.2Degermer fines 79.8 -1.0 20.9

'\6 G -J O U FINAL OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

HIGH-LYSINE CO RN CH ARACTERISTICS - 1 6 7

a s s i g n e d s c o r e s o f 5 a n d b e l o w . S t a t i s t i c a l e v a l u a t i o n s w e r e m a d e o n t h e e x p e r i m e n t a l d a t a a f t e r c o m p l e t i o n o f e a c h t a s t e p a n e l s e r ie s . A n a l y s i s o f v a r i a n c e , r e g r e s s i o n a n a l y s i s ( S n e d e c o r a n d C o c h r a n , 1 9 6 8 ) , a n d D u n c a n ’s m u l t i p l e r a n g e t e s t ( D u n c a n , 1 9 5 5 ) w e r e u s e d t o e x a m i n e r e s u l t s . S t a t i s t i c a l s i g n i f i c a n c e is a t t h e 5 % p r o b a b i l i t y - o f - e r r o r l e v e l w h e r e v e r t h e t e r m “ s i g n i f i c a n t ” a p p e a r s i n t h i s p a p e r .

P r o d u c t r e l a t e d t e s t s

T h e h i g h - l y s i n e a n d d e n t c o r n f r a c t i o n s w e r e c o m p a r e d in s e l e c t e d e x a m p l e s w h i c h r e p r e s e n t m a j o r f o o d u s e s ( S e n t i a n d S c h a e f e r , 1 9 7 2 ) . T h e f r a c t i o n s w e r e c o o k e d in a W e n g e r X -5 e x t r u d e r a c c o r d i n g t o t h e m e t h o d d e s c r i b e d b y C o n w a y a n d A n d e r s o n ( 1 9 7 3 ) . W a t e r w a s a d d e d a t t h e r a t e o f 0 . 5 —0 . 7 g a l / h r , t h r o u g h p u t w a s 6 0 I b / h r , t e m p e r a t u r e w a s 1 3 8 - 1 4 9 ° C a n d a 1 / 8- in . d i e w a s u s e d . T h e e x t r u d e d f r a c t i o n s w e r e g r o u n d t o - 6 0 m e s h in p r e p a r a t i o n f o r c h e m i c a l , p h y s i c a l a n d f e r m e n t a t i o n t e s t s .

F e r m e n t a b i l i t y c o m p a r i s o n s w e r e m a d e o n u n e x t r u d e d a n d e x t r u s i o n - p r o c e s s e d f r a c t i o n s

c o n t a i n i n g 0 .1 % a d d e d d i a s t a t i c m a l t b y A A C C m e t h o d 2 2 -1 1 ( 1 9 6 2 ) . G a s p r o d u c t i o n w a s m e a s u r e d d u r i n g a 5 - h r f e r m e n t a t i o n . T h i s t e s t w a s i n c l u d e d t o c o m p a r e h i g h - l y s i n e w i t h o r

d i n a r y d e n t c o r n a s a s u b s t r a t e f o r f e r m e n t a t i o n p r o c e s s e s . T h i s a s p e c t c o u l d b e o f i n t e r e s t t o b r e w e r s a n d d i s t i l l e r s f r o m t h e s t a n d p o i n t o f

i n a d v e r t e n t m i x i n g o f h i g h - l y s i n e w i t h o r d i n a r y c o r n , r a w m a t e r i a l a v a i l a b i l i t y w h i c h m i g h t d i c t a t e u s i n g h i g h - l y s i n e c o r n o r a p o s s i b l e n o v e l t y f e a t u r e f o r p r o m o t i o n a l p u r p o s e s .

H i g h - l y s i n e a n d d e n t m e a l f r a c t i o n s w e r e c o m p a r e d in a n o r t h e r n c o r n b r e a d f o r m u l a t i o n d e s c r i b e d b y B o o k w a l t e r e t a l . ( 1 9 7 1 b ) . H ig h -

l y s i n e a n d y e l l o w d e n t f r a c t i o n s a l s o w e r e c o m p a r e d in p a n c a k e s , a s f o l l o w s :

F o r m u l a t i o nP a n c a k e I n g r e d i e n t s (

C o r n f l o u r o r f i n e s 7 0W h e a t f l o u r ( a l l - p u r p o s e ) 7 0B a k i n g p o w d e r 6.1B a k i n g s o d a 1 .5S a l t 3 .0M a r g a r in e 1 7 .5B u t t e r m i l k 1 5 0W h o l e e g g 2 8W a te r 100

P r o c e d u r e . A d d a l l i n g r e d i e n t s t o t h e b o w l

o f a H o b a r t M o d e l G m i x e r . B l e n d 2 m i n . A d d

w a t e r . B le n d 2 m i n ( m e d i u m s p e e d f o r a l l b l e n d i n g ) . B a k e f o r 1 m i n o n e a c h s id e a t

2 0 4 ° C o n a p a n c a k e g r i d d l e .

RESULTS & DISCUSSIONCorn fractions

T h e f rac t io n s , p a r t ic le size ranges, y ields a n d a n a ly t ic a l values are sh o w n in Tab le 1. A l th o u g h th e y ie lds o f all f rac t io n s w ere lo w e r t h a n u su a l ly o b ta in e d in c o m m e rc ia l mil l ing , th e a m o u n t s were c o n s id e re d s a t i s f a c to ry b e cau se fa t c o n t e n t s w ere n o rm a l . Because y ie lds o f low- fa t f lo u r a n d d e g e rm e r fines f rac t io n s f ro m y e l lo w d e n t w ere so lo w , m ate r ia ls w ere in a d e q u a te to c o m p le te all tests . T h e s o f t e r k e rn e l o f o p a q u e -2 m ak e s the ge rm eas ie r to rem o v e w i t h o u t b re ak in g th a n in th e d e n t c o rn a n d li t t le ge rm c o n t a m in a te s th e o p a q u e -2 f r a c t io n s , a c o n d i t io n w h ic h a c c o u n ts fo r th e lo w e r fa t a n d p r o te in values. B efore m ill ing, p ro te in c o n te n t o f h igh-lys ine c o rn was 10.3%, w h e reas t h a t o f y e l lo w d e n t was 11.6%.

Table 3—Mean flavor scores of cooked gruels containing stored corn fractions3

Corn fractions

Temperature (°C) Days

0 28 56 90 182 182 365—18°C 49° C —18°C 49° C — 18°C 37° C —18°C 37° C —18°C 25° C —18°C 25° C

High-lysineGrits (1st, 2nd,

3rd break) 8.0 8.0 8.0 7.6 7.6 8.0 8.2 7.7 8.2 G 8.3 8.1 8.2 8.0Low-fat meal 7.6 7.8 7.5 7.8 B 7.8 B 8.2 D 7.9 D 8.0 H,l 7.3 H,l X 8.2 8.0 8.1 M 7.5 MXLow-fat flour 7.2 7.3 Aa 6.0 AX 6.8 C 5.9 CX 7.4 E 5.6 EX,FX 7.3 J,K 6.0 JX 7.7 L 6.8 LX 7.4 P 6.3 NX,PXDegermer fines 7.3 7.7 A 6.6 A 7.1 C 6.6 C 7.4 E 6.3 EX,F 7.3 J,K 6.1 KX 7.1 LX 6.7 LX 7.6 N 6.9 NX

Yellow dentGrits (1st, 2nd,

3rd break) 7.9 7.5 7.6 7.9 7.5 7.9 7.6 8.2 G 7.5 GX 7.9 8.1 7.8 7.8Low-fat meal 7.8 7.6 7.3 7.5 B 6.4 BX 7.8 7.4 DX 7.8 H 5.9 HX 8.2 8.1 8.1 M 7.3 MXLow-fat flour - - - - - - - - - - - 6.5 Q,PX 5.6 OX,PX

a Means with same letter designation, A,A and B,B etc. indicate fractions tasted in the same trial; "X ” denotes significantly lower (5% level) flavor score.

Table 4—Available lysine and fat acidity values of stored corn fractions

0 days56 days,

49° C182 days,

37° C182 days,

25° C365 days,

25° c

Corn fractions ALa FAb AL FA AL FA AL FA AL FA

High-lysine Grits (1st, 2nd,

3rd break) 3.5 10.6 3.1 18.6 3.5 29.5 3.2 27.3 3.4 29.7Low-fat meal 3.9 16.0 3.4 31.1 3.4 50.3 3.3 45.5 3.6 48.9Low-fat flour 3.7 18.3 3.7 35.3 3.5 42.9 3.7 65.0 3.5 56.8Degermer fines 3.8 23.6 3.6 33.2 3.2 41.6 3.5 62.4 3.7 56.7

Yellow dent Grits (1st, 2nd, 3rd break) 1.9 13.1 2.0 42.9 2.2 69.0 1.9 46.8 2.0 60.7

Low-fat meal 2.2 13.3 2.5 65.5 2.4 107.6 2.4 73.9 2.5 97.0Low-fat flour 3.1 26.6 - - - - - - 2.8 206Degermer fines 3.1 - - - - - - — -

a AL = available lysine, grams per 16g N.b FA = fat acidity, milligrams KOH per 1 OOg sample, dry basis.

1 6 8 - JO U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

A fte r mil l ing , p ro te in va lues fo r the high- lysine f r a c t io n s w ere 3 .1 —4.5 p e rce n ta g e p o in ts lo w e r , w h e rea s th o se o f the y e l lo w d e n t f r a c t io n s were 0 . 9 —4 .6 p e rcen tag e p o in ts lo w e r t h a n in th e w h o le co rn . B efo re m ill ing, fa t c o n te n t o f high-lysine was 5 .7% an d 4 .8 % fo r d e n t c o rn . F a t c o n te n t o f th e m il led h igh-lys ine f rac t io n s was 0 .3 an d 0 .4% , w h e reas t h a t o f y e l lo w d e n t was 0 .6 to 5.4%. A sh an d c ru d e f iber values w ere s im ila r fo r all f ra c t io n s e x c e p t t h a t th o se in y e l lo w d e n t d e g e rm er fines w ere th e h ighes t .

Color propertiesB y visual o b se rv a t io n , h igh-lys ine f rac

t io n s are l ig h te r an d less y e l lo w in co lo r t h a n d e n t c o rn f rac t io n s . T h is obse rv a t io n is in a g re e m e n t w i th H u n te r c o lo r m ea s u re m e n ts . T h e L ( l igh tness) , a (+ red , —g reen ) a n d b (+ y e l lo w , - b l u e ) values a p p e ar in T ab le 2. L va lues fo r high-lys ine f ra c t io n s were a b o u t 4 to 13 u n i ts h igher th a n th o se o f y e l lo w d e n t . T h e a values fo r d e n t grits a n d m eal w ere h ig h er th a n th o se o f h igh-lys ine , w h ich range suggests a re d d ish cast. T h e b values fo r d e n t f rac t ions w ere h igher th a n th o se o f high- lysine. Gri ts a n d m eal f rac t io n s w ere the m o s t d i f f e re n t ; f lo u r a n d fines, the least . Storage stability

M ean flavor scores o f c o o k e d gruels c o n ta in in g s to r e d c o rn f rac t io n s are re c o rd e d in Tab le 3. F lavor scores w i th th e sam e l e t t e r d e s ig n a t io n , A ,A and B,B, i n d ica te f r a c t io n s t h a t w ere c o m p a r e d fo r f lavor in the sam e tas te panel . T h e l e t te r “ X ” in d ic a te s a s ig n if ican tly lo w e r flavor score . A least s ign if ican t d i f fe re n ce fo r c o m p a r in g tw o m ean f lavor scores was a p p r o x im a te ly 0 .7 . T h e genera lly h ig h er f lavor scores fo r h igh-lys ine f rac t io n s th an fo r d e n t co rn f rac t io n s i n d ic a te d b e t t e r overall s ta b i l i ty a n d re f le c te d the p resence o f g re a te r a m o u n t s o f ge rm fragm en ts in o rd in a ry co rn f rac t io n s . Grits a n d m ea l f r a c t io n s o f b o t h c o rn ty p es ch an g e d the least in f lavor d u r in g s to rage , a l th o u g h grits w ere sc o red h ig h er th a n meal. In h igh-lys ine c o rn , d e g e rm er fines rece ived b e t t e r sco res t h a n did low -fa t f lour . A d e q u a te s ta b i l i ty is i n d ic a te d fo r all h igh-lys ine a n d d e n t c o rn f rac t ions .

In T ab le 4 are sh o w n the va lues fo r available lysine a n d fa t a c id i ty w h ich were d e te r m in e d d u r in g s to rage . Available lysine was su b s ta n t ia l ly h ig h er fo r high- lysine t h a n y e l lo w d e n t c o rn f rac t ions . T h e a m o u n t s o f available lysine in the va r ious y e l lo w d e n t f rac t io n s in c reased w i th d ec re ased p a r t ic le size f ro m grits to f lou r ; in h igh-lys ine c o rn f rac t io n s lysine was m o re u n i f o r m ly d i s t r ib u te d . During s to rage, th e levels o f available lysine in b o t h h igh-lys ine an d d e n t c o rn f rac t io n s r e m a in e d re la tive ly c o n s ta n t . F a t a c id i ty values w ere h ig h e r w i th sm a lle r pa r tic le size f r a c t io n s fo r b o t h d e n t a n d high- lysine c o rn . F a t a c id i ty values fo r high- lysine c o rn w ere lo w e r t h a n fo r y e l lo w

d e n t b o t h in i t ia l ly a n d a f t e r s to rage . These lo w e r fa t a c id i ty va lues w ere asso c ia ted w i th lo w e r fa t c o n te n t o f the

high-lys ine f rac t io n s a n d h ig h e r f lavor scores t h a n d e n t c o rn f r a c t io n s d u r in g s to rage.

Table 5—Amylograph characteristics of unextruded and extruder-processeda corn fractions

Corn fractions

Initialpaste

viscosity at 29° C (BUb)

Pastingtemp(°C)

Peakheight(BU)

Peaktemp(°C)

Peak height after

16 min, 95°C (BU)

Setback viscosity at 50° C

(BU)

High-lysine Grits (1st, 2nd, 3rd break) 5 66.5

Unextruded

970 86.8 590 1230Low-fat meal 5 68.0 920 88.2 550 1170Low-fat flour 5 68.0 1100 93.5 760 1350Degermer fines 0 70.2 915 94.2 630 1325

Yellow dent Grits (1st, 2nd, 3rd break) 0 71.0 295 95.0 295 710

Low-fat meal 0 68.0 675 89.0 470 970Low-fat flour 0 67.3 930 89.0 585 1230Degermer fines 0 68.0 530 84.5 335 660

High-lysine Grits (1st, 2nd, 3rd break) 175

Extruder processedb

c ( 20)c c 15 40Low-fat meal 110 c (10)c c 20 30Low-fat flour 200 c ( 20)K c 10 35Degermer fines 100 c (10)c c 10 25

Yellow dent Grits (1st, 2nd, 3rd break) 140 c (30)c c 25 60

Low-fat meal 140 c ( 20)c c 20 50Low-fat flour 140 c ( 20)c c 15 50Degermer fines 160 c (10)' c 10 25

a Wenger X-5 extruder cookiing conditions: 138 — 149°C, 0.5 -0.7 gal/hr water, 60 Ib/hrthroughput, 1/8-in. die

k BU = Brabender Visco-Amylograph unitsc No peaks during heating; values in parentheses are curve heights at 95°C.

Table 6—Gas production after 5-hr fermentation of unextruded and extruder-processeda corn fractions

Standard procedure13 Modified procedure

Corn fractions

Extruderprocessed

(mm)Unextruded

(mm)

Additionaldiastatic

malt/sample(g)

Unprecooked(mm)

High-lysine Grits (1st, 2nd, 3rd break) 500 165 0.25 460

Low-fat meal 500 135 0.25 430Low-fat flour 560 60 0.35 420Degermer fines 560 90 0.30 425

Yellow dent Grits (1st, 2nd, 3rd break) 430 460

Low-fat meal 425 430 — —Low-fat flour 430 150 0.20 440Degermer fines 490 190 0.51 430

a For extruder cooking conditions see footnote a, Table 5. k AACC Method 22-1 1 (0.1% diastatic malt).

HIGH-LYSINE C O R N CH ARACTERISTICS - 1 6 9

Table 7—Properties of extrusion-processed2 corn fractions

Before cooking After cooking

Available Expansion Availablelysine (X-die lysine

Com fractions WAIb WSIC (g/16g N) diameter) WAI WSI (g/16g N)

High-lysine Grits (1st, 2nd,

3rd break) 2.2 1.0 3.3 2.6 2.9 50.9 3.2Low-fat meal 2.2 1.3 3.4 2.0 3.6 40.3 3.0Low-fat flour 2.2 1.0 3.5 1.8 3.8 38.1 3.4Degermer fines 2.2 1.4 3.8 1.7 2.6 51.7 3.8

Yellow dent Grits 2.1 1.4 1.9 3.3 5.3 23.2 1.9Low-fat meal 2.3 1.4 2.4 3.2 5.2 27.6 2.4Low-fat flour 2.4 1.4 3.0 1.9 4.6 30.8 2.7Degermer fines 2.7 3.8 2.6 1.6 5.8 27.7 2.6

a For extruder cooking conditions see footnote a, Table 5 b WAI = water absorption index, grams gel/grams sample c WSI - water solubility index, percent solubles

A m y lo g ra p h c h arac te r i s t ic sB ra b e n d e r V isc o -A m y lo g ra p h values

are l is ted in T ab le 5 fo r h igh-lys ine and y e l lo w d e n t f ra c t io n s b o t h as-is a n d e x t r u d e r p rocessed . U n e x t r u d e d high-lys ine f rac t io n s gave h ig h e r v iscosi ty va lues at pas t ing t e m p e ra tu r e s , a f t e r 16 m in at 9 5 ° C a n d a t 5 0 °C , t h a n u n e x t r u d e d d e n t c o m f rac t io n s . D e n t grits r e q u i r e d th e h ighes t pa s t ing t e m p e r a t u r e , w h e reas high-lys ine grits re q u ire d th e lo w es t . This d e m o n s t r a te s th e e f fe c t o f h o r n y e n d o sp e rm c o n ta in e d in o r d in a r y d e n t co rn grits a n d its g re a te r res is tance to gelatin ize t h a n h igh-lys ine c o rn grits . L o w e r v iscosi ty values f o r o rd in a ry d e n t c o rn f rac t io n s w ere p r o b a b ly a s so c ia ted w i th e n d o s p e rm h a rd n ess a n d res is tance o f

h o r n y pa r t ic les t o swell fu l ly d u r in g c o o k ing. T h e e x t r u d e r -p ro c e s s e d f rac t io n s o f th e tw o co rn s h a d sim ila r va lues fo r in it ia l p as te v iscosi ty , p e a k h e ig h t a n d s e tb a c k viscosi ty . E v id e n t ly , f r a c t io n s o f b o th co rns r e ac t s im ila r ly t o th e sam e e x t r u d e r c o n d i t io n s even t h o u g h d if fe ren ces in a m y lo g ra p h c h arac te r i s t ic s w ere d e m o n s t r a te d by c o m p a ra b le f r a c t io n s t h a t h ad n o t b e en e x t r u d e d .

F e r m e n t a t i o n ch a ra c te r i s t ic sC o m p ara t iv e gas p r o d u c t i o n values fo r

e x t ru d e r -p ro c e s s e d a n d u n e x t r u d e d high- lysine a n d d e n t f r a c t io n s a f te r 5-hr fe r m e n ta t io n are sh o w n in T ab le 6. O n the basis o f th e s t a n d a r d p ro c e d u re t h a t adds 0 .1% d ia s ta t ic m al t to f rac t io n s , d i f fe r

ences in gas p r o d u c t i o n w ere fo u n d . F e r m e n ta t io n s w ere act ive in th e e x t ru d e r - p ro cessed f r a c t io n s ; 7 0 —130 m m m o re gas was p r o d u c e d w i th high-lys ine f rac t io n s t h a n w i th d e n t f rac t io n s . These d a ta suggest lesser p r e c o o k in g r e q u i r e m e n ts fo r h igh-lys ine t h a n o r d in a r y d e n t c o rn to a t t a in th e sa m e f e r m e n ta t io n ra tes , a d i f fe ren ce w h ic h m a y be a d v a n tag e o u s . Less active f e r m e n ta t io n s w ere o b se rv ed w i th f ra c t io n s n o t e x t r u d e d ; o n ly th e d e n t grits a n d m ea l f r a c t io n s w ere as act ive as c o m p a ra b le e x t ru d e r -p ro c e s s e d f rac t io n s . T he m u c h m o r e act ive f e r m e n ta t io n s p r o d u c e d w i th th e u n e x t r u d e d d e n t f rac t io n s th a n w i th t h e h igh -lys ine ones , w h e n all w ere c o m p a r e d u n d e r t h e sa m e c o n d i t io n s , p r o b a b ly re f lec ts m o re s ta r c h dam age d u r in g m il l ing o f o r d in a ry d e n tcorn . A d d i t io n a l d ia s ta t ic m a l t was re q u i r e d f o r u n e x t r u d e d h igh-lys ine a n d tw o o f th e n o r m a l c o rn f r a c t io n s as sh o w n u n d e r “ M o d if ied P r o c e d u r e ” in T ab le 6. Th is a d d e d m al t was n ecessa ry to o b ta in s im ila r gas p r o d u c t i o n fo r all f rac t io n s d u r in g th e 5-hr f e r m e n ta t io n . T he d a ta suggest t h a t e x t r u d e r -p ro c e s s e d high-lys ine c o rn w o u ld be read i ly a d a p t able as a su b s t r a te f o r f e r m e n ta t io n p ro c esses i f a n e e d ever d ev e lo p ed .

E x t r u s io n c o o k in gP ro p e r t ie s o f h igh-lys ine a n d d e n t co rn

f rac t io n s b e fo re a n d a f t e r e x t r u s io n c o o k ing are sh o w n in T ab le 7. Available lysine values o f f r a c t io n s o f th e t w o co rn s were essen t ia l ly u n c h a n g e d a f te r e x t r u s io n c o o k in g . W a te r - a b so rp t io n va lues fo r th e y e l lo w d e n t in c re a sed to a m u c h g rea te r e x t e n t a f t e r c o o k in g th a n th o se o f th e h ig h - ly s in e f rac t io n s . W ater-so lub i l i ty values fo r t h e h igh-lys ine in c re ased to a m u c h g re a te r e x t e n t t h a n th o se o f the

Table 8—Baking tests with corn fractions

Corn fraction ApplicationHandling

properties

Loaf volume

(center ht, in.) Crumb color

Grain and texture

Meanflavorscore

High-lysineLow-fat meal Corn bread Normal 3.0 99.5 Light yellcw Medium cells,

slightly open8.0

Yellow dentLow-fat meal Corn bread Slightly

thin2.8 100 Yellow Medium cells,

slightly open8.0

High-lysineLow-fat flour Pancakes Normal 1.1a 99.5 Light yellow Small cells,

fine8.1

Degermer fines Pancakes Normal 1.1a 99.0 Slightly gray yellow

Medium cells, slightly open

7.9

Yellow dentLow-fat flour Pancakes Normal 1.1a 100 Yellow Small cells,

fine8.1

Degermer fines Pancakes Normal 1.2a 98 Dull gray yellow Medium cells, slightly open

7.4

a Four pancakes stacked


d e n t f r a c t io n s . U n d e r t h e sam e c o o k in g c o n d i t i o n s , a p p a r e n t ly s t a r c h f ro m high- lys ine a p p ea rs t o ge la t in ize m o re c o m p le te ly t h a n t h a t f r o m d e n t c o rn . G re a te r e x p a n s io n was o b se rv ed in th e grits a n d m ea l f r a c t io n s o f y e l lo w d e n t t h a n th o se o f h igh-lys ine . H o r n y e n d o s p e rm , as in d e n t c o rn , m a y be r e q u i r e d t o p ro d u c e a h ig h ly p u f fe d p r o d u c t a l t h o u g h this c h a ra c te r i s t ic sh o u ld be c o n f i rm e d by f u r th e r w o rk .

B aking tes tsT h e b a k in g c h arac te r i s t ic s o f meal,

f lo u r a n d d e g e rm er fines f rac t io n s o f high-lys ine a n d o r d in a r y d e n t c o rn are c o m p i le d in T ab le 8. C o m b re ad c o n ta in ing h igh-lys ine c o m m ea i h a d s l igh tly b e t t e r h a n d l in g p ro p e r t i e s a n d lo a f v o lu m e th a n c o m b re a d c o n ta in in g o r d in a r y d e n t co rn meal . C r u m b c o lo r was a slightly l ig h te r y e l lo w in th e c o rn b re ad c o n ta in ing h igh-lys ine m eal as well as in p an ca k es m a d e w i th h igh-lys ine f lou r . G ra in a n d t e x tu r e a n d m e a n f lavor scores were a lm o s t id e n t ica l in co rn b re ad c o n ta in in g meals o f e i t h e r c o rn ty p e . G ra in a n d t e x tu re a n d m e a n f lavor sco res w ere a lm o s t id en t ica l w h e n th e f lo u r f rac t io n s o f the t w o w ere c o m p a r e d in p a n ca k es . W hen th e d e g e rm e r f ines f rac t io n s o f t h e tw o

c o rn ty p e s w ere c o m p a r e d in p an ca k es , h ig h e r f lav o r scores a n d a m o re n o rm a l y e l lo w c r u m b c o lo r re su l te d f ro m high- lysine. H igh-lysine c o rn f rac t io n s re q u ir e d n o spec ia l t r e a tm e n t t o p r o d u c e h igh ly a c c e p ta b le c o rn b re ad a n d p an cak es .

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S c r im s h a w , N . S . 1 9 7 1 . P r o t e i n v a lu e o f

C o l o m b i a n o p a q u e - 2 c o r n f o r y o u n g a d u l t

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7 / 3 1 / 7 3 ._________________________________________________

P r e s e n t e d a t t h e 3 3 r d A n n u a l M e e t i n g o f t h e

I n s t i t u t e o f F o o d T e c h n o l o g i s t s in M i a m i

B e a c h .

D r . W . F . K w o l e k , B i o m e t r i c i a n , N o r t h C e n

t r a l R e g i o n , U . S . D e p a r t m e n t o f A g r i c u l t u r e ,

s t a t i o n e d a t t h e N o r t h e r n L a b o r a t o r y , p r o v id e d

s t a t i s t i c a l e v a lu a t io n s ; M r . H . F . C o n w a y , C h e m

i c a l E n g in e e r , p r o v id e d e x t r u s i o n - c o o k e d m a t e

r ia ls ; a n d M r . A . J . P e p l i n s k i , C h e m i c a l E n g in e e r ,

a s s is t e d w i t h p r e p a r a t i o n o f t h e d r y - m i l l e d f r a c

t i o n s .

T h e m e n t i o n o f f i r m n a m e s o r t r a d e p r o d

u c t s d o e s n o t i m p l y t h a t t h e y a r e e n d o r s e d o r

r e c o m m e n d e d b y t h e D e p a r t m e n t o f A g r i c u l

t u r e o v e r o t h e r f i r m s o r s i m i l a r p r o d u c t s n o t

m e n t i o n e d .

S. MANEEPUN,' B.S. LUH and R.B. RUCKERDept, of Food Science & Technology and Dept, of Nutrition, University of California, Davis, CA 95616

AMINO ACID COMPOSITION AND BIOLOGICAL QUALITY OF LIMA BEAN PROTEIN

INTRODUCTIONT H E L E G U M E S are an i m p o r t a n t sou rce o f p lan t p ro te in s fo r h u m a n c o n s u m p t io n . T h o u g h ce r ta in v eg e tab le p ro te in s are lo w in so m e essen t ia l a m in o acids (O rr an d W att , 1 9 5 7 ) , th e y are th e m ain source o f p r o t e in i n ta k e in c e r ta in pa r ts o f the w o r ld w h e re ava i lab i li ty o f an im a l p ro te in is scarce . Bressani a n d Elias( 1 9 6 8 ) a n d D eans ( 1 9 5 8 ) have rev iew ed the l i t e ra tu re o n th e use a n d p rocess ing o f p rocessed p la n t p ro te in s as h u m a n fo o d . A v ar ie ty o f i so la t io n p ro c e d u re s a p p e a r successful.

O w en an d C h ich e s te r ( 1 9 7 1 ) i so la ted p ro te in f ro m rap eseed p resscake a n d f o u n d t h a t g r in d in g th e m eal p r io r t o e x t r a c t io n in c reased p ro te in y ie ld by 10%. Hang e t al. ( 1 9 7 0 ) s tu d ie d the so lub ili t ie s o f n i t ro g e n o u s c o n s t i t u e n t s o f m u n g beans , pea b e an s a n d red k id n e y b ean s in so lu t io n s o f d iso d iu m p h o s p h a te , t r iso d i u m c i t ra te a n d so d iu m c a rb o n a te . By in creasing the c o n c e n t r a t io n o f these salts , th e re was a g radual increase in the a m o u n t o f n i t ro g e n e x t r a c t e d f ro m the beans u n t i l the so lu b i l i ty r e ac h ed a m a x i m u m . P a n t a n d T uls ian i ( 1 9 6 9 ) su c c ee d ed in e x t r a c t in g seed m eals ( P h a s e o l u s m u n g a s ) w i th so d iu m c h lo r id e so lu t io n w h ich so lu b i l iz ed 74 t o 80% o f th e to ta l n i t ro g en . W olf a n d T a m u r a ( 1 9 6 9 ) s t u d ied th e e f fec t o f h e a t t r e a tm e n t o n so lub i l i ty o f s o y b e a n p ro te in s . W hen the p ro te in was h e a te d to 1 0 0 ° C a b o u t one- h a lf o f th e p r o te in p re c ip i t a te d , while the re m a in in g p r o te in s s e d im e n te d slowly .

B ender ( 1 9 7 2 ) a n d L ien er ( 1 9 5 8 ) have rev iew ed th e e f fe c ts o f h e a t in g to im prove the n u t r i t iv e value o f legum e p r o te in . M o d e ra te h e a t t r e a tm e n t usua l ly im p ro v es th e n u t r i t iv e va lue o f m an y legum e p ro te in s b y in a c t iv a t io n o f h ea t- labile c o m p o u n d s w h ic h are d e le te r io u s , b u t o v e r-h ea t in g o f t e n causes a decrease in t h e i r n u t r i t iv e va lue . Kose e t al. ( 1 9 4 8 ) s tu d ie d a l im a bean p r o te in f ra c t io n a n d c o n c lu d e d t h a t h e a t t r e a tm e n t co u ld i m prove its n u t r i t iv e va lue b e cau se o f the rem ova l o f heat- lab i le t ry p s in - in h ib i to r p re sen t in raw beans .

1 P r e s e n t a d d r e s s : I n s t i t u t e o f F o o d R e - s e a r c h & I n d u s t r i a l D e v e lo p m e n t , K a s e t s a r t

U n iv e r s i t y , B a n g k o k , T h a i l a n d

In th e p re se n t in v es t ig a t io n , d ry large l im a b ean s w ere used fo r p ro te in e x t r a c t io n . A m in o acid c o m p o s i t i o n a n c p r o te in e f f ic ien cy r a t io ( P E R ) o f f reeze-dr ied l im a bean p r o te in c o n c e n t r a t e (L PC ) were c o m p a re d w i th th a t o f casein and so y bean p ro te in s . T he e f fe c t o f fo r t i f ic a t io n w i th essen tia l a m in o acids on P E R o f LPC is p re sen te d .

EXPERIMENTALD r y l i m a b e a n s

1 0 0 lb o f d r y l a r g e l i m a b e a n s (P h a s e o lu s l u n a tu s L . v a r B C 6 ) g r o w n in K i n g C i t y , C a l i f . , d u r i n g t h e 1 9 7 1 s e a s o n w e r e u s e d f o r t h e p r o t e i n e x t r a c t i o n s t u d y . T h e d r y b e a n s c o r t a m e d 1 2 .1 7 % m o i s t u r e a n d 2 1 . 3 7 % p r o t e i n . S u f f i c i e n t q u a n t i t y o f t h e d r y b e a n s w a s g r o u n d p r i o r t o e x t r a c t i o n in a M I A G B r a u n s c h w e i g h a m m e r m i l l w i t h a n 8 - m e s h s ie v e .

E f f e c t o f p H a n d t e m p e r a t u r e o n p r o t e i n c o a g u l a t i o n

200g o f g r o u n d d r y l i m a b e a n p o w d e r w e r e s o a k e d w i t h 6 0 0 m l o f d i s t i l l e d w a t e r a t 2 2 ° C f o r 1 2 h r . T h e p H o f t h e s l u r r y w a s i n i t i a l l y 6 .3 . I t w a s a d j u s t e d t o p H 7 .2 b y a d d i n g 0 .1 M N a j P O , s o l u t i o n . T h e r e s u l t i n g p r o d u c t w a s m i x e d in a W a r i n g B l e n d o r f o r 1 0 m i n a n d t h e n a l l o w e d t o s t a n d f o r a n h o u r w i t h f r e q u e n t s t i r r i n g . C h e e s e c l o t h w a s u s e d t o r e m o v e t h e c o a r s e p a r t i c l e s . T h e p r o t e i n e x t r a c t w a s p a s s e d t h r o u g h a n 8 0 - m e s h s i e v e , a n d c e n t r i f u g e d a t

1 0 0 0 r p m f o r 1 5 m i n in a n I n t e r n a t i o n a l C e n t r i f u g e t o r e m o v e s t a r c h a n d r e s i d u e s . T h e s u p e r n a t a n t f r a c t i o n w a s d i v i d e d i n t o a l i q u o t s o f 2 5 - m l e a c h . T h e a l i q u o t s w e r e a d j u s t e d w i t h 0 .2 M H 3 P O , t o p H 4 . 0 a n d 5 . 0 r e s p e c t i v e l y . T h e e f f e c t o f h e a t i n g f o r 3 m i n a t v a r i o u s t e m p e r a t u r e s o n p r o t e i n y i e l d w a s d e t e r m i n e d . A f t e r c e n t r i f u g a t i o n a n d f r e e z e d r y i n g , t h e p r o

t e i n c o n t e n t a n d y i e l d o f t h e v a r i o u s p r e p a r a

t i o n s w e r e d e t e r m i n e d .

P r e p a r a t i o n o f l i m a b e a n p r o t e i n c o n c e n t r a t e ( L P C )

2 k g o f d r y l a r g e l i m a b e a n s w e r e u s e d f o r p r e p a r a t i o n o f p r o t e i n c o n c e n t r a t e . T h e e x t r a c t i o n p r o c e d u r e w a s t h e s a m e a s t h a t d e s c r i b e d a b o v e . T h e s u p e r n a t a n t f r a c t i o n , a f t e r s e p a r a t i n g t h e r e s i d u e a n d s t a r c h , w a s t r e a t e d w i t h 0 .2 M H 3 P 0 4 t o p H 5 . 0 . T h e m i x t u r e w a s h e a t e d a t 1 0 0 ° C in a s t e a m - j a c k e t e d k e t t l e f o r 1 0 m i n . T h e c o a g u l a t e d p r o t e i n s w e r e s e p a r a t e d f r o m t h e s u p e r n a t a n t b y c e n t r i f u g a t i o n . T h e c o a g u l a t e d p r o d u c t w a s a d j u s t e d w i t h 0 .1 N a O H t o p H 6 . 4 , a n d t h e n f r e e z e d r i e d i n a S t o k e s F r e e z e d r i e r , m o d e l 2 0 0 4 L a t 1 3 0 p H g f o r 4 8 h r . T h e d r i e d L P C w a s k e p t i n t i g h t l y c o v e r e d g la s s b o t t l e s .

C h e m i c a l a n a l y s i s

P r o t e i n n i t r o g e n . T h e K j e l d a h l m e t h o d w a s

u s e d f o r d e t e r m i n a t i o n o f t o t a l n i t r o g e n in t h e d r y b e a n a n d t h e f r e e z e - d r i e d L P C ( A O A C ,

1 9 7 0 ) . T h e s a m p l e s iz e s w e r e 0 . 5 - 0 . 6 g f o r L P C a n d 1 . 0 - 2 . 0 g f o r d r y b e a n p o w d e r . T h e p r o t e i n c o n t e n t w a s o b t a i n e d b y m u l t i p l y i n g p e r c e n t N b y 6 . 2 5 , s i n c e t h i s v a l u e a p p l i e s f o r m a n y p r o t e i n f r a c t i o n s f r o m b e a n s ( J o n e s , 1 9 3 1 ) , a n d a p p e a r e d t o b e a n a d e q u a t e c o n v e r s i o n f a c t o r f o r l i m a b e a n p r o t e i n b a s e d o n i t s a m i n o a c id c o m p o s i t i o n .

M o i s t u r e c o n t e n t . 2 g o f t h e g r o u n d s a m p l e w a s w e i g h e d a c c u r a t e l y i n t o a l u m i n u m d i s h e s in d u p l i c a t e . T h e s a m p l e s w e r e d r i e d a t 1 3 0 ° C in a n o v e n f o r 1 h r ( A O A C , 1 9 7 0 ) .

S t a r c h . T h e a n t h r o n e c o l o r i m e t r i c m e t h o d d e s c r i b e d b y M c C r e a d y e t a l . ( 1 9 5 0 ) w a s u s e d .

C r u d e f i b e r . T h e m e t h o d f o r c r u d e f i b e r d e s c r i b e d in A O A C ( 1 9 7 0 ) w a s u s e d .

A s h . T h e A O A C ( 1 9 7 0 ) m e t h o d f o r a s h d e t e r m i n a t i o n w a s u s e d .

F a t . T h e s o x h l e t p e t r o l e u m e t h e r e x t r a c t i o n m e t h o d ( A O A C , 1 9 7 0 ) w a s u s e d .

A m i n o a c i d a n a l y s e s . A p p r o x i m a t e l y 2 5 0 m g o f t h e g r o u n d s a m p l e w a s w e i g h e d a c c u r a t e ly i n t o a 5 0 0 m l r o u n d b o t t o m f l a s k . T h e s a m p l e w a s r e f l u x e d w i t h 2 5 0 m l 6 N H C 1 f o r 2 0 h r o n a n e l e c t r i c a l h e a t e r , c o o l e d a n d f i l t e r e d t h r o u g h a s i n t e r e d g la s s f i l t e r . T h e f i l t r a t e w a s e v a p o r a t e d t o a l m o s t d r y n e s s in a f l a s h e v a p o r a t o r . T h e r e s i d u e w a s d i s s o l v e d in 5 0 m l v o l u m e t r i c f l a s k a n d d i l u t e d t o v o l u m e w i t h d i s t i l l e d w a t e r . T h e T e c h n i c o n a m i n o a c i d a n a l y z e r w a s u s e d f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f a m i n o a c i d s ( H a m i l t o n , 1 9 6 3 ; R o a c h , 1 9 6 6 ) .

B i o l o g i c a l e v a l u a t i o n o f p r o t e i n q u a l i t y

4 0 m a l e a l b i n o r a t s a t t h e a g e o f 2 8 d a y s ( w e i g h t r a n g e 4 1 - 5 4 g ) w e r e r a n d o m i z e d i n t o

f iv e g r o u p s . E a c h g r o u p ( 8 r a t s ) w a s f e d a d i f f e r e n t a s s a y d i e t . R a t s w e r e h o u s e d i n d i v i d u a l l y i n s t a i n l e s s s t e e l c a g e s i n a n a i r - c o n d i t i o n e d r o o m a t 2 3 ° C a n d 4 5 - 5 5 % r e l a t i v e h u m i d i t y . A s s a y d i e t a n d w a t e r w e r e o f f e r e d a d l i b i t u m . T h e a n i m a l s w e r e w e i g h e d e v e r y o t h e r d a y a n d t h e i r f o o d i n t a k e w a s m e a s u r e d e v e r y 4 d a y s . T h e t o t a l t e s t p e r i o d w a s 2 1 d a y s . T h e p r o t e i n e f f i c i e n c y r a t i o ( P E R ) m e t h o d r e c o m m e n d e d b y A O A C ( 1 9 7 0 ) w a s f o l l o w e d e x c e p t t h a t g l u c o s e w a s r e p l a c e d b y c o r n s t a r c h . T h e m a t e r i a l u n d e r t e s t w a s f e d a s t h e s o l e s o u r c e o f p r o t e i n a t t h e 1 0 % l e v e l . T h e b a s a l d i e t w a s c o m p o s e d o f t h e f o l l o w i n g : ( i n % ) p r o t e i n , 1 0 ; c o r n o i l , 8 ; c e l l u l o s e , 2 ; c h o l i n e c h l o r i d e , 0 . 3 ; v i t a m i n p r e m i x , 0 . 3 ; m i n e r a l p r e m i x , 5 ; a n d e n o u g h c o r n s t a r c h t o m a k e 1 0 0 . V i t a m i n s w e r e a d d e d s o t h a t e a c h lO O g o f d i e t c o n t a i n e d : ( i n m g ) n i a c i n , 2 0 ; C a - p a n t o t h e n a t e , 1 0 ; r i b o f l a v i n , 1 .5 ; t h i a m i n e - H C l , 0 . 9 1 ; p y r i d o x i n e - H C l , 1 .5 ; f o la - c i n , 0 . 3 ; b i o t i n , 0 . 1 ; B 1 2 , 0 . 0 2 ; v i t a m i n A p a l - m i t a t e , 1 0 0 0 I . U . ; v i t a m i n D 3 , 2 0 0 I .U . ; D L - a - t o c o p h e r o l a c e t a t e , 1 0 ; m e n a d i o n e , 0 . 0 6 ; a n d b u t y l a t e d h y d r o x y l t o l u e n e , 1 0 . M in e r a l s w e r e a d d e d s o t h a t e a c h lO O g o f d i e t c o n -

Volume 3 9 (1 9 7 4 )-J O U R N A L OF FOOD S C ! E N C E - \T \

M 2 - J 0 U R N A L OF FOOD S C IE N C E -V o lu m e 3 9 (1974)

t a i n e d : ( i n g ) C a C O , , 1 .5 ; K 2 H P 0 4 , 1 . 6 2 5 ; C a H P 0 4 , 0 . 3 ; N a C l , 0 . 5 ; F e S O „ • 7 H , 0 , 0 . 1 2 5 ; M g S 0 4 , 0 . 3 2 4 ; K I , 0 . 0 1 3 ; Z n C 0 3 , 0 . 0 0 4 ; C u S 0 4 • 5 H 2 O , 0 . 0 0 1 5 ; a n d M n S 0 4 • H 2 0 , 0 . 0 1 1 5 . T h e f o l l o w i n g d i e t s c o m p r i s e d t h e f iv e

e x p e r i m e n t a l g r o u p s :G r o u p A . P r o t e i n - f r e e d i e t . S t a r c h ( 1 0 % )

w a s s u b s t i t u t e d f o r p r o t e i n in t h e b a s a l d i e t .G r o u p B. C a s e i n d i e t . C a s e i n o b t a i n e d f r o n t

N u t r i t i o n B i o c h e m i c a l C o r p . . C l e v e l a n d , O h i o , ( S u p e r i o r g r a d e - 8 7 % p r o t e i n ) w a s a d d e d a t 1 1 .5 % in t h e b a s a l d i e t .

G r o u p C . S o y b e a n d i e t . R P - 1 0 0 . S o y p r o t e i n ( 9 0 % ) f r o m P u r i n a C o m p a n y w a s a d d e d a t 11.1 % in t h e b a s a l d i e t .

G r o u p D. L i m a b e a n c o n c e n t r a t e d i e t . L i m a b e a n p r o t e i n c o n c e n t r a t e ( 5 4 . 3 1 % p r o t e i n ) w a s

a d d e d a t 1 8 .4 1 % in t h e b a s a l d i e t .

G r o u p E . S u p p l e m e n t e d l i m a b e a n c o n c e n

t r a t e d i e t . T h e b a s a l d i e t w i t h l i m a b e a n p r o t e i n c o n c e n t r a t e a s t h e p r o t e i n s o u r c e w a s f o r t i f i e d w i t h e s s e n t i a l a m i n o a c i d s b a s e d o n t h e a m i n o a c i d a n a l y s i s d a t a a n d t h e m i n i m u m a m o u n t s r e q u i r e d f o r t h e r a t ( N A S - N R C , 1 9 6 2 ) . T h e f o l l o w i n g a m i n o a c i d s w e r e a d d e d : ( % i n d i e t ) L - l y s i n e , 0 . 3 ; p h e n y l a l a n i n e . 0 . 5 ; L - m e t h i o n i n e ,

0 .5 a n d L - v a l in e , 0 . 3 .T h e f iv e d i e t s w e r e f e d s e p a r a t e l y t o e a c h

g r o u p o f r a t s f o r 21 d a y s , f r o m t h e g a i n in w e i g h t a n d f e e d c o n s u m p t i o n d a t a t h e p r o t e i n e f f i c i e n c y r a t i o ( P E R ) a n d f e e d e f f i c i e n c y r a t i o ( F E R ) w e r e c a l c u l a t e d . F o r n e t p r o t e i n u t i l i z a t i o n ( N P U ) , t h e m e t h o d r e c o m m e n d e d b y M i l l e r a n d B e n d e r ( 1 9 5 5 ) w a s u s e d . T h e m e t h o d is b a s e d o n c o n s t a n c y o f t h e N / H , 0 c o n t e n t r a t i o i n t h e r a t . T h e t o t a l N o f t h e b o d y c a n b e d e t e r m i n e d f r o m t h e w a t e r c o n t e n t . B o d y w a t e r w a s d e t e r m i n e d a f t e r k i l l i n g t h e r a t s w i t h e t h e r , o p e n i n g t h e b o d y c a v i t i e s a n d d r y i n g t h e c a r c a s s e s ( 1 0 0 ° C f o r 4 8 h r ) t o a c o n s t a n t w e i g h t i n a F r e a s o v e n , M o d e l 8 4 5 - A . T h e e q u a t i o n f o r t h e c a l c u l a t i o n is :

l o g ( 4 . 8 - y ) = 0 . 4 3 7 - 0 . 0 1 2 3 x

w h e r e x = a g e i n d a y s a n d y = N / H 2 O X 1 0 0 .B y u s i n g t h e e q u a t i o n , t h e N - c o n t e n t v a l u e

c a n b e u s e d t o c a l c u l a t e t h e n e t p r o t e i n u t i l i z a t i o n b y a p p l y i n g t h e e q u a t i o n :

N P U = (B - B k + l k ) / I

w h e r e B a n d B k a r e t h e t o t a l b o d y N o f t h e a n i m a l s o n t h e t e s t a n d n o n p r o t e i n d i e t s r e s p e c t i v e l y a n d I a n d l k a r e t h e i n t a k e o f N in t h e t w o g r o u p s .

Table 1 —Effect of pH and temperature on the yield of the protein preparation form large lima beans (Phaseolus lunatus L. var BC6 )

pH 4.0 pH 5.0

Yield dry Yield dryprotein Protein Yield of protein Protein Yield ofcurd content protein curd content protein

Temp g/100g in dry g/100g g/100g in dry g/100g°C beans curd,% beans beans curd,% beans

43.3 7.17 49.63 3.55 7.80 52.50 4.0948.9 7.67 49.81 3.82 8.05 51.38 4.1354.4 7.67 49.56 3.80 8.56 51.38 4.4060.0 10.07 49.81 5.02 8.93 52.56 4.6965.6 17.62 50.31 5.03 9.06 52.75 5.2871.1 17.87 47.63 8.51 10.44 52.25 5.4576.7 18.87 46.60 8.79 18.99 51.38 9.7482.2 18.28 48.63 8.89 20.13 51.00 10.2687.8 18.37 48.56 8.92 20.89 51.63 10.7993.3 20.25 49.06 9.93 27.81 52.57 14.62

was o b ta in e d in the t r e a tm e n t a t p H 5.0 a n d 9 3 .3 ° C fo r 3 m in . T h e p ro te in c o n te n ts in the d r ied p ro te in cu rd s var ied b e tw een 4 6 .6 % a n d 5 0 .3 1 % in th e series c o ag u la te d a t p H 4 .0 , a n d b e tw e e n 5 1 .0 a n d 5 2 .5 7 % in th e series c o ag u la te d a t pH 5.0. In o r d e r t o ge t th e h ig h e r y ie ld o f l im a b e an p ro te in , i t was n ecessa ry to ad ju s t th e p H o f the p h o s p h a te e x t r a c t to pH 5.0, fo l lo w ed by h e a t in g th e e x t r a c t w i th s t i r r in g to a boil. T he m a jo r i ty o f the p r o t e in was p re c ip i t a te d by h e a t in g at pH 5 .0 , while so m e re m a in e d in the s u p e r n a t a n t f rac t io n u n d e r these c o n d i t ions . Th is e f fe c t is s im ila r to t h a t p re viously r e p o r t e d by W olf an d T a m u ra( 1 9 6 9 ) s tu d y in g s o y b e a n p ro te in . T he f o r m a t io n o f lo w -m o le cu la r w e ig h t a n a lo gous m a te r ia ls was f o u n d w h e n so y b e a n p ro te in s w ere acid p re c ip i t a te d . I n f o r m a t io n g a th e re d in th e p re se n t in v es t ig a t io n p o in ts to a c o n d i t i o n o f m o re p ro te in p re c ip i ta t io n a t h ig h er t e m p e ra tu re s .

T h e i m p o r t a n t in te r fe r in g su b s tan ces in th e e x t r a c t io n o f LPC w ere s ta rch a n d c ru d e f iber . A large p o r t io n o f t h e s ta r c h re m a in e d in the res idue o f th e p h o s p h a te

e x t r a c t . In p re l im in a ry e x p e r im e n t s , e x t r a c t io n o f p ro te in s f ro m the bean p o w der was n o t success fu l due to th e p ro b le m o f s ta rch ge la t ion w h e n s t ro n g a lka l ine salt was a d d e d su d d e n ly . I t was f o u n d t h a t th e p h o s p h a te m u s t be a d d e d s lo w ly w i th s t i r r in g t o o v e rc o m e th e d if f icu l t ies .

I t was n ecessa ry t o s t i r th e c o n te n t s in the s te a m - ja c k e te d k e t t l e to a b o i l fo r 10 min to c o ag u la te th e p ro te in s a n d to also in ac t iv a te the t ry p s in i n h ib i t o r (see be low ).

T h e p ro te in c u rd was b r o u g h t t o pH6.4 ± 0 .2 w i th 0 .1 N N a O H a n d th e n freeze dried . It was o b se rv ed t h a t c o n s id erab le b ro w n in g r e a c t io n t o o k place w h e n th e p ro te in c o n c e n t r a t e was d r ied in an oven a t 70°C . T h e f reeze -d r ied p r o d u c t was m u c h b e t t e r in q u a l i ty . It was l igh t in co lo r , so f t in t e x tu r e , a n d l ight in w e igh t .

C hem ica l ana lyses o f va r ious f rac t io n s

C hem ica l ana lyses o f w h o le d ry l im a beans , th e v a r ious f rac t io n s p r e p a re d f ro m th e m , an d w h e a t f l o u r fo r c o m p a r i son are p re s e n te d in T ab le 2.


E ffec t o f pH a n d t e m p e ra tu r e on p r o t e in co ag u la t io n

P re l im in ary e x p e r im e n t s in d ic a te d th a t l im a b e an p r o te in s in th e p h o s p h a te e x t rac t w ere ra p id ly in so lu b i l iz e d by h e a t ing to a boil. It was also o bse rved th a t h igher y ie lds o f l im a b e an p ro te in c o n c e n t r a te (L PC ) w ere o b ta in e d at p H 4 .0 an d5.0 th a n a t o t h e r values. E x p e r im e n t s w ere car r ied o u t to s t u d y th e e f fe c t o f h e a t in g a t p H 4 .0 a n d 5 .0 fo r 3 m in on the y ie ld a n d p u r i ty o f p ro te in s . T h e re su l ts are p r e se n te d in Tab le 1.

I t is a p p a r e n t t h a t th e y ie ld o f p ro te in increased w h e n th e t e m p e ra tu r e was ra ised f r o m 4 3 .3 t o 9 3 .3 °C . Highest yield

Table 2—Chemical analyses of various fractions prepared from lima beans (Phaseolus lunatus L. var BC6) and wheat flour

SampleMoisture

%

Totalsolids

%Starch

%Fat%

Protein%

Crudefiber

%Ash%

Whole dry lima beans 12.17 87.83 47.67 3.24 21.37 7.35 4.46

Freeze-dried protein preparation 8.66 91.34 9.73 0.82 54.31 0 10.18

Residue 4.20 95.8 36 12 1.25 6.62 8.49 3.24Starch preparation 3.60 96.4 80.66 1.12 4.62 0.83 1.38Precooked, freeze-dried

lima bean powder 3.20 96.8 50.72 3.12 20.67 5.25 2.56Wheat flour 9.15 90.85 77.2 1.00 9.47 0 0.50

LIMA BEAN P R O T E IN -1 7 3

Table 3—Comparison of the amino acid contents3 of whole largelima beans, freeze-dried protein concentrate and wheat flour

Whole lima beanFreeze-dried protein

concentrateWheatflour

Amino acidg/100g

of dry bean g/16g Ng/100g

dry basis g/16g N g/16g NAspartic acid 3.63 17.51 8.93 17.06 4.17Th reonine 0.71 3.43 2.21 9.84 2.41Serine 1.55 7.48 3.74 7.15 4.52Glutamic acid 2.96 14.78 7.41 14.15 34.24Proline 0.73 3.67 2.21 4.22 13.21Glycine 1.09 5.26 2.08 3.97 3.64Alanine 0.90 4.34 2.48 4.74 2.77Valine 0.79 3.81 2.17 4.15 2.87Cystine 0.32 1.54 0.55 2.65 2.94Methionine 0.19 0.92 0.59 1.13 0.79Isoleucine 0.74 3.57 2.35 4.49 2.65Leucine 1.55 7.48 4.09 7.81 6.41Tyrosine 0.74 3.57 2.03 3.88 2.96Phenylalanine 0.97 4.68 2.47 4.72 4.71Lysine 1.36 6.56 3.46 6.61 2.13Histidine 0.57 2.75 1.53 2.92 1.89Arginine 0.90 4.34 2.62 5.01 3.50

3 Tryptophan was not determined because it was destroyed during acidhydrolysis of the proteins.

T h e d ry b ean s c o n ta in e d 12 .17% m ois tu re a n d 2 1 .3 7 % p ro te in s . C o m p a r e d w i th w h e a t f lo u r , d ry l im a b e an s c o n ta in e d tw ice as m u c h p ro te in . T h e resu l ts f ro m a m in o acid ana lyses are p r e se n te d in T ab le 3. W hen e x p re ssed as g / 16g N, the d if fe ren ces in t h e in d iv id u a l a m in o acid c o n te n t o f w h o le l im a b e an c o m p a r e d to LPC was n o t g reat , w i th th e e x c e p t io n o f th reo n in e a n d c y s t in e w h ic h w ere in c reased a p p r o x im a te ly 3 a n d 1.5 t im es , re spec t ive ly . T r y p to p h a n was n o t d e te r m in e d becau se o f d e s t r u c t io n d u r in g acid h y d ro ly s is . I f s im ila r t o o t h e r b e an p r o te in , h o w e v e r , t r y p t o p h a n is p r o b a b ly p re sen t on the o r d e r o f 1 — 1 -5g p e r 16gN. F o r c o m p a r is o n , th e a m in o acid c o n t e n t o f a w h e a t f l o u r sa m p le is a lso p re se n te d in T ab le 3. T h e m o s t n u t r i t i o n a l ly l im it ing a m in o acids in l im a bean p ro te in , based o n a n a ly t ica l ev idence , a p p e a r e d to be th e su l fu r c o n ta in in g a m in o acids.

B io lo g ica l e v a lu a tio n o f p ro te in q u a lity

R esu l ts o f th e feed in g tes ts u s ing lima bean p ro te in c o n c e n t r a t e (L PC ), i so la ted s o y b e a n p ro te in , a n d casein are p re sen te d in T ab le 4.

G r o u p A was fed a n o n p r o t e i n diet . T he w e ig h t o f ra ts d e c reased d u r in g the w h o le fe ed in g p e r io d , m in u s 0 .5 4 ± O . lg / r a t / d a y . This g ro u p was u sed as a c o n t r o l t o ca lcu la te th e d if fe rence b e tw e e n th e w e igh t an d n i t ro g e n c o n te n t o f th e t e s t g ro u p s ( B e n d e r a n d Doell, 1957a , b). T h e ra ts in g ro u p B w ere fed a 10% casein p ro te in in the d ie t . T h e a p p r o x im a te w e igh t gain was 3 .2 3 ± 0 .2 6 g / r a t / d a y . T h is g ro u p was u se d to o b ta in re fe re n ce F E R , N P U an d P E R values u n d e r te s t c o n d i t i o n s fo r c o m p a r i son w i th the o t h e r g roups . T h e P E R was d e te r m in e d to be 2 .9 3 ± 0 .0 8 a n d the F E R 3 .3 7 ± 0 .1 4 . N e t p ro te in u t i l iz a t io n

was e q u a l to 6 2 .5 ± 0 .25% .G r o u p C was fed a so y b e a n p ro te in

die t . T h e ra te o f g ro w th was n o t as goo d as casein. S o y b e a n p ro te in s are k n o w n to be d e f ic ie n t in so m e essen t ia l a m in o acids, i .e . , ly s ine , t h r e o n in e a n d m e t h i o n ine , w h ic h l im it i ts n u t r i t iv e value (B lock a n d M itche ll , 1 9 4 6 ; G u g g e n h e im a n d F r i e d m a n n , 1960) . T h e P E R and F E R values o f s o y b e a n p r o te in s w ere2 .3 4 ± 0 .0 8 a n d 4 .3 0 ± 0 .2 0 , re sp ec t iv e ly , a n d th e N P U value was 5 0 .9 7 ± 2 .19% . L ikew ise , M u s ta k a s e t al. ( 1 9 6 4 ) ev a lu a te d 10 c o m m e rc ia l p r o d u c t s o f soy f lo u rs by fe e d in g th e m t o ra ts . T h e y o b ta in e d P E R values in th e range 2 .0 9 —2 .4 6 , a n d N P U values 5 5 —62 . In the p r e se n t in v es t ig a t io n , th e w e ig h t gain f o r t h e s o y b e a n d ie t was 2.5 ±0 .3 g / r a t / d a y . S o y b e a n p r o t e i n is o n e o f th e l eg u m e p r o te in s t h a t can be u se d in d ie ta ry fo o d s a n d f o r p r o t e in fo r t i f i c a t io n .

In G r o u p D, th e ra ts were fed d iets c o n ta in in g 10% LPC. T h e average w e igh t gain f o r t h e l im a b e an p r o te in c o n c e n t r a te was 1 .28 ± 0 .2 g / r a t / d a y . T h e P E R a n d F E R w e r e 1 . 6 8 ± 0 . 0 7 an d5 .9 4 ± 0 .2 5 , re sp ec t iv e ly . T h e N P U value was 48 .4 1 ± 1 .97 (T ab le 4) . P rev iously , R o c k la n d a n d M e tz le r ( 1 9 6 7 ) s tu d i e d the n u t r i t i o n a l va lue o f p r e c o o k e d a n d f reeze-d r ied l im a beans . T h e y o b ta in e d a P E R va lue o f 1.83 w h ic h is very c lose t o the va lue fo r LPC.

In G r o u p E , t h e ra ts w ere fed w i th th e sa m e f ree z e -d r ied LPC, b u t fo r t i f i e d w i th essen tia l a m in o ac ids , as c o m p u t e d f ro m a m in o acid analysis o f l im a bean p ro te in c o n c e n t r a t e (T ab le 3) a n d the N u t r i e n t R e q u i r e m e n t o f L a b o r a to r y A n im als (N A S -N R C , 1 9 6 2 ) . T h e c a lcu la t io n was based on th e m in im u m a m o u n t s o f n u t r i en ts r e q u i r e d f o r ra t g r o w th . All o f the e s s e n t i a l a m in o acids (m e th io n in e , p h e n y la la n in e , valine and lys ine) t h a t w e re l im i t in g w ere a d d e d so t h a t g ro w th a n d o t h e r p a ra m e te r s w o u ld be m a x i m iz e d . T h is w as d o n e p r im a r i ly to te s t fo r th e p re sen ce o f g r o w th in h ib i to r s , su c h as t ry p s in in h ib i to r s . I t is c lear t h a t a d d i t io n o f t h e essen t ia l a m in o acids re su l te d in s ign if ican t in c rease in g r o w th w h ic h was s im ila r t o t h a t o f ra ts fed th e casein die t . In th is reg ard , m e t h io n in e a n d lysine were p r o b a b ly m o s t i m p o r t a n t in s t im u la t in g g ro w th . T he g r o w th ra te fo r ra ts fed d ie t E was 3 .2 8 ± 0 .4 8 g / r a t / d a y . T h e P E R an d F E R values o f d ie t E w ere 3 .0 4 ± 0 .0 9 a n d 3 .4 2 ± 0 .0 3 , re sp ec t iv e ly , a n d th e N P U value was 7 2 .7 0 ± 3 .3 8 . T h e n u t r i t iona l q u a l i ty o f l im a bean p ro te in th u s can be im p r o v e d b y f o r t i f i c a t io n w i th the a p p r o p r i a te a m in o acids.

M o s t l eg u m e seeds are d e f ic ie n t in lysine a n d su l fu r - c o n ta in in g a m in o acid. In genera l, th e q u a l i ty o f legum e p ro te ins is n o t as g o o d as t h a t o f a n im a l p ro te in in t e rm s o f e ssen t ia l a m in o acids, b u t p ro te in q u a l i ty can be im p ro v e d by th e i r

Table 4—Determination of protein efficiency ratio (PER) and feed efficiency ratio (FER) of lima bean protein concentrate by feeding to Albino rats

Group Type of dietFood consumption

g/rat/dayaGain in wt

(g) 21 days3 PER3 FER!a

A Nonprotein 4.73 + 0.38 -11.25 ± 2.09 - -

B Casein 10.91 ± 1.36 68 ± 5.49 2.93 ± 0.08 3.37 ± 0.14C Soybean 10.74 ± 1.66 52.50 ± 6.31 2.34 ± 0.08 4.30 ± 0.20D Lima bean protein 7.61 ± 0.82 26.88 ± 4.15 1.68 ± 0.07 5.94 ± 0.25E Lima bean protein

+ methionine, lysine phenylalanine, valine 10.72 ± 1.99 68.38 ± 10.43 3.04 ± 0.09 3.42 ± 0.03

3 x ± S.E. = Mean ± Standard error

1 74-JOURNAL OF FOOD SCIENCE-Volume 39 (1974)

Table 5—Net protein utilization (NPU) by Albino rats fed with various protein types3

GroupType of

dietAvg initial

v/t (g)b

Wt lost after drying in oven (g)b

Nitrogenconsumption

g/rat/dayb

Body nitrogen content

(g)b

Net protein utilization

(%)b

A Nonprotein 48.13 ± 4.13 24.83 ±2.14 — 1.02 ± 0.08 -B Casein 45.88 ± 3.52 80.81 ± 8.26 3.63 ± 0.49 3.67 ± 0.37 62.50 + 0.25C Soybean 48.25 ± 3.49 69.26 ±7.10 2.85 ± 0.30 3.59 ± 0.58 50.97 ±2.19D Lima Bean

protein 46.75 ± 3.93 53.44 ± 6.54 2.20 ± 0.27 2.54 ± 0.29 48.41 ± 1.97E Lima bean

protein + methionine, lysine, phenylalanine, valine 46.75 ± 3.60 88.12 ± 8.44 3.63 ± 0.35 3.60 ± 0.67 72.70 ± 3.38

a C o rre la tio n betw een the ra tio o f body N to Body H 2 O, th e e q u a tion is log (4 .8 —y ) = 0 .4 3 7 —0 .0 1 2 3 x w here x = age o f rats = 28 days (w ean ling period ) + 21 days (feed ing period) = 49 days. By using above equ a tion y = 4 .12 = N /H 20 X 100.

b x ± S .E . = Mean ± S tandard e rro r.

a d d it io n . D a ta p re se n te d in T ab le 2 in d ic a te th a t lim a b e an s are re a so n a b ly h igh in p ro te in . D ue to th e lo w e r level o f m e th io n in e , an im p o r ta n t su lfu r -c o n ta in in g a m in o a c id , i ts n u tr i t io n a l v a lu e as a to ta l so u rc e o f h u m a n d ie ta ry p ro te in is l im ite d . In te rm s o f h u m an re q u ire m e n ts , h o w e v e r, c o n su m p tio n o f LPC a t levels n o g re a te r th a n 5 —6% o f th e n o rm a l da ily c a lo ric in ta k e (2 1 0 0 to 2 5 0 0 c a lo rie s) p ro v id es all o f th e o th e r e sse n tia l a m in o acid s w ith th e e x c e p tio n o f m e th io n in e at th e ir levels o f m in im u m re q u ire m e n t fo r m a in te n a n c e in a d u lts .

T h e re a re a n u m b e r o f a p p ro a c h e s in w h ich th e n u tr i t io n a l q u a lity o f lim a b ean s m ay be im p ro v e d . P la n t b re ed in g p ro g ram s to im p ro v e p ro te in q u a li ty an d c o n te n t c o u ld be su c c ess fu l. T h e re is a lso a n e e d fo r in v e s tig a tio n on th e e f fe c t o f e n v iro n m e n ta l v a riab les th a t m ig h t in f lu en ce th e p ro te in a n d a m in o acid c o n te n t , su c h as seed q u a li ty , w a te r s tre ss , so il p ro p e rt ie s a n d m in e ra l n u tr ie n ts fo r th e p la n ts . I t is a lso p o ss ib le to im p ro v e th e n u tr i t iv e v a lu e o f lim a b ean p ro te in s b y fo r t if ic a t io n w ith m e th io n in e in fo o d

p re p a ra tio n s . T h e lim a b ean c o n c e n tra te p re p a ra tio n in c o m b in a tio n w ith o th e r p ro te in s c o u ld c e r ta in ly b e u se d to o b ta in a b a la n ce d a m in o acid d is tr ib u tio n a n d sh o u ld be c o n s id e re d in th is a p p lic a tio n .

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Ms received 6/28/73; revised 8 /2 1 /73; accepted 8/24/73,

The senior author thanks the U.S. Agency of International Development for providing the fellowship which made this study possible. We are grateful to Mr. Carl Tucker of the Dept, of Agronomy for supplying the lima beans used in this study. The advice of Messrs. Amara Bhumiratana and H.N. Daoud is gratefully acknowledged.

D. D. C R EN W E LG E, C. W. D IL L , P. T. T Y B O R a n d W. A . L A N D M A N ND epartm ents o f A n im a l Science a n d B iochem istry , Texas A & M U niversity, College S ta tio n , T X 7 7 843

A COMPARISON OF THE EMULSIFICATIOM CAPACITIES OF SOME PROTEIN CONCENTRATES

INTRODUCTIONT H E F U N C T IO N A L p ro p e rt ie s o f p ro te in iso la te s is a p r im a ry fa c to r d e te r m in ing th e ir u t i l i ty in fo o d p ro d u c ts . S o lu b ili ty , w h ip p in g a b ili ty , e m u ls if ic a t io n c a p a c ity an d s im ilar p ro p e rt ie s are u sed in d e te rm in in g p o te n tia l fo o d syste m s in w h ic h a g iven iso la te m ay len d its d esirab le p ro p e rtie s .

T he e m u ls if ic a tio n c a p a c ity o f p ro te in s is o f u tm o s t im p o r ta n c e to th e ir u t i l iz a t io n in sa lad d ressin g s an d c o m m in u te d m ea t p ro d u c ts (B o r to n e t a l.,1 9 6 8 ). T h e a c t io n o f p ro te in s as em u lsif ie rs is in f lu e n c e d b y p ro te in c o n c e n tra t io n , speed o f m ix in g , ty p e o f o il, an d ty p e o f e m u ls if ic a tio n sy s te m .

T h e m e th o d s o f d e te c tin g e m u ls io n in v e rs io n have b e e n su b je c t to a la c k o f p re c is io n . Several m e th o d s fo r th e d e te rm in a t io n o f th e e n d p o in t , o r co llap se o f e m u ls io n s , have b e en e m p lo y e d . S w ift e t al. (1 9 6 1 ) , C a rp e n te r an d S affle (1 9 6 4 ) , P ea rso n e t al. (1 9 6 5 ) , B o rto n e t al.(1 9 6 8 ) an d Ivey e t a l. (1 9 7 0 ) v isua lly o b served th e a b ru p t d ec rease in v isco sity a s s o c ia te d w ith e m u ls io n in v ers io n . G o u ld e n (1 9 6 1 ) e m p lo y e d in fra -red a b s o rp tio n to a n a ly ze th e n a tu ra l em u ls io n in m ilk , b u t w as l im ite d to d is c o n tin u o u s ph ase p a r tic le s o f u n ifo rm a n d lim ite d size. E le c tr ica l c o n d u c tiv ity w as u sed by W ebb et al. (1 9 7 0 ) , to d e te rm in e e m u lsio n e n d p o in ts b u t w as e ffe c tiv e o n ly in d ilu te p ro te in so lu tio n s . A c to n an d Saffle(1 9 7 2 ) d e te rm in e d th a t a p h ase v o lu m e fa c to r e x e r te d so m e c o n tro l o v er th e m ax im u m level o f o il in c o rp o ra tio n in to e m u ls io n s using m ea t p ro te in s as th e em u lsifie r.

T h is in v e s tig a tio n w as u n d e r ta k e n to c o m p a re th e e m u ls if ic a tio n c a p a c itie s o f fo u r p ro te in s u n d e r s ta n d a rd iz e d c o n d it io n s using a m o re sen sitiv e a n d o b jec tiv e m e th o d th a n p rev io u s ly used fo r e n d p o in t d e te rm in a tio n .

EXPERIMENTALM a t e r i a l s

T h e e m u l s i f i c a t i o n c a p a c i t y o f f o u r p r o t e i n p r e p a r a t i o n s w a s c o m p a r e d o v e r a r a n g e o f p r o t e i n c o n c e n t r a t i o n s . T h e t w o p l a n t p r o t e i n s s t u d i e d w e r e a g l a n d l e s s c o t t o n s e e d f l o u r ( 5 7 . 5 % p r o t e i n ) a n d a s o y b e a n c o n c e n t r a t e ( 6 7 . 5 % p r o t e i n ) . T h e t w o p r o t e i n s o f a n i m a l o r i g i n u s e d w e r e a d e c o l o r i z e d b o v i n e h e m o g l o b i n ( T y b o r e t a l . , 1 9 7 3 ) ( 9 0 . 1 % p r o t e i n ) a n d a l o w - h e a t n o n f a t d r y m i l k ( 3 5 . 4 % p r o t e i n ) .

P r o t e i n d e t e r m i n a t i o n s w e r e m a d e b y t h e K je l - d a h l p r o c e d u r e ( A O A C , 1 9 7 0 ) .

T h e p r o t e i n p r e p a r a t i o n s w e r e s u s p e n d e d in d i s t i l l e d - d e i o n i z e d w a t e r t o p r o d u c e a s t o c k s u s p e n s i o n c o n t a i n i n g lO g p r o t e i n p e r l i t e r . D i l u t i o n s o f t h i s s u s p e n s i o n w e r e m a d e b y t r a n s f e r r in g t h e r e q u i r e d v o l u m e s t o 1 0 0 m l f l a s k s a n d b r i n g i n g t o v o l u m e . T h e e n t i r e 1 0 0 m l v o l u m e w a s u s e d in t h e d e t e r m i n a t i o n o f e m u l s i f i c a t i o n c a p a c i t y . C o r n o i l w a s c h o s e n a r b i t r a r i l y a s t h e o i l p h a s e f o r t h i s s t u d y .

M e t h o d o l o g y

E m u l s i o n s w e r e f o r m e d in a W a r in g B l e n d o r e q u i p p e d w i t h a w a t e r - j a c k e t e d s t a i n l e s s s t e e l c u p . T h e B l e n d o r w a s o p e r a t e d i n s e r i e s w i t h a v a r i a b l e a u t o t r a n s f o r m e r a n d a m i c r o - a m m e t e r w h i c h c o u l d b e a t t e n u a t e d f o r s e n s i t i v e m e a s u r e m e n t o f p e a k a m p e r a g e r e q u i r e m e n t s . T h e d i a g r a m o f t h e a t t e n u a t i o n c i r c u i t i s p r e s e n t e d in F i g u r e 1.

T h e 1 0 0 - m l a l i q u o t s o f p r o t e i n s u s p e n s i o n w e r e m i x e d in t h e B l e n d o r f o r 1 m i n a t a t r a n s f o r m e r s e t t i n g o f 3 0 t o t h o r o u g h l y d i s p e r s e t h e p r o t e i n s . T h e t r a n s f o r m e r t h e n w a s s e t a t t h e d e s i r e d s p e e d a n d o i l a d d e d u n t i l e m u l s i o n c o l l a p s e o c c u r r e d . T h e i n c r e a s i n g v i s c o s i t y d u r i n g e m u l s i o n f o r m a t i o n c a u s e d a s t e a d y r i s e in a m p e r a g e r e q u i r e m e n t s o f t h e B l e n d o r m o t o r . T h e s u d d e n d r o p in v i s c o s i t y a t i n v e r s i o n r e s u l t e d in a s h a r p d r o p in a m p e r a g e , a l l o w i n g a p r e c i s e

d e t e r m i n a t i o n o f t h e i n v e r s i o n p o i n t . T h e v o l u m e o f o i l r e q u i r e d t o r e a c h t h e i n v e r s i o n p o i n t w a s e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l e m u l s i o n v o l u m e ( v o l u m e o f p r o t e i n s o l u t i o n p l u s o i l a d d e d ) a n d t h i s f i g u r e u s e d f o r c o m p a r i n g t h e e m u l s i f i c a t i o n c a p a c i t y o f t h e p r o t e i n s s t u d

i e d . T h i s m e t h o d h a s r e c e n t l y b e e n r e p o r t e d b y A c t o n a n d S a f f l e ( 1 9 7 2 ) a s a n e f f e c t i v e o n e f o r c o m p a r i s o n o f e m u l s i f i c a t i o n c a p a c i t y .

P r o t e i n s o l u b i l i t y m e a s u r e m e n t s w e r e m a d e b y a m o d i f i c a t i o n o f t h e m e t h o d o f L a w h o n a n d C a t e r ( 1 9 7 1 ) u t i l i z i n g t h e L o w r y e t a l . ( 1 9 5 1 ) m e t h o d f o r d e t e r m i n i n g s o l u b l e p r o t e i n .

RESULTS & DISCUSSIONT H E F IR S T ST E P in th e s tu d y w as to

o p tim iz e th e e f fe c ts o f b le n d o r sp e e d , oil a d d it io n ra te an d pH o n e m u ls if ic a tio n c a p a c ity o f th e p ro te in s . S ince p ro te in s o f d if fe re n t o rig in s w e re to be c o m p a re d , m ea n in g fu l in te r p r e ta t io n re q u ire d o p t i m iz a tio n fo r e ach in d iv id u a l p ro te in in te rm s o f fa c to rs a ffe c tin g its e m u ls if ic a t io n c ap a c ity .

Blendor speedT h e e f fe c t o f B len d o r sp eed o n th e

e m u ls if ic a tio n c a p a c ity o f th e p ro te in s

Fig. 1 —Diagram o f the attenuation circuit used with the m icroammeter.



Table 1 —Effect of Blendor speed on the oil phase volume at inversion for the four protein isolates

Sample

Autotransformer setting

40 50 60 70 80 90 100% Oil phase volume

S o y 8 6 . 7 7 4 . 6 7 4 .1 7 3 . 8 7 1 . 5 6 7 . 4 6 5 . 3

G l o b i n — - - - 8 5 . 9 5 8 . 7 5 6 . 5

C o t t o n s e e d — - 8 1 . 5 7 5 . 7 7 3 . 5 6 5 . 0 6 3 . 9

N F D M - 8 1 . 5 8 2 . 5 8 1 . 3 7 6 . 3 7 0 . 0 5 2 . 8

w as d e te rm in e d a t a u to tra n s fo rm e r se ttin g s f ro m 4 0 to 100 . T h e re su lts a re p re se n te d in T ab le 1. In c reasin g B len d o r sp eed d ecreased th e oil p h a se v o lu m e at in v ers io n fo r all sam p les, co n firm in g th e re su lts o f Ivey e t al. (1 9 7 0 ) an d S w ift et al. (1 9 6 1 ) . In su ff ic ie n t m ix in g u su a lly w as o b se rv ed a t t r a n s fo rm e r se ttin g s b e lo w 8 0 , re su ltin g in e r ro n e o u s ly h igh oil p h ase v o lu m e p e rce n ta g e s . T h is e ffe c t is e v id en ced a t th e 4 0 se ttin g fo r soy p r o te in , and is re sp o n sib le fo r th e o th e r m issing d a ta w h ic h w ere a rb itra r ily e x c lu d e d . T h e e f fe c t w as n o te d p a r tic u la r ly fo r th e g lo b in f ra c t io n , an d w as lea s t im p o r ta n t fo r th e so y p ro te in fo r w h ic h se ttin g s as low as 50 c o u ld be u sed w ith sa tis fa c to ry in c o rp o ra tio n o f th e fa t. T h e m ax im u m o il ph ase v o lu m e fo r th e m ilk p ro te in s w as a tta in e d a t a se ttin g o f 6 0 . H ow ev er, m o st c o m p le te a n d ra p id in c o rp o ra tio n o f fa t w as a cc o m p lish e d a t a se ttin g o f 7 0 . T h e o p t im u m a u to tra n s fo rm e r se ttin g s w ere d e te rm in e d to be 80 fo r th e so y , c o tto n s e e d and g lo b in p ro te in s , an d 70 fo r th e m ilk p ro te in s . T h e u se o f th ese o p tim u m b le n d in g sp eed s m ay n o t a llow e x ac t c o m p a riso n w ith re su lts o b ta in e d u n d e r c o n d it io n s o f c o n s ta n t b len d in g sp eed s p a r tic u la r ly w h en ra te o f o il a d d it io n is o p tim iz e d .

R a te o f o il a d d itio nT he e ffe c t o f th e ra te o f oil a d d it io n

w as d e te rm in e d b y a d ju s tin g th e a d d it io n ra te s b e tw e e n 0 .4 0 m l/sec an d 2 .0 0 m l/ sec . F ig u re 2 su m m ariz e s th e re su lts o f d if fe re n t oil a d d it io n ra te s .

T h e oil a d d it io n ra te s d id n o t a p p re c iab ly a ffe c t th e e m u ls if ic a tio n c a p a c ity o f th e so y , g lo b in an d c o tto n s e e d p ro te in s . A ra te o f 0 .6 7 m l/se c w as d e te rm in e d to b e o p t im u m w ith re sp ec t to a c c u ra c y o f o il v o lu m e d e te rm in a tio n a t in v ers io n an d th e t im e re q u ire d fo r to ta l o il a d d itio n . T h e o p tim u m ra te fo r th e m ilk p ro te in s w as 1 .00 m l/se c a f te r w h ic h a sh a rp d e crease in e m u ls if ic a tio n c a p a c ity w as n o te d . C a rp e n te r an d Saffle (1 9 6 4 ) c o n c lu d e d th a t v a ry in g th e ra te o f oil a d d it io n had no e ffe c t a lth o u g h S w ift e t al.(1 9 6 1 ) in d ic a te d a p o sitiv e lin ea r re sp o n se . It w as c o n c lu d e d fro m th ese f in d ings th a t th e ra te o f oil a d d it io n has an e ffe c t o n c e r ta in p ro te in s w h ile o th e rs are u n a f fe c te d .

pH e ffe c tT h e e ffe c t o f pH o n e m u ls if ic a tio n

c a p a c ity w as d e te rm in e d o v er th e range o f pH 3 to 10. T h e re su lts p re se n te d in F ig u re 3 in d ic a te th a t at low pH v alues, a re la tiv e ly h igh e m u ls if ic a tio n c a p a c ity w as o b ta in e d fo r all b u t th e c o tto n se e d p ro te in . W ith in creas in g pH , a d ec rease to a m in im u m p o in t w as n o te d fo llo w ed by an in c rease in em u ls ify in g c a p a c ity . T h e re su ltin g cu rv es re sem b le ty p ic a l p ro te in so lu b ili ty cu rves. S w ift an d S u lz b ac h e r(1 9 6 3 ) lik ew ise d e m o n s tra te d th a t e m u ls if ic a tio n c a p a c ity c o rre la te d w ith th e pH o f th e p ro te in su sp en s io n .

P ro te in so lu b ili tyT he so lu b ili ty o f th e p ro te in s o v er a

range o f pH v alues w as d e te rm in e d to re la te p ro te in so lu b ility to e m u ls if ic a tio n c a p a c ity . S o lu b ility as a f u n c tio n o f pH w as d e te rm in e d over th e ran g e o f pH 3 to 10 using th e L aw h o n a n d C a te r (1 9 7 0 ) m e th o d . T he re su lts a re p re se n te d in F ig u re 4 . S o lu b ility o f th e p ro te in s w as h ig h e st a t lo w pH v a lu es an d d ecreased to a m in im u m a f te r w h ich so lu b ili ty o n ce again in c re a sed . A g en era l c o r re la t io n b e

tw e e n e m u ls if ic a tio n c a p a c ity a n d so lu b ili ty w as e v id e n t, p a r tic u la r ly b e tw e e n th e m in im u m p o in ts fo r e m u ls if ic a tio n c a p a c ity (F ig . 3 ) an d s o lu b ili ty (F ig . 4 ). P ea rso n e t al. (1 9 6 5 ) o b se rv ed s im ila r re su lts b e tw e e n e m u ls ify in g c a p a c ity an d p ro te in so lu b ili ty . B ased o n th e se so lu b ili ty d a ta , th e o p t im u m p H v a lu es to be u sed fo r e m u ls if ic a tio n c a p a c ity c o m p a r isons o f th e p ro te in sa m p le s w e re 9 .4 fo r th e soy p ro te in , 3.1 fo r th e g lo b in , 8 .9 fo r th e c o tto n s e e d , an d pH 7.1 fo r th e m ilk p ro te in s . T hese pH v a lu es w ere ch o se n to m a in ta in a h igh level o f p ro te in so lu b ili ty .

In in s ta n c e s o f e m u ls io n f o rm a t io n w ith o u t re g ard to p H , p ro te in s o lu b ili ty m ig h t n o t be o p tim iz e d . F ig u re 5 sh o w s th e o il ph ase v o lu m e s o f soy p ro te in s and c o tto n s e e d p ro te in a t pH 9 .4 a n d 8 .9 re sp ec tiv e ly fo r o p t im u m p ro te in so lu b ili ty a n d a lso n e a r n e u tr a li ty (p H 7 .3 a n d 6 .5 re sp ec tiv e ly ) . W hen p ro te in so lu b ili ty is o p tim iz e d , a lo w er p ro te in c o n c e n tr a t io n is re q u ire d to o b ta in th e o il p h ase v o lu m es sim ila r to th o se a t n e u tra li ty .

P ro te in c o n c e n tr a t io nB echer (1 9 6 5 ) in d ic a te d th a t th e e m u l

s if ic a tio n c a p a c ity o f several e m u ls if ie rs is r e la te d to th e e m u ls if ie r c o n c e n tr a t io n . Such a re la tio n s h ip fo r th e p ro te in s u n d e r s tu d y w o u ld in d ic a te valid c o m p a r iso n o n ly a t th e o p tim u m c o n c e n tr a t io n fo r each . E m u ls if ic a tio n c a p a c itie s w e re d e te rm in e d fo r p ro te in c o n c e n tr a t io n s b e tw e e n 0 .2 0 0 g /1 0 0 m l a n d 1 .7 0 g /1 0 0 m l o f a q u e o u s p h ase . T h e re su lts a re p re se n ted in F ig u re 5. In c re a se in c o n c e n tr a t io n re su lte d in in c re a sed e m u ls if ic a tio n , c o rro b o ra t in g th e d a ta o f C a rp e n te r an d Saffle (1 9 6 4 ) f ro m e x tra c te d m e a t p ro te in s a n d W ebb e t al. (1 9 7 0 ) f ro m sea bass e x tra c ts .

Fig. 2 —E ffect o f oil addition rate on percent o il phase volume o f em ulsion a t inversion.

EMULSIFICATION CAPACITIES OF PROTEIN CONCENTRATES- 1 7 7

Fig. 3 —Effect o f p H on the percent oil phase volume o f emulsion at inversion.

pH

Fig. 4 —E ffect o f p H on the solubility o f the soy, globin, cottonseed and m ilk proteins.

T he o p tim u m c o n c e n tr a t io n w as la rg e ly d e p e n d e n t u p o n th e p ro te in ty p e . T he soy p ro te in a tta in e d o p tim u m e m u ls ify ing c a p a c ity a t a c o n c e n tr a t io n o f 0 .9 8 6 g /1 0 0 m l. T h e g lo b in a tta in e d o p t i m u m em u ls ify in g c a p a c ity a t 0 .4 0 4 g /1 0 0m l. A g lo b in c o n c e n tr a t io n g re a te r th a n 0 .4 5 1 re su lte d in in a d e q u a te m ix in g d u e to th e e m u ls io n v isco sity . C o tto n s e e d p ro te in c o n c e n tr a t io n w as o p t im u m a t 0 .8 8 4 g /1 0 0 m l, w h ile th e c o n c e n tr a t io n

o f m ilk p ro te in s w as o p tim u m at 1 .1 9 g / 100 m l.

V alid c o m p a riso n s o f th e em u ls if ic a t io n c ap a c itie s o f th e p ro te in s c o u ld be m ad e w h en th e B len d o r sp e e d , p ro te in c o n c e n tr a t io n , ra te o f o il a d d it io n a n c pH fo r m a x im u m p ro te in p e p tiz a t io n w ere o p tim iz e d fo r eac h p ro te in sam p le . O f th e fo u r p ro te in s s tu d ie d , th e b o v in e g lo b in e x h ib ite d th e g re a te s t e m u ls if ic a tio n ca p a c ity , in v e rtin g a t an o il p h ase v o lu m e o f

.2 .4.......................

.6 .8 1.0 1.2 1.4 1.6 1.8 2.0PROTEIN , g / 100 ml

Fig. 5—Effect o f protein concentration in the aqueous phase on the percent oil phase volume o f emulsion at inversion.

8 3 .9 % a t a c o n c e n tr a t io n o f 0 .4 0 4 g p ro t e i n / 100 m l. T h e so y b e a n c o n c e n tra te a tta in e d an o p tim u m e m u ls if ic a tio n ca p a c ity (2 8 .7 % o il p h a se v o lu m e a t a c o n c e n tr a t io n o f 0 .9 8 6 g /1 0 0 m l. T h e m ax im u m o il p h ase v o lu m e o f 7 5 .8 % fo r th e c o tto n s e e d p ro te in re q u ire d a c o n c e n tra t io n o f 0 .8 8 4 g /1 0 0 m l. T h e m ilk p ro te in s gave a n o p tim u m re sp o n se o f 7 5 .5 % oil p h a se v o lu m e a t a c o n c e n tr a t io n o f 1 .1 9 0 g /1 0 0 m l. T his e m u ls io n also e x h ib ite d a lo w e r v isco sity a t in v e rs io n th a n th e g lo b in e m u ls io n .

T h e g lo b in p ro d u c e d th e b es t e m u ls io n o f th e fo u r p ro te in p re p a ra tio n s s tu d ie d w h en th e y w ere c o m p a re d o n a u n it p ro te in basis. H o w ev e r, it m u s t b e n o te d , p a r tic u la r ly fo r th e so y a n d c o tto n se e d p ro te in s , th a t several m e th o d s o f iso la tio n are p o ss ib le an d th e fu n c tio n a l p ro p e rt ie s m ay v ary w ith th e m e th o d o f iso la tio n .

REFERENCESActon, J.C. and Saffle, R.L. 1972. Emulsifying

capacity of muscle protein: Phase volumes at emulsion collapse. J. Food Sci. 37: 904.

AOAC. 1970. Protein m easurement with the folin phenol reagent. In “ Official Methods of Analysis,” 12th ed., Association of Official Analytical Chemists, Washington, D.C.

Becher, P. 1965. “ Emulsions: Theory and Practice ,” 2nd ed., Reinhold Publishing Corporation, New York.

Borton, R .J., Webb, N.B. and Bratzler, L.J. 1968. Emulsifying capacities and emulsion stability of dilute m eat slurries from various m eat trimmings. Food Technol. 22: 506.

Carpenter, J.A. and Saffle, R.L. 1964. A simple m ethod of estimating the emulsifying capacity of various sausage meats. J. Food Sci. 29: 774.

Goulden, J.D.S. 1961. Quantitative analysis of milk and o ther emulsions by infra-red absorption. Nature 191: 905.

Ivey, F .J., Webb, N.B. and Jones V.A. 1970. The effect of disperse phase droplet size and interfacial film thickness on the emulsifying capacity and stability of m eat emulsions. Food Technol. 24: 1279.

Lawhon, J.T. and Cater, C.M. 1971. Effect of processing m ethod and pH of precipitation on the yields and functional properties of protein isolates from glandless cottonseed. J. Food Sci. 36: 372.

Lowry, O.H. Rosebrough, N.J., Farr, A.L. and Randall, R .J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265.

Pearson, A.M. Spooner, M.E., Hegarty, G.R. and Bratzler, L.J. 1965. The emulsifying capacity and stability of soy sodium pro- teinate, potassium caseinate and non-fat dry milk. Food Technol. 19: 1841.

Swift, C.E., L ockett, C. and Fryar, A.J. 1961. Com minuted m eat emulsions—The capacity of meats for emulsifying fat. Food Technol. 15: 468.

Swift, C.E. and Sulzbacher, W.L. 1963. Comm inuted meat emulsions: Factors affecting m eat proteins as emulsion stabilizers. Food Technol. 17: 224.

Tybor, P.T., Dill, C.W. and Landm ann, W.A.1973. Effect of decolorization and lactose incorporation on the emulsification capacity of spray-dried blood protein concentrates. J. Food Sci. 38: 4.

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Ms received 7/2 /73; revised 9 /7 /73 ; accepted9/11/73.____________________________________

Technical Paper No. 10600 of the Texas Agricultural Experim ent Station, College Station, Texas. This research was supported by a grant from the Fats & Proteins Research Foundation, Inc., Des Plaines, 111.

S. H. C. L IN , J. T. L A W H O N , C. M. C A T E R a n d K. F. M A T T ILF o o d Protein R& D C enter, Texas A & M U niversity , College S ta tio n , T X 7 7 8 4 3

COMPOSITION AND CHARACTERISTICS OF GLANDLESS AND LIQUID CYCLONE PROCESS DEGLANDED COTTONSEED WHEYS

INTRODUCTIONP R E P A R A T IO N o f c o tto n se e d p ro te in c o n c e n tra te s o r iso la te s f ro m c o tto n se e d f lo u r has m e rit in p ro m o tin g n u tr i t io n a l as w ell as fu n c tio n a l uses o f c o tto n se e d p ro d u c ts in fo o d (M a rtin e z a n d B erard i,1 9 7 1 ). In o rd e r to ach ieve th is , n o n p r o te in c o n s t i tu e n ts o f th e c o tto n s e e d f lo u r m u s t b e re m o v e d b y p ro p e r m ean s so th a t th e p ro te in level can be in c re a se d . V a rio u s a q u e o u s e x tr a c t io n p ro c e d u re s have b e en d e v e lo p ed b y th e U S D A S o u th e rn M a rk e tin g & N u tr i t io n D iv., N ew O rlean s to e f f ic ie n tly iso la te v a rio u s c o tto n se e d p ro te in f ra c tio n s fo r fo o d uses. T h e d e ta ile d a c c o u n ts o f th e se iso la tio n p ro c e d u re s hav e b e en r e p o r te d b y B erard i e t al. (1 9 6 9 , 1 9 7 2 ) a n d M artin ez e t al. (1 9 7 1 ) . I t can be seen th a t c o tto n s e e d o ffe rs a g re a t p o te n t ia l fo r a v a r ie ty o f p ro te in p ro d u c ts d e s ired b y th e fo o d in d u s tr ie s . In th e n e a r f u tu re , th e c o tto n s e e d p ro te in iso la tio n p la n ts w ill p ro b a b ly in c rease in size a n d n u m b e r in o rd e r to fu lly u tilize c o tto n s e e d a n d to m e e t th e d e m a n d fo r c o tto n s e e d p ro te in to be u sed in v a rio u s fo o d p ro d u c ts . C o n se q u e n tly , we w ill be c o n f ro n te d w ith p ro b le m s c o m m o n ly a sso c ia ted w ith p ro te in iso la tio n p ro c esses w h ic h p ro d u c e a liq u id b y -p ro d u c t (S m ith , 1 9 5 8 ). O b v io u sly , th e p ro b le m g row s w ith in c re a s in g p ro d u c tio n . U nless th is liq u id b y -p ro d u c t can be p ro p e rly tre a te d o r again u t il iz e d , it m ay h in d e r th e p ro d u c tio n o f c o tto n s e e d p ro te in iso la te s , e sp ec ia lly in o u r e n v iro n m e n t- sen sitiv e so c ie ty .

T h e liq u id b y -p ro d u c t o b ta in e d fro m c o tto n s e e d p ro te in iso la tio n p ro cesses is c a lled c o tto n s e e d w h ey . W hat a c o t to n seed w h ey is to c o tto n s e e d m ay be l ik e n e d to w h a t a ch eese w h ey is to c o w ’s m ilk . H o w ev e r, th e p ro b le m re la te d to th e fo rm e r can be q u ite d if fe re n t f ro m th a t o f th e la t te r in te rm s o f its o rig in an d ch em ica l n a tu re . C o tto n s e e d w h ey is c o n v e n tio n a lly p re p a re d fro m d rie d c o t to n seed f lo u r to w h ich w a te r is a d d e d to fa c i li ta te p ro te in e x tr a c t io n , w h e reas , ch eese w h e y is m ere ly th e “ le f t-o v e r” f ro m c o w ’s m ilk in ch eese m a n u fa c tu r in g (L a w h o n e t a l., 1 9 7 3 ). G re a te r v a r ia tio n s a re th e re fo re e x p e c te d in c o tto n se e d w h e y s th a n in m ilk w h ey since th e d if

fe re n t m e th o d s a n d w ide v a rie ty o f c o tto n s e e d s u se d in th e p ro te in iso la tio n p ro c e d u re s d e te rm in e w h ich a n d h o w m u c h o f th e c o tto n s e e d c o n s t itu e n ts are e x tr a c te d a n d f in a lly re m a in in th e liq u id w h ey s. I t is n e ce ssa ry to a n a ly ze an d c h a ra c te riz e v a rio u s c o tto n s e e d w heys n o t o n ly to b e t te r u n d e rs ta n d w h a t c o tto n s e e d w h e y co n sis ts o f b u t a lso to in v es tig a te th e p o te n t ia l d isp o sa l p ro b le m it p re se n ts a n d /o r th e o p p o r tu n ity fo r re co v e rin g v a lu ab le w h ey c o n s t i tu e n ts fo r f u r th e r u til iz a t io n . T h is re p o r t w ill d iscuss th e c h em ica l c o m p o s it io n a n d c h a ra c te r is tic s o f c o tto n s e e d w h e y s an d p ro v ide in fo rm a t io n fo r p o ss ib le e c o n o m ic b y -p ro d u c t re tr iev a l a n d d isp o sa l.

MATERIALS & METHODSF l o u r s

G l a n d l e s s c o t t o n s e e d f l o u r w a s p r e p a r e d f r o m R o g e r s G L - 7 g l a n d l e s s c o t t o n s e e d a s d e

s c r i b e d b y L a w h o n e t a l . ( 1 9 7 2 a ) . T h e l i q u i d c y c l o n e p r o c e s s ( L C P ) d e g l a n d e d c o t t o n s e e d f l o u r w a s p r e p a r e d a t a n d p r o v i d e d b y t h e U S D A S o u t h e r n M a r k e t i n g & N u t r i t i o n R e s e a r c h D iv . , N e w O r l e a n s . T h e p r o c e s s h a s b e e n

d e s c r i b e d in t h e l i t e r a t u r e ( V i x e t a l . , 1 9 7 1 ) .

W h e y p r e p a r a t i o n

V a r i o u s c o t t o n s e e d w h e y s w e r e p r e p a r e d f r o m c o t t o n s e e d f l o u r s b y u s i n g t h e p r o t e i n i s o l a t i o n p r o c e d u r e s r e p o r t e d b y B e r a r d i e t a l . ( 1 9 6 9 , 1 9 7 2 ) a n d M a r t i n e z e t a l . ( 1 9 7 1 ) . M o d

i f i c a t i o n s w e r e m a d e t o c a r r y o u t p r o t e i n e x t r a c t i o n a n d p r e c i p i t a t i o n in a s m a l l e r s c a l e in t h e r e s e a r c h l a b o r a t o r y . T h e p r o t e i n i s o l a t i o n p r o c e d u r e s a n d t h e i r c o r r e s p o n d i n g m a j o r w h e y s a r e s u m m a r i z e d b e l o w :( a ) P r o c e s s B w h e y , f r o m s i n g l e - s t e p e x t r a c

t i o n a t p H 1 0 w i t h t w o - s t e p s e l e c t i v e p r e c i p i t a t i o n s a t p H 7 a n d 4 .

( b ) P r o c e s s C - N S P a n d C - S P w h e y s , f r o m t w o -

s t e p s e l e c t i v e e x t r a c t i o n s w i t h w a t e r a n d a t p H 1 0 f o l l o w e d b y c o r r e s p o n d i n g p r e

c i p i t a t i o n s a t p H 4 a n d 7 , r e s p e c t i v e l y . H e r e N S P a n d S P d e s i g n a t e t h e e x t r a c t i o n p r o c e d u r e s y i e l d i n g n o n s t o r a g e a n d s t o r a g e p r o t e i n i s o l a t e s , r e s p e c t i v e l y .

( c ) P r o c e s s F w h e y , f r o m t o t a l e x t r a c t i o n o f s e e d p r o t e i n s w i t h 0 . 3 5 M C a C l 2 f o l l o w e d

b y s e l e c t i v e p r e c i p i t a t i o n a t p H 3 .I n e a c h e x t r a c t i o n s t e p , 4 0 0 g o f f l o u r w e r e

e x t r a c t e d f o r 3 0 m i n w i t h 4 l i t e r s o f t h e s o l v e n t , e x c e p t t h e f i r s t e x t r a c t i o n s t e p o f P r o c e s s C in w h i c h 6 l i t e r s o f w a t e r w e r e u s e d . S e p a r a t i o n o f f l o u r r e s i d u e f r o m e x t r a c t o r p r o t e i n c u r d f r o m l i q u i d w h e y w a s a c h i e v e d b y c e n t r i f

u g a t i o n a t 1 0 0 0 X G f o r 1 5 m i n . T h e s e l i q u i d

w h e y s w e r e e i t h e r d i r e c t l y a n a l y z e d o r f r e e z e d r i e d f o r f u r t h e r i n v e s t i g a t i o n .

A n a l y t i c a l p r o c e d u r e s

T o t a l s o l i d s i n l i q u i d w h e y s w e r e d e t e r

m i n e d b y e v a p o r a t i n g l i q u i d s a m p l e s t o d r y n e s s o n a s t e a m b a t h a n d t h e n d r y i n g in a v a c u u m

o v e n a t 9 5 ° C f o r 4 h r . N i t r o g e n w a s d e t e r m i n e d b y m i c r o - K j e l d a h l m e t h o d ( A O A C , 1 9 7 0 ) . M o i s t u r e , n i t r o g e n , a s h a n d g o s s y p o l c o n t e n t s o f t h e f r e e z e - d r i e d w h e y s o l i d s w e r e d e t e r m i n e d a c c o r d i n g t o s t a n d a r d A O C S m e t h o d ( A O C S .

1 9 7 1 ) .P r o t e i n s w e r e c a l c u l a t e d b y N x 6 . 2 5 o r d e

t e r m i n e d b y t h e L o w r y m e t h o d ( L o w r y e t a l . , 1 9 5 1 ) . N o n p r o t e i n n i t r o g e n w a s d e t e r m i n e d a s s o l u b l e n i t r o g e n in 1 0 % t r i c h l o r o a c e t i c a c i d ( T C A ) s o l u t i o n . A m i n o a c i d a n a l y s i s w a s q u a n t i t a t i v e l y d e t e r m i n e d b y t h e s a m e p r o c e d u r e s d e s c r i b e d in a p r e v i o u s p u b l i c a t i o n ( L a w h o n e t a l . , 1 9 7 2 b ) . C a r b o h y d r a t e s w e r e m e a s u r e d b y p h e n o l - s u l f u r i c a c i d m e t h o d o f D u b o i s e t a l .( 1 9 5 6 ) u s i n g g l u c o s e a s s t a n d a r d . T h i n - l a y e r

c h r o m a t o g r a p h y o n S i l i c A R T L C 7 G F ( M a l l i n c - k r o d t , S t . L o u i s , M o . ) w a s a d o p t e d t o d e t e r m i n e s e m ¡ q u a n t i t a t i v e l y t h e d i s t r i b u t i o n o f c a r b o h y d r a t e s ( J e f f r e y e t a l . , 1 9 6 9 ) . P e n t o s e s

o r r i b o s e in n u c l e i c a c i d s w e r e d e t e r m i n e d b y O r c i n o l m e t h o d ( V o l k i n a n d C o h n , 1 9 5 4 )

u s in g r i b o s e o r a d e n o s i n e m o n o p h o s p h a t e a s s t a n d a r d s .

T o t a l p h o s p h o r u s w a s a c i d - d i g e s t e d a n d i n o r g a n i c p h o s p h o r u s w a s d i r e c t l y t a k e n f o r a n a l y s i s b y a c o l o r i m e t r i c m e t h o d ( S u m n e r ,

1 9 4 4 ) . P h y t a t e p h o s p h o r u s w a s d e t e r m i n e d b y

T C A e x t r a c t i o n f o l l o w e d b y f e r r i c c h l o r i d e p r e

c i p i t a t i o n a n d p h o s p h o r u s a n a l y s i s o f t h e f e r r i c - p h y t a t e c o m p l e x ( O b e r l e a s , 1 9 7 1 ) . M in e r a l a n a l y s i s w a s c a r r i e d o u t b y a t o m i c a b s o r p

t i o n s p e c t r o s c o p i c t e c h n i q u e ( A O A C , 1 9 7 0 ) .V i t a m i n s w e r e a n a l y z e d b y L i f e S c i e n c e

D iv . , T e c h n o l o g y I n c . , H o u s t o n . V i t a m i n A a s

v i t a m i n A p a r m i t a t e , c a r o t e n e a s ( 3 - c a r o t e n e , a n d a s c o r b i c a c i d w e r e d e t e r m i n e d a c c o r d i n g t o

p r o c e d u r e s p u b l i s h e d b y F r e e d ( 1 9 6 6 ) . R i b o f l a v i n , t h i a m i n e , p a n t o t h e n i c a c i d , c h o l i n e a n d p y r i d o x i n e w e r e a n a l y z e d b y s t a n d a r d A O A C

m e t h o d s ( A O A C , 1 9 7 0 ) . a - T o c o p h e r o l w a s d e t e r m i n e d a c c o r d i n g t o A m e s ( 1 9 7 1 ) .

G e l f i l t r a t i o n c h r o m a t o g r a p h y

S e p h a d e x G 7 5 c o l u m n , 2 .4 x 4 0 c m , w a s e l u t e d w i t h 0 .0 5 M c a r b o n a t e b u f f e r a t p H 1 0 . 2 c o n t a i n i n g 0 .5 M N a C l a n d 0 . 0 2 % s o d i u m a z i d e . 4 m l o f 5% f r e e z e - d r i e d w h e y s a m p l e , d i s s o l v e d in t h e s a m e b u f f e r a n d f i l t e r e d t h r o u g h a 5 - g m p o r e s iz e f i l t e r w e r e t h e n a p p l i e d t o t h e c o l u m n . T h e e l u e n t s a t a b o u t 2 0 m l / h r f l o w

r a t e w e r e c o n t i n u o u s l y m o n i t o r e d a t 2 8 0 n m

a n d c o l l e c t e d i n t o 4 - n i l f r a c t i o n s f o r f u r t h e r a n a l y s i s .


COTTONSEED WHEYS- 1 7 9

Table 1—Estimated yields of cottonseed whey solids and nitrogen from cottonseed flours

WheySolids

<%)

Yield in solids

(% Flour)

Yield in nitrogen

(% Flour N)

GlandlessProcess B 3.5 25 15Process C-NSP 2.0 26 14

C-SP 1.2 8.2 7.8(Combined) - (34) (22)

Process F 7.7 53 21

LCP deglandedProcess B 2.9 22 12Process C-NSP 1.8 24 9.5

C-SP 1.3 12 13(Combined) - (36) (23)

Process F 7.6 52 -

Table 2—Proximate analyses of cottonseed flours and wheys (% dry wt basis)

SampleProtein

(N X 6.25) Carbohydrates Ash

GlandlessFloura 64 15 7.4B whey 36 31 18C-NSP whey 32 41 14C-SP whey 57 13 13F whey 24 13 47

LCP deglandedFlourb 69 12 9.5B whey 35 23 26C-NSP whey 27 33 27C-SP whey 71 9.7 17

a 1.2% o il and 2.4% crude fib e r b 1.4% o il and 2.1 % crude fib e r

RESULTS & DISCUSSIONYields

C o tto n s e e d w h ey s c o n ta in e d w a te r so lu b le c o tto n s e e d f lo u r m a te ria ls w h ich w ere e x tra c te d b u t n o t p re c ip ita te d by th e p ro te in iso la tio n p ro cesses . T o ta l so lid s in w h ey s ran g ed fro m 1 . 2 —1 .1 % d e p e n d in g u p o n w h ich p ro te in iso la tio n p ro c e d u re was u sed as sh o w n in T ab le 1.2 2 —36% o f th e o rig ina l f lo u r so lid s , o r12 —23% o f th e f lo u r n itro g e n re m a in e d in P ro cess B a n d C w h e y s , in d ic a tin g a su b s ta n tia l loss o f th e f lo u r m a te ria ls as w ell as a p o te n t ia l d isp o sa l p ro b le m . P ro c ess F y ie ld e d a w h e y w h ich c o n ta in e d n o t o n ly c o tto n se e d f lo u r c o n s t itu e n ts b u t also a large a m o u n t o f c a lc iu m c h lo rid e used in th is p a r tic u la r p ro te in iso la tio n p ro cess . T h e a m o u n ts o f f lo u r m ate ria ls reco v e red in to w h ey f ra c tio n s varied g reatly a m o n g v a rio u s p ro c e d u re s b u t o n ly slig h tly b e tw e e n d if fe re n t ty p e c o tto n se e d flo u rs . I t w o u ld be e x p e c te d th a t h ig h e r p e rce n ta g e o f th e f lo u r m ateria ls m ay e n d u p in th e fin a l w h ey fra c tio n s in a larg e r p i lo t-p la n t p ro cess w here f lo u r is e x tr a c te d m o re th a n o n e tim e an d p ro te in iso la tio n by c o n tin u o u s c e n tr if u g a tio n is n o t as c o m p le te as a sm alle r la b o ra to ry ru n . O b v io u sly , su ch h igh y ie ld s o f so lid s a n d n itro g e n in w h ey s as liq u id b y -p ro d u c ts w ill pose a m a jo r c h a llenge in e c o n o m ic re tr iev a l a n d d isp o sa l to fu tu re c o tto n se e d p ro te in iso la te p rocesso rs .Proximate analyses

T ab le 2 su m m ariz e s th e p ro x im a te analyses o f th e freeze -d ried w h ey so lid s on a m o is tu re -free basis. U n d e rs ta n d a b ly , th e c o n s t itu e n ts sh o w n do n o t a d d u p to 100% b ecau se o f th e in d ire c t m e th o d s used in q u a n ti ta t io n . D a ta on th e o rig ina l f lo u rs a re a lso lis ted fo r c o m p a riso n . T h e re su lts again revea led to so m e e x te n t th e c h a ra c te ris tic s o f each w h ey a n d its p ro c

ess d e p e n d e n c y . P ro cess B w h ey m ig h t be c o n s id e re d as a c o m b in a tio n o f C-NSP a n d C-SP w h ey s based on th e n a tu re o f e ac h p ro cess . P ro cess C -N SP w h ey was fro m th e f irs t-s tep e x tr a c t io n , th u s i t c o n ta in e d m o re c a rb o h y d ra te s a n d less p ro te in (N x 6 .2 5 ) th a n C-SP w h ey w h ich w as fro m th e se c o n d -s te p a lk a lin e e x tr a c t io n . T h e re la tio n sh ip a m o n g th e m will be f u r th e r d iscu ssed la te r.

ELUT IO N VOLUME ------)

Fig. 1 —Absorption profiles o f glandless cottonseed wheys chromatographed on Sephadex G-75 column. (A ) Process B; (B) Process C-fJSP; (C) Process C-SP; (D ) Process F ; and (E) D ialyzed Process B whey. I, I I and I I I designate the three portions described in the text.

T he d ry so lid s f ro m P ro cess F w h ey w ere a lm o s t o n e -h a lf ash w h ic h w as, o f c o u rse , f ro m C aC l2 used in th e e x tra c t in g so lv e n t as m e n tio n e d e a rlie r . C o n sid e rab le d if f ic u lty w as e x p e r ie n c e d in freeze d r y in g a n d a n a ly z in g i t d u e to i ts h igh C aC l2 c o n te n t . T h e re fo re , P ro cess F w h e y fro m L C P f lo u r w as n o t p re p a re d a n d n o fu r th e r ana ly sis o f th is w h ey w as a t te m p te d .

O il, c ru d e f ib e r a n d to ta l o r free gossy- p o l c o n te n ts o f w h e y so lid s w ere neg lig ib le since th e w h e y s c o n s is te d o f o n ly w a te r-so lu b le su b s ta n c e s . As e x p e c te d , a sh c o n te n ts w ere h igh a n d w ere large ly c o n tr ib u te d by so d iu m h y d ro x id e an d p h o sp h o r ic ac id u sed in th e p ro te in e x t r a c tio n a n d p re c ip ita tio n s te p s o f th e iso la t io n p ro c e d u re s . T h is w as su b s ta n tia te d by m in e ra l a ssays to be d iscu ssed se p a ra te ly . E ac h m a jo r a n d so m e im p o r ta n t m in o r w h ey c o n s t i tu e n ts w ill be a lso fu r th e r d iscu ssed la te r .

Whey fractionationS e p h a d e x gel f i l t r a tio n c h ro m a to g

ra p h y was u sed to f ra c t io n a te a n d to b e t te r c o m p a re v a rio u s c o tto n s e e d w h ey s. T h e c h ro m a to g ra m s sh o w n in F ig u re 1 d e p ic t th e m a jo r d iffe re n ce a m o n g g lan d less c o tto n s e e d w h ey s a cc o rd in g to th e ir U V -a b so rp tio n p ro file s . P o r tio n s o f th e c h ro m a to g ra m s , I , II a n d III , re p re se n t re sp e c tiv e ly th e large m o lecu la r-w e ig h t c o m p o u n d s to ta l ly e x c lu d e d fro m th e gel, in te rm e d ia te f ra c t io n s c o n ta in in g m ajo r w h ey p ro te in s , a n d th e lo w m o lecu la r- w e ig h t w h ey c o n s t i tu e n ts su c h as sa lts , c a rb o h y d ra te s , a m in o ac id s , p e p tid e s an d p ig m e n ts . P ro cess B w h ey w as a ty p ic a l c o tto n s e e d w h ey a n d P ro cess C -N SP an d SP w h ey s w ere s im p ly p o r t io n s o f th e B w h ey i f th e f irs t p e ak (I in F ig . 1, C ) was e x c lu d e d . It w as o b se rv ed la te r th a t th is p e ak d isa p p e a re d a n d c o r re sp o n d e d to a p ro te in f ra c t io n w h ic h co u ld be p re c ip i ta te d fro m P ro cess C-SP w hey b y a d ju s tin g its p H fro m 7 to 4 . T h u s, P rocess C-SP


Table 3—Amino acid composition of cottonseed wheys (g/16g l\l)

Amino acidsGlandless wheys LCP deglanded whey

B Dialyzed B C-NSP C-SP F B C-NSP C-SP

Lysine (to ta l) 5.5 6.9 6.0 4.2 4.9 4.7 4.1 4.2(avail.) 5.2 6.9 5.6 4.1 3.9 4.5 4.1 4.1

H istid ine 2.0 2.1 1.8 2.3 2.2 1.7 1.1 2.4A rg in ine 12.7 12.3 12.4 13.0 13.5 13.3 12.6 11.0T ryp top han 1.0 1.4 1.2 1.2 1.3 1.0 1.2 1.6C ystine 5.0 5.0 4.4 3.0 3.8 5.2 4.1 3.5A spartic acid 7.1 7.4 7.0 6.7 6.7 8.2 9.5 8.1Threon ine 2.5 2.7 2.5 2.8 2.1 2.2 2.4 2.9Serine 2.4 2.8 1.2 3.2 2.7 2.3 2.1 3.7G lu tam ic acid 24.3 26.8 17.3 21.5 21.8 24.9 19.1 21.9Proline 3.0 3.3 4.1 3.1 2.3 3.0 2.7 3.8G lycine 3.6 3.8 3.3 4.2 3.4 3.4 3.3 3.9Alanine 2.6 3.0 2.9 3.1 2.2 2.1 2.4 3.3Valine 1.7 2.2 2.1 2.9 2.1 1.4 1.5 3.6M eth ion ine 0.9 1.3 0.8 1.8 1.3 1.2 0.9 1.8Isoleucine 1.1 1.4 1.3 2.1 1.3 1.0 0.9 2.7Leucine 2.1 2.6 2.5 4.4 2.7 2.0 2.7 5.0Tyrosine 2.7 3.1 2.5 2.4 2.3 2.6 2.2 3.1Pheylalanine 2.0 2.0 2.0 3.1 2.2 1.8 1.8 4.2

% A m in o acid N 78 84 74 81 77 78 76 82

w h e y a t pH 4 b e ca m e s im ila r to e ith e r P ro cess C -N SP o r P ro cess B w h ey s. T h is re la tio n s h ip can a lso be e x p la in e d by th e d iffe re n c e in th e p ro cesses u sed in p re p a rin g c o tto n s e e d p ro te in iso la te s as d e sc rib ed ea rlie r . P ro cess F w h ey was u n iq u e d u e to th e use o f C aC l2 in th e p ro te in iso la tio n p ro c e d u re . R e la tiv e ly less o f th e lo w m o le c u la r-w e ig h t c o m p o u n d s (III in F ig u re 1, D) w ere o b se rv ed in th is w h e y , in d ic a tin g th e p re sen ce o f c a lc iu m re n d e r in g th ese c o m p o u n d s less so lu b le . G el f i l t r a tio n c h ro m a to g ra m s o f L C P d e g la n d ed c o tto n s e e d w h e y s , n o t sh o w n h e re , w ere very s im ila r to th o se fro m g lan d less w heys.

T h e re su lts o b se rv ed in gel f i l t r a tio n o f c o tto n s e e d w h ey s w ere h ig h ly re p ro d u c ib le . V a lu ab le in fo rm a tio n w as p ro v id ed in a d d it io n to f r a c t io n a t io n o f w h ey c o m p o n e n ts a c c o rd in g to th e ir m o le c u la r w e ig h ts . L aw h o n e t al. (1 9 7 3 ) u sed i t in e v a lu a tin g m em b ran e p ro cess in g s o f c o tto n se e d w h ey s. F u r th e r in v es tig a tio n o f w h ey c o n s titu e n ts , e sp e c ia lly p ro te in s , w ill be c a rr ied o u t b y e m p lo y in g th is te c h n iq u e .

I t w as n o te d th a t g land less c o tto n s e e d w h e y s w ere s lig h tly b ro w n ish y e llo w in c o lo r a n d L C P c o tto n s e e d w h ey s w ere d a rk e r th a n g land less w h ey s. T hese co lo rs w ere in te n s if ie d o r d a rk e n e d b y in c re a s in g pH . T h e y se e m e d to a sso c ia te w ith p o r t io n s III o f th e gel f i l t r a tio n c h ro m a to g ram s in d ic a tin g p o ss ib ility o f rem o v a l th ro u g h u ltr a f i l t r a t io n o r d ia lysis.

W hey p ro te in sP ro te in p ro b a b ly is o n e o f th e m o st

im p o r ta n t c o n s t i tu e n ts o f c o tto n se e d

w h ey . As c o m m o n ly p ra c t ic e d , i ts d e te r m in a tio n has b een based u p o n to ta l n i t r o gen m u ltip lie d by 6 .2 5 . T h is req u ire s fu r th e r ju s t if ic a t io n b ecau se so m e c o t to n seed w h ey n itro g e n m ay n o t be re la te d to p ro te in s , p e p tid e s , o r a m in o acid s. N o n p ro te in n itro g e n (N P N ) as d e te rm in e d by so lu b le n itro g e n in 10% T C A so lu tio n ran g ed fro m 1 5 - 8 0 % o f th e to ta l n i t r o gen. N ev erth e le ss , m o st o f th is N PN was a m in o acid n itro g e n since a b o u t 80% (T ab le 3 ) o f th e to ta l n itro g e n w as in d ic a te d to be a m in o acid n i tro g e n . A fte r e x h au s tiv e d ia lysis versus d is tilled w a te r fo r 3 d ay s a t 0 -5 °C , P ro cess B w h ey re su lte d in th e gel f i l t r a tio n c h ro m a to g ra m sh o w n in F ig u re 1, E , an d w as neg lig ib le in N PN . T h u s , N PN c o rre sp o n d e d to p o r tio n 111 o f th e c h ro m a to g ra m s an d co u ld a lso p o ss ib ly re p re se n t so m e d ia ly zab le lo w m o le c u la r w e ig h t p ro te in s w h ich m ig h t n o t have th e sam e fu n c tio n a l c h a ra c te r is tic s o f m u c h la rger m o le c u la r w e ig h t w hey p ro te in s o r m a jo r p ro te in iso la te s . T h e m o le c u la r w e ig h ts o f th e m a jo r c o tto n s e e d w h ey p ro te in s (in P o rt io n II, F ig u re 1) w ere in th e ran g e fro m4 .5 x 10 3 to 1.6 x 104 a cc o rd in g to S e p h a d ex gel f i l t r a tio n c h ro m a to g ra p h y . R esu lts o f m o re e x ten siv e s tu d y w ill be p u b lish ed in a se p a ra te p a p e r.

A m in o acid c o m p o s itio n s o f th e u n f ra c t io n a te d w h ey s are lis ted in T ab le 3. C o m p are d to th e ir p a re n t flo u rs (L aw h o n e t a l., 1 9 7 2 b ) , i t can be seen th a t lysine a n d cy stin e c o n te n ts in P ro cess B and C -N SP w h ey s w ere su b s ta n tia lly in creased a t th e e x p en se o f slig h t re d u c tio n s in se rin e , v a lin e , iso le u c in e , and p h e n y la la n in e c o n te n ts . T h ese in c reases w ere also

n o tic e d w h en th ese w h ey s w ere c o m p a re d to th e m a jo r c o tto n s e e d p ro te in iso la te (Iso la te II o r SP Iso la te ) re p o r te d by M artin ez e t al. (1 9 7 0 ) . S im ila r re su lts w ere a lso o b se rv ed in LC P d e g la n d ed c o tto n se e d w h ey s e x c e p t th a t ly s in e w as n o t in c re a sed as su c h . D ialysis f u r th e r in creased ly sin e c o n te n t to a b o u t 7 g /1 6 g N as sh o w n by an e x am p le given in T ab le 3 fo r th e dialyz.ed B w h ey . S ince th e d e fic ien cy o r lack o f a v a ilab ility o f ly sin e and su lfu r a m in o acid s is o f p r im a ry c o n cern in m o st leg u m es a n d cerea ls , h ig h e r ly sin e an d su lfu r a m in o acid s (c y s tin e p lu s m e th io n in e ) in c o tto n s e e d w h ey s w ill be o f in te re s t to fo o d u se rs in te rm s o f th e ir n u tr i t io n a l s ig n ifican ce .

W hey p ro te in s , by d e f in i t io n , are w a te r-so lu b le p ro te in s a t all pH ranges used in th e p ro te in iso la tio n p ro cess . T h e re fo re , n itro g e n so lu b ili ty o f w h ey so lid s w as n e a r 100% as e x p e c te d , e x c e p t P rocess C-SP w h ey w h ic h c o n ta in e d a pre- c ip itab le p ro te in f ra c tio n a t pH 4 as m e n tio n e d ea rlie r . T h is p ro te in f ra c t io n c o u ld be re la te d to th e n o n -s to ra g e p ro te in f ra c tio n o f c o tto n s e e d b y p re p a ra tio n (B e ra rd i e t a l., 1 9 7 2 ), r a th e r th a n to be c o n s id e re d as o n e o f th e w h ey p ro te in s by d e f in i t io n . C h illing a t r e fr ig e ra te d te m p e ra tu re im p a r te d tu r b id i ty to th e w h ey s an d p re c ip ita te d a sm all f ra c t io n o f c o t to n se e d w h ey p ro te in . H o w ev e r, th is e f fe c t w as to ta l ly rev ersib le a n d c o u ld be varied b y pH a n d io n ic s tr e n g th . I t c o u ld be su sp e c te d th a t th is c o ld -p re c ip ita b le p ro te in o f c o tto n s e e d m ig h t have so m e th in g in c o m m o n w ith a c o ld -p re c ip ita b le p ro te in f ra c t io n o f so y b e a n (B riggs and M an n , 195 0 ).

COTTONSEED WHEYS- 181

Table 4—Mineral analysis of cottonseed flours and wheys on dry weight basis

MineralGlandless

flourGlandless wheys LCP

FlourLCP wheys

B C-NSP C-Sp B C-NSP C-SP

Ash 6.58 21.2 16.2 14.5 9.2 25.6 26.7 16.6K 1.82 3.04 4.15 2.38 1.94 3.63 4.63 2.68P (total) 1.64 6.64 4.78 3.46 1.70 7.50 7.60 4.20

(inorg.) 0.16 5.90 4.02 1.80 0.11 6.70 4.80 1.90Mg 0.74 0.78 0.86 0.74 0.89 1.20 1.19 0.89Ca 0.51 0.24 0.20 0.24 0.23 0.15 0.18 0.18Na 0.036 1.60 0.02 1.83 0.005 2.60 0.021 3.07

Fe 130 20 9 110 104 9 24 97Zn 120 82 80 70 75 54 54 41Pb 0.32 < 0.1 0.4 0.4 0.5 1.3 2.3 1.2

Heat stability of whey proteins was preliminarily examined by heating reconstituted Process B whey at 2% protein level. Heatings at 63 and 100°C for 30 min did not cause any appreciable precipitation. Utilization of cottonseed whey proteins for food may take advantage of their high water solubility and heat stability. More comprehensive study in these areas will be carried out in the future. Carbohydrates

Water-soluble carbohydrates of cottonseed flours were recovered into wheys. Analysis of glandless cottonseed Process B whey indicated that raffinose and sucrose were the major carbohydrates representing about one-half and one-fourth of the total carbohydrates, respectively. A small amount of stachyose and some other hexoses and pentoses were also present in this whey. Mixture of several carbohydrates of high water solubility makes it impossible to recover carbohydrates from cottonseed whey through concentration and crystallization as is done with lactose from milk whey (Jenness and Patton, 1959). Recovery and utilization of cottonseed whey carbohydrates will pose entirely different problems and be more complicated.

When cottonseed Process B whey was fractionated by gel filtration (Fig. 1, A), 95% of the carbohydrates were found in portion III as simple carbohydrates, whereas, the remaining 5% were mostly associated with portion I. Further examination of both carbohydrates (Volkin and Cohn, 1954; Dubois et al., 1956) and proteins (Lowry et al., 1951) in portions 1 and II revealed that they had protein- carbohydrate ratios of 1:0.46 and 1:0.014, respectively. The carbohydrates in portion I were mainly pentoses. It is therefore concluded that the major cottonseed whey proteins (in II) were associated with small amounts of carbohydrates and there was a large molecular-weight fraction (in I) soluble in water and con

Table 5—Vitamin assays of cottonseed flours and wheys (per 100g dry solids)

VitaminGlandless

Flour Process B WheyLCP deglanded

Flour Process B Whey

Vitamin A (IU) 446 66.4 170 156Carotene (mg) NDa — ND -a-tocopherol (mg) 1 57 0.5 0.5 0.5Thiamine (mg) 2 16 3.98 2.47 2.50Riboflavin (mg) 0 41 0.98 0.39 1.05Niacin (mg) 4 32 8.78 6.18 10.0Pyridoxine (mg) 0 79 0.76 0.77 1.10Pantothenic acid (mg) 0 46 3.12 1.81 4.88Choline (mg) 442 268. 446. 363.Total ascorbic acid (mg) 2 45 ND 4.31 4.80

N D : none detected

taining almost equal amounts of carbohydrates and proteins. The pentoses in l might be very likely associated with pentose protein fraction of cottonseed (Fontaine, 1948), and less likely with soluble ribonucleic acids if present. Similar results were also obtained from other wheys. Since all soybean proteins contained some carbohydrates (Wolf et al.,1966), it was not surprising to see cottonseed whey proteins also containing some carbohydrates. Interaction between cottonseed whey proteins and carbohydrates still requires further investigation.

Minerals and vitaminsMinerals and vitamins are of particular

interest here to determine whether their presence in cottonseed wheys have any significant value. Analyses are shown in Table 4 and 5, respectively, for minerals and vitamins of cottonseed wheys. Data obtained from their parent flours are also listed for comparison. Obviously, cottonseed wheys can be considered as concentrates of minerals and stable water-soluble vitamins from cottonseed flours, as reflected by increasing ash and potassium,

and most of the water-soluble vitamins in wheys. High inorganic phosphorus content in all wheys and high sodium content in B and C-SP wheys clearly indicated that they were contributed by the phosphoric acid and sodium hydroxide used in the protein isolation processes. The low sodium content in C-NSP wheys occurred because these wheys were prepared from water extractions without any addition of alkaline.

Since phytate phosphorus accounted for more than 70% of the total phosphorus in cottonseed (Dollear and Mark- ley, 1948; Pons et al., 1953), the fate of phytic acid throughout the Process B was surveyed. It was found that most of the phytate phosphorus was not extracted but remained in the residue fraction. Only a negligible amount was detected in whey. Therefore, there was no need to consider in this study the problems usually associated with phytic acid (Fontaine, 1948; Cosgrove, 1966).

It was noted that vitamin A, thiamine, niacin, pyridoxine and choline contents in cottonseed B whey were higher than those of dried milk whey solids (Hartman

1 8 2-JOURNAL OF FOOD SCIENCE- Volume 39 (1974)

and Dryden, 1965). High vitamin A content in Process B whey solids as determined by Carr-Price reaction (Freed, 1966) was rather unique, especially when the presence of carotene was not detectable. However, this result has been confirmed by the same colorimetric measurement but not by a biological assay.Future outlook

The results of this study have demonstrated the variations among different type cottonseed wheys according to their chemical compositions and characteristics. However, a more extensive study of each major whey component will be conducted.

It can be seen that recovery of the utilizable whey constituents by proper fractionation techniques will not only reduce processing loss but also minimize disposal problem. Investigations in these areas are currently in progress. Process B whey is very similar to a whippable extract, Product C, reported by Lawhon et al. (1972a), and also possesses whipping characteristic. Cottonseed whey proteins may be incorporated into baked products and beverages as being assessed by Lawhon et al. (1974). Cottonseed carbohydrates may be used as a carbon source for the growth of yeast for food or feed purposes (Dollear and Markley, 1948). Finally, recycling of liquid effluents after recovering most of the utilizable whey constituents (Lawhon et al., 1973) may leave no significant amount of cottonseed wheys to be discharged into domestic water bodies.

REFERENCESAmes, S.R. 1971. Determ ination of vitamin E

in foods and feeds—A collaborative study. J.AOAC 54: 1.

AOAC. 1970. “ Official Methods of Analysis,” 11th ed. Assoc, of Offic. Anal. Chem., Washington, D.C.

AOCS. 1971. “ Official and Tentative M ethods,” 3rd ed. Am. Oil Chem. Soc., Chicago,111.

Berardi, L.C., Fernandez, C.J. and Martinez, W.H. 1972. Cottonseed protein isolates: Selective precipitation procedure. Presented at the 32nd Annual Meeting of the Institute of Food Technologists, May 22—24, Minneapolis, Minn.

Berardi, L.C., Martinez, W.H. and Fernandez,C.J. 1969. Cottonseed protein isolates: T w o -s te p extraction procedure. Food Technol. 23(10): 75.

Briggs, D.R. and Mann, R.L. 1950. An electrophoretic analysis of soybean protein. Cereal Chem. 27: 243.

Cosgrove, D.J. 1966. The chemistry and biochemistry of inositol polyphosphates. Rev. Pure & Appl. Chem. 16: 209.

Dollear, F.G. and Markley, K.S. 1948. Miscellaneous constituents. In “ Cottonseed and Cottonseed Products,” p. 466, Ed. Bailey, A.E. Interscience Publishers, Inc., New York, N.Y.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. 1956. Colorimetric m ethod for determ ination of sugars and related substances. Anal. Chem. 28: 350.

Fontaine, T.D. 1948. Cottonseed proteins. In “ Cottonseed and Cottonseed Products,” p. 409, Ed. Bailey, A.E. Interscience Publishers, Inc., New York, N.Y.

Freed, M. 1966. “ Method of Vitamin Assay,” 3rd ed. Assoc, of Vitamin Chem., Inc., Interscience Publishers, New York, N.Y.

Hartman, A.M. and Dryden, L.P. 1965. “ Vitamins in Milk and Milk Products.” Am. Dairy Sci. Assoc.

Jeffrey, D.C., Arditti, J. and Ernest, R. 1969. Determ ination of di-, tri- and tetrasaccha- rides in m ixtures with their com ponent moieties by th in layer chrom atography. J. Chromatog. 41: 475.

Jenness, R. and Patton, S. 1959. “ Principles of Dairy Chemistry.” John Wiley & Sons, Inc., New York, N.Y.

Lawhon, J.T ., Cater, C.M. and Mattil, K.F. 1972a. A whippable extract from glandless cottonseed flour. J. Food Sci. 37: 317.

Lawhon, J.T ., Rooney, L.W., Cater, C.M. and Mattil, K.F. 1972b. Evaluation of a protein concentrate produced from glandless co ttonseed flour by a wet-extraction process. J. Food Sci. 37: 778.

Lawhon, J.T ., Lin, S.H.C., Cater, C.M. and Mattil, K.F. 1973. Recycling of effluent from membrane processing of cottonseed

wheys. Food Technol. 27(2): 26.Lawhon, J.T ., Lin, S.H.C., R ooney, L.W.,

Cater, C.M. and Mattil, K.F. 1974. Utilization of cottonseed whey protein concentrates produced by ultrafiltration . J. Food Sci. 39: 183.

Lowry, O.H., Rosebrough, N .J., Farr, A.L. and Randall, R.J. 1951. Protein m easurem ent with the Folin phenol reagent. J. Biol. Chem. 193: 265.

Martinez, W.H. and Berardi, L.C. 1971. The technology of cottonseed proteins. In “ Proceedings of the 20th Oilseed Processing Clinic,” p. 51. USDA, ARS 72—93, New Orleans, La.

Martinez, W.H., Berardi, L.C. and Fernandez, C.J. 1971. Method for preparation of p ro tein isolates from calcium chloride extracts of defatted oilseeds. Presented at the 56th Annual Meeting, Am. Assoc. Cereal Chem., Oct. 10—14, Dallas, Tex.

Martinez, W.H., Berardi, L.C. and G oldblatt, L.A. 1970. Cottonseed protein products— Com position and functionality . J. Agr. Food Chem. 18: 961.

Oberleas. D. 1971. The determ ination of phytate and inositol phosphate. In “ M ethods of Biochemical Analysis,” Vol 20, p. 87, Ed. Glick, D. Interscience Publishers, New York. N.Y.

Pons, W.A. Jr., Stansbury, M.F. and Hoffpauir, C.L. 1953. An analytical system ‘for determining phosphorus com pounds in plant materials. J. AOAC 36: 492.

Smith, A.K. 1958. Vegetable protein isolates. In “Processed Plant Protein Foodstuffs,” p. 249, Ed. Altschul, A.M. Academic Press, New York, N.Y.

Sumner, J.B .1944. A m ethod for the colorim etric determ ination of phosphorus. Science 100: 413.

Vix, H.L.E., Eaves, P.H., Gardner, H.K. and Lambou, M.G. 1971. Degossypolyzed co ttonseed flour—The liquid cyclone process. J. Am. Oil Chem. Soc. 48: 611.

Volkin, E. and Cohn, W.E. 1954. Estim ation of nucleic acid. In “ Methods of Biochemical Analysis,” Vol 1, p. 287, Ed. Glick, D. Interscience Publishers, New York, N.Y.

Wolf, W.J., Sly, D.A. and Kwolek, W.F. 1966. Carbohydrate content of soybean proteins. Cereal Chem. 43: 80.


This research was funded in parts by USDA Research Agreement No. 12-14-100-11021 (72) and by the Natural Fibers & Food Protein Comm ittee of Texas. Appreciation is expressed to Jack Pirson for his technical assistance.

J. T. L A W H O N , S. H. C. U N , L . W. R O O N E Y , C. M . C A T E R a n d K. F. M A T T IL

F o o d P ro te in R & D C en te r, Texas A & M U n iv e rs ity , C o llege S ta tio n , T X 7 7 8 4 3

UTILIZATION OF COTTONSEED WHEY PROTEIN CONCENTRATES PRODUCED BY U LTRA FILTRA TIO N

INTRODUCTION

O I L S E E D P R O T E I N i s o l a t i o n p r o c e s s e s r e s u l t i n a w h e y - t y p e l i q u i d b y - p r o d u c t a s d o c h e e s e m a n u f a c t u r i n g p r o c e s s e s . T h e p e r e n n i a l p r o b l e m o f c h e e s e w h e y u t i l i z a t i o n a n d / o r d i s p o s a l i s w e l l k n o w n ( G r o v e s a n d G r a f , 1 9 6 5 ) . T h i s i s s u e i s p o t e n t i a l l y t h e p r o b l e m o f f o o d p r o c e s s o r s i s o l a t i n g p r o t e i n f r o m c o t t o n s e e d a n d o t h e r o i l s e e d s .

P r o c e s s i n g o f c h e e s e w h e y w i t h s e m i

p e r m e a b l e m e m b r a n e s h a s p r o v e n t o b e e c o n o m i c a l l y a t t r a c t i v e o n a c o m m e r c i a l

s c a l e ( E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , P r o j . # 1 2 0 6 0 D X F , 1 9 7 1 ) . A l s o , t h e f e a s i

b i l i t y o f t r e a t i n g s o y w h e y b y m e m b r a n e

p r o c e s s e s h a s b e e n d e m o n s t r a t e d ( G o l d s m i t h e t a l . , 1 9 7 2 ) . T h e o p e r a t i o n u s e d

w a s a l m o s t i d e n t i c a l t o t h a t u s e d c o m m e r c i a l l y f o r t h e t r e a t m e n t o f c h e e s e

w h e y . B y s e l e c t i n g m e m b r a n e s h a v i n g p r o p e r p o r e s i z e s , s o l u b l e w h e y c o n s t i t u

e n t s c a n b e f r a c t i o n a t e d a n d c o n c e n t r a t e d w i t h o u t h e a t i n g .

I n r e s e a r c h p r e s e n t l y u n d e r w a y a t t h e F o o d P r o t e i n R e s e a r c h & D e v e l o p m e n t C e n t e r ( F P R D C ) , T e x a s A & M U n i v e r s i t y ,

c o t t o n s e e d w h e y s a r e b e i n g m e m b r a n e p r o c e s s e d t o r e c o v e r v a l u a b l e p r o t e i n c o n

s t i t u e n t s ( c o t t o n s e e d w h e y s m a y c o n t a i n f r o m 2 1 - 3 1 % o f t h e n i t r o g e n i n t h e s o u r c e f l o u r e x t r a c t e d ) a n d t o s i m u l t a n e o u s l y p r e v e n t t h e p o l l u t i o n w h i c h w o u l d e n s u e i f w h e y s w e r e d i s c a r d e d i n t o d o m e s t i c w a t e r b o d i e s .

I n t h e w o r k r e p o r t e d h e r e , t h r e e g l a n d l e s s c o t t o n s e e d p r o t e i n p r o d u c t s o b t a i n e d b y u l t r a f i l t r a t i o n ( U F ) w e r e e v a l u a t e d f o r p o t e n t i a l u s e i n p r o t e i n f o r t i f i c a t i o n o f b r e a d a n d n o n c a r b o n a t e d , “ a d e ” - t y p e b e v e r a g e s o r i n s t a n t f r u i t d r i n k s , a n d a l s o a s w h i p p i n g p r o d u c t s . T h e a m i n o a c i d c o m p o s i t i o n o f e a c h U F p r o d u c t w a s d e t e r m i n e d a n d c o m p a r e d w i t h t h e a m i n o a c i d c o m p o s i t i o n o f t h e p a r e n t g l a n d l e s s

c o t t o n s e e d f l o u r .

EXPERIMENTAL

Preparation o f u ltrafiltration products

Three d ifferent glandless co tton seed wheys were prepared in the F P R D C p ilot p lant. Tw o protein isolation procedures were used. Multiple runs were made w ith each procedure. In each run protein was extracted from 4 0 lb o f Rogers G L-7 glandless cottonseed flour.

O ne process (designated Process B) co n sisted essentially o f extractin g flour w ith tap

w ater (1 0 :1 solvent-to-solids ratio by w eight) adjusted to pH 10 with sodium hydroxide, separating the solubilized protein and other soluble com ponen ts from an insoluble residue by cen trifugation and then dividing the isolated protein in to storage (SP ) and nonstorage p*o- tein (N SP) curds by sequential precip itations at pH 7 and pH 4 , respectively. Process B produces one m ajor w hey.

T he second iso lation process used (designated Process C) was the tw o step, two solvent procedure previously described in the literature (Berardi e t a l., 1 9 6 9 ) . This la tter procedure yields tw o m ajor wheys. B oth isolation procedures were developed at the U SD A Southern Regional Research C en ter, New Orleans.

Each w hey was pasteurized by heating to 6 3 °C for 30 min before m em brane processing with a R O pak Single-Core Reverse Osmosis (R O ) Machine m anufactured by Ray Pak, In c ., W estlake Village, C alif. This U F /R O m achine was equipped w ith 24 sq ft o f tubular type U F m em branes ( 5 ,0 0 0 - 1 0 ,0 0 0 M.W. cut o ff) and 24 sq f t o f R O m em branes (90% NaCl re jection ). T he cellulose acetate m em branes are supported on the exterio r o f 5 /8 in. diam ceram ic cores. Pasteurized, un filtered whey was circulated through the annular space surrounding the rod-like m em brane cores centered in 6 ft long stainless steel tubes. T h e U F m em brane allowed passage o f salts and carboh ydrates in to the U F filtrate (perm eate) and retained and co n cen trated nitrogenous com pounds. U F perm eate was then fed in to the second stage, i.e ., RO m em branes, which concen trated salts and carbohyd rates passing ca. 10% o f the salts.

U F co n cen trate from each w hey was spray dried with an A nhydro Spray Dryer Typ e III-A No. 2. An inlet air tem perature o f 1 4 9 - 1 5 4 ° C and an ou tle t tem perature o f 8 5 - 9 1 ° C were used.

The product from ultrafiltration o f Process B Whey is designated herein as U F B product. Products from Process C W heys are designated herein as U F C-NSP product and U F C-SP product.

A nalytical procedures

A m ino acid analyses o f glandless cotton seed flour and spray-dried U F products obtained in the study (with the excep tion o f tryptophan and cystine) w ere qu antitatively determ ined by the procedure developed by Spackm an et al.(1 9 5 8 ) . Tryptoph an was determ ined by the m ethod o f K oh ler and Palter ( 1 9 6 7 ) . Cystine was measured using a m od ification o f the procedure by Schram et al. (1 9 5 4 ).

Sam ples were hydrolyzed for determ ination o f all am ino acids excep t cystine and try p tophan in constant-boiling HC1 for 24 hr under a nitrogen flush. Procedures for preparing protein hyd rolysate for cystine and tryptophan are specified in the m ethods cited.

M oisture, oil, crude fiber, ash and free and tota l gossypol w ere determ ined according to

standard AO CS m ethod s (A O C S, 1 9 7 1 ). N itrogen was determ ined by the m icro-K jeldahl m ethod. C arbohydrates in term s o f glucose were measured co lorim etrica lly by the pheno- su lfuric acid m ethod o f D ubois e t al. ( 1 9 5 6 ) . T o ta l and inorganic phosphorus were determined by the m eth od according to Sum ner (1 9 4 4 ) .

Protein fo rtifica tio n o f breads

The U F products were tested for protein enrichm ent o f w heat flour breads in w heat flour- whey protein blends proportioned to contain 17 .5% protein on a 14% m oisture basis. G landless co tton seed flour was also blended with w heat flour in the sam e m anner and included in the statistically designed experim ent for com parative purposes. An all-w heat flour (1 2 .4 % protein in the flour) bread was baked as a co n trol.

A random ized com p lete-b lock design was follow ed in cond ucting baking trials. Ten treatm ents consisting o f the five m aterials or blends, m entioned above, each baked with and w ithout 1.5% sodium stearoyl-2 lacty la te (S S L ) added as a dough cond ition er were com pared in the design. One com plete b lo ck o f ten treatm ents (on e lo a f per treatm en t) was baked on a given day. Three b lo ck s o f treatm en ts were baked during the experim en t.

Dough m ixing properties

M ixing properties o f the m aterials tested in 17 .5% protein blends were evaluated with a Brabender Farinograph according to the standard AACC ( 1 9 7 0 ) procedure. T h e instrum ent was equipped with a 50g stainless steel bow l. W ater absorption was measured as the am ount o f w ater required to cen ter the curve on the 5 0 0 Brabender unit line, corrected to 14% flour m oisture.

Baking procedure

Pound loaves were baked w ith a short-tim e dough system w hich required high yeast and brom ate co n cen tratio n s, low sugar and sh ortened ferm entation tim e. T he form ula on a lOOg flour basis was: yeast, 3g; sugar, 5g ; salt, 2g; shortening, 3g. S S L , when used, was added at a 1.5% level. Each dough was m ixed with a Model 1 20-A H obart M ixer equipped w ith a M cD uffy bow l. Each dough was given optim um w ater absorption , ox id ation level and m ixing tim e. T he optim um s were established by prelim inary baking evaluation o f several doughs from each flour blend. Each dough was scaled to 540g and ferm ented for 4 0 m in at 3 2 °C and 95% relative hum idity. T h ey w ere sheeted through a N ational sheeter a t 9 /3 2 in. and 7 /3 2 in. settings and m oulded through a N ational m oulder. Each dough was proofed to height, and baked for 25 min at 2 1 8 ° C. L o a f volum e was determ ined by rape seed displacem ent. C olor m easurem ents on breads were made with a Hunterlab Digital C olor and C olor D ifference M eter M odel D 25D .

Volume 3 9 (1 9 7 4 )—JO U RN AL OF FOOD SCIENCE- 183

1 8 4 - JO U R N A L OF FOOD S C IE N C E - Volume 3 9 (1974)

Table 1—Analytical data on ultrafiltration products and the glandless cottonseed flour extracted for protein isolation(%.dry weight basis)

Product Mois- Crude Gossypol Nitrogen Protein Phosphorous Carbo-Identifications ture Ash fiber Oil Total Free Total Nonprotein (N X 6.25) Total Inorg. hydrates

UF B 6.1 8.4 0.01 0.1 0.02 0.00 11 .6 3.6 72.3 2.30 2.08 23.3UF C-NSP 6.7 6.2 0.08 0.6 0.02 0.01 10.3 2.6 64.3 1.49 1.17 33.8UF C-SP 5.5 6.5 0.01 3.4 0.02 0.01 12 .1 1.4 75.9 1.47 0.74 8.3Glandless co tton

seed flour 6.9 7.4 2.50 1.2 0.02 0.02 10.3 0.28 64.3 1.47 0.16 14.2

3 Products designated by membrane type, isolation process and whey type.

Protein fo rtifica tio n o f noncarbonated beverages

T he U F B protein product was selected for fortify in g an “ ade”-type beverage and an instant fruit drink w hich are sold as powder to be reconstitu ted with water. U F B product was dispersed in distilled w ater and the resulting protein solution-suspension centrifuged to obtain a clear supernatant for use (instead o f w ater) in recon stitu tion . An orange-flavored “ ade” beverage and an orange-flavored fruit drink were su bjected to organoleptic testing. Supernatants were prepared w ith su fficien t protein to give 1%, 2% and 3% protein levels in the test beverages. T h e co n tro l or 0% protein sam ple was prepared according to instructions on a presw eetened package or ja r o f com m ercial beverage powder.

A panel o f ten untrained taste judges each scored four samples (a co m p lete b lock o f treatm ents in the random ized b lock design used) at three judging sessions. T he same procedure was follow ed in separate tests o f the “ ade” drink and the instant fruit drinks. T he four treatm ents consisted in each instan ce o f beverage fortified with 0% , 1%, 2% , or 3% protein .

Sam ples were served each judge in coded paper cups with covers. Drinking straws were inserted through the covers for tasting w ithout observing co lo r d ifferences, should any be discernible. Sam ples were scored using a linear scale with possible values ranging from 1 for “ d istastefu l” to 10 for “ ex ce llen t” . An analysis o f variance was perform ed on each set o f test data.

W hipping-property m easurem ents

T he general procedure used in m aking w hipping tests was as follow s:(a) Sam ples o f dry product to be w hipped were

added to 10 0 ml o f distilled w ater, stirred gently to disperse and the solution-suspension adjusted to eith er pH 4 .5 or pH 7 w ith H 3PO „.

(b ) T h e m ixture was then whipped for 6 min at a speed for heavy beating (M ixm aster speed12).

(c ) A fter 6 m in foam viscosity was measured with a Model L V T -E B rookfield viscom eter, using appropriate T -bar spindles at 3 or 6 rpm.

(d) 75g o f sugar were then added to the whipped m aterial, and whipping resumed for an additional 2 min.

(e) M easurem ents in (c ) were repeated.V olum es be fo re and after whipping were

recorded and the percent o f volum e increase calcu lated as follow s:

V o l a fter V o l before % V o l _ whipping whipping ^increase y 0 | before whipping


P R O X I M A T E A N A L Y S E S o f t h e U F

p r o d u c t s a n d p a r e n t g l a n d l e s s c o t t o n s e e d f l o u r a r e i n T a b l e 1 . T h e p r o t e i n c o n t e n t ( N x 6 . 2 5 ) o f U F p r o d u c t s r a n g e d f r o m

6 4 . 3 % ( d r y w t b a s i s ) f o r U F C - N S P t o 7 5 . 9 % f o r U F C - S P . A s m e m b r a n e t e c h

n o l o g y a d v a n c e s a n d s a l t s a n d s u g a r s a r e m o r e c o m p l e t e l y s e p a r a t e d f r o m t h e p r o t e i n b y U F m e m b r a n e s t h e s e p r o t e i n l e v e l s c a n b e f u r t h e r e l e v a t e d . A p p r o x i -

Table 2—Am ino acid analysis of glandless cottonseed flou r andglandless cottonseed whey u ltra filtra tion protein products

Am ino acids

Glandless cottonseed

flou r g/16g N

UF B g/16g N

UF C-NSP g/16g N

UF C SP g/16g N

Lysine 4.0 6.1 a 9 5,9Histidine 2.6 3.2 1.7 2.7Ammonia 1.8 1.8 1.7 1.8Arginine 11.7 12.8 12.3 10.6Tryptophan 1.5 1.3 1.3 1.6Cystine 2.4 5.7 5.2 2.3Aspartic acid 9.1 6.3 6.6 8.4Threonine 3.2 2.5 2.8 3.9Serine 4.3 2.5 2.4 4.1Glutamic acid 20.5 26.3 23.7 19.3Proline 3.8 3.1 2.9 3.9Glycine 4.2 3.4 3.1 4.6Alanine 3.9 2.4 2.9 4.6Valine 4.5 1.9 1.9 4.5Methionine 1.4 1.4 1.2 2.2Isoleucine 3.1 1.2 1.1 3.4Leucine 6.0 2.3 2.2 6.5Tyrosine 3.3 3.1 2.8 3.6Phenylalanine 6.0 1.9 1.9 4.7

Totals 97.2 88.3 84.6 98.6Available lysine 3.8 6.1 6.4 5.6— — —

Table 3—Farinograph water absorption, m ixing time and stab ility of the wheat-cottonseed protein material blends3

WaterCottonseed

materialsabsorption

i%)bMixing time

(min)Stability

(min)

controlglandless

61.8 7.5 10.0

cottonseed flou r 66.9 5.0 5.5UF B 55.9 6.5 3.5UF C-SP 67.0 6.5 8.0UF C-NSP 52.5 4.5 5.0

3 17.5% protein in blend b Expressed on 14% moisture basis

PROTEIN CO N CE NTR ATES FROM COTTONSEED W H E Y S -ÌZ 5

m a t e l y o n e - f i f t h o f t h e n i t r o g e n w a s s o l u

b l e i n a 1 0 % s o l u t i o n o f t r i c h l o r o a c e t i c a c i d .

A s h w a s r e d u c e d f r o m 2 4 . 3 % i n u n

f r a c t i o n a t e d w h e y s o l i d s t o 8 . 4 % i n U F B p r o d u c t o n a d r y w e i g h t b a s i s . P r o d u c t

U F C - N S P w a s c o n s i s t e n t l y h i g h e r i n c a r

b o h y d r a t e s d u e t o c h a r a c t e r i s t i c d i f f e r e n c e s i n e x t r a c t i o n p r o c e d u r e s u s e d .

A m i n o a c i d a n a l y s e s o f t h e U F p r o d

u c t s a n d p a r e n t f l o u r a r e i n T a b l e 2 . V a l

u e s s h o w n a r e m e a n s o f d u p l i c a t e a n a l y s e s . V a l u e s w h i c h w e r e i n c r e a s e d o r

d e c r e a s e d m o r e t h a n 3 3 % f r o m t h o s e o f t h e s o u r c e f l o u r b y U F f r a c t i o n a t i o n a r e

u n d e r l i n e d .

Protein fortification of breadsM i x i n g p r o p e r t i e s o f d o u g h s p r e p a r e d

f r o m w h e a t - c o t t o n s e e d p r o d u c t b l e n d s a s

Fig . 1 —P o u n d loaves o f b re a d b a k e d w ith b le n d s o f w h e a t f lo u r a n d U F p r o te in p r o d u c t o f

g land less c o tto n s e e d f lo u r w ith o u t SSL added . L o a f A c o n ta in s 100% w h e a t f lo u r . L o a f B

c o n ta in s R ogers G L -7 g land less c o tto n s e e d f lo u r , L o a f C c o n ta in s U F B , L o a f D co n ta in s U F C-SP, a n d L o a f E c o n ta in s U F C-NSP.

F ig . 2 —P o u n d loaves o f b re a d b a k e d w ith b le n d s o f w h e a t f lo u r a n d U F p r o te in p ro d u c ts o r g land less c o tto n s e e d f lo u r w ith a n d w i th o u t 1.5% S SL added . Loaves A th ro u g h E c o n ta in m a te ria ls as in d ic a te d b e n e a th F ig u re 1. The u p p e r ro w c o n ta in s n o SSL a n d th e lo w e r ro w

has S SL added.

m e a s u r e d b y a F a r i n o g r a p h a r e g i v e n i n

T a b l e 3 . T h e p r e s e n c e o f U F B a n d U F

C - N S P i n t h e d o u g h s d e c r e a s e d w a t e r a b s o r p t i o n c o n s i d e r a b l y . B u t t h e r e w a s a n

i n c r e a s e i n w a t e r a b s o r p t i o n o v e r t h a t o f t h e c o n t r o l w h e n U F C - S P a n d g l a n d l e s s

c o t t o n s e e d f l o u r w e r e p r e s e n t i n t h e

d o u g h . M i x i n g t i m e a n d s t a b i l i t y a r e a l s o p r e s e n t e d i n T a b l e 3 . T h e s t a b i l i t y o f t h e d o u g h d e c r e a s e d m a r k e d l y w h e n U F B a n d U F C - N S P w e r e p r e s e n t a n d w a s s l i g h t l y l e s s t h a n t h e c o n t r o l w h e n U F C - S P w a s p r e s e n t .

D o u g h s b e c a m e v e r y s t i c k y w h e n f a r i n o g r a p h w a t e r a b s o r p t i o n s f o r U F B a n d U F C - N S P ( T a b l e 3 ) w e r e u s e d f o r b a k i n g . O p t i m u m b a k i n g w a t e r a b s o r p t i o n w a s d e t e r m i n e d f o r e a c h m a t e r i a l a n d a l l o p t i m u m b a k i n g w a t e r a b s o r p t i o n s w e r e l e s s t h a n t h e f a r i n o g r a p h a b s o r p t i o n s .

P r o o f i n g t i m e s o f d o u g h s w i t h U F B a n d U F C - N S P w e r e m e a s u r a b l y h i g h e r t h a n f o r d o u g h s w i t h U F C - S P a n d g l a n d

l e s s c o t t o n s e e d f l o u r ( T a b l e 4 ) .L o a f v o l u m e s w e r e a n a l y z e d s t a t i s t i

c a l l y b y t h e a n a l y s i s o f v a r i a n c e . T r e a t m e n t m e a n s f r o m t h r e e r e p l i c a t i o n s d i f f e r e d s i g n i f i c a n t l y a t 5 % a n d 1 % l e v e l s o f s i g n i f i c a n c e b y F - t e s t . L o a f v o l u m e m e a n s s h o w n t o b e s i g n i f i c a n t l y d i f f e r e n t b y D u n c a n ’s m u l t i p l e r a n g e t e s t a t t h e 5 % l e v e l a r e i n d i c a t e d i n T a b l e 4 .

C r u s t c o l o r o f b r e a d s m a d e w i t h U F B a n d U F C - N S P w a s d a r k e r t h a n t h o s e o f b r e a d s m a d e w i t h U F C - S P a n d g l a n d l e s s f l o u r ( T a b l e 4 , F i g . 1 ) . T h i s m a y b e d u e

t o t h e h i g h e r s u g a r c o n t e n t s o f U F B a n d

U F C - N S P ( 2 3 . 3 a n d 3 3 . 8 % , r e s p e c t i v e l y ) . R e l a t i v e l o a f v o l u m e s o f b r e a d s a r e a p p a r e n t i n F i g u r e 2 .

A s u f f i c i e n t a m o u n t o f e a c h c o t t o n

s e e d p r o d u c t w a s a d d e d t o r a i s e t h e p r o t e i n c o n t e n t o f t h e f l o u r u s e d i n b a k i n g

b y 4 1 % . T h e a c t u a l f i n a l p r o t e i n c o n t e n t s o f t e s t l o a v e s a r e g i v e n i n T a b l e 4 . T h e p r o t e i n c o n t e n t o f f o r t i f i e d b r e a d s w a s i n

c r e a s e d b y 2 8 . 3 % o v e r t h a t o f t h e a l l w h e a t c o n t r o l .

Protein fortification of beverages

M e a n s c o r e s f o r e a c h b e v e r a g e t e s t e d a r e g i v e n i n T a b l e 5 . A n a n a l y s i s o f v a r i a n c e s h o w e d n o s i g n i f i c a n t d i f f e r e n c e b e

t w e e n t r e a t m e n t s i n e i t h e r s e t o f v a l u e s a t t h e 1 % l e v e l . T h e a d d i t i o n o f t h e U F B p r o t e i n p r o d u c t d u l l e d t h e o r a n g e c o l o r o f e a c h d r i n k s l i g h t l y . N o c o m p e n s a t i o n

w a s m a d e i n t h e i n g r e d i e n t s o f t h e c o m

m e r c i a l p o w d e r s f o r t h e p r e s e n c e o f t h e p r o d u c t a d d e d .

A l t h o u g h d r i n k s w i t h p r o t e i n l e v e l s i n e x c e s s o f 3 % w e r e n o t o r g a n o l e p t i c a l l y t e s t e d , s u p e r n a t a n t s c o n t a i n i n g 3 . 5 , 4 . 5 , 6 a n d 7 % w e r e p r e p a r e d f o l l o w i n g t h e p r o c e d u r e u s e d t o p r e p a r e s u p e r n a t a n t s f o r u s e i n t e s t b e v e r a g e s t o d e m o n s t r a t e t h a t i t w a s p o s s i b l e t o i n c o r p o r a t e h i g h e r l e v e l s o f p r o t e i n i f d e s i r e d .

A n i n - d e p t h s t u d y o f t h e t h e r m a l s t a -


Table 4-P roperties o f bread baked w ith wheat flo u r blended w ith cottonseed u ltra filtra tion products or cottonseed flo u r to 17.5% protein level

Materials

Material in blends3

(g)

Prooftime(min)

Loafvolume^

(cc)

Specific loaf volume (cc/g)

Protein0 (N X 6.25)

(%)Crustcolor

100% wheat flou r 0 81 2975 be 6.15 15.9 28.3100% wheat flou r + SSL - 72 3217 a 6.72 - -UF B 9.4 130 2569 de 5.42 20.4 24.7UF B + SSL - 125 2817 cd 5.91 - -UF C-NSP 10.9 150 2508 e 5.13 20.1 23.1UF C-NSP + SSL - 143 2792 cd 5.85 - -UF C-SP 8.8 70 2958 be 6.09 20.7 25.9UF C-SP + SSL - 82 3033 b 6.24 - -Glandless cottonseed flou r 11.3 88 2633 de 5.49 20.4 26.7Glandless cottonseed flou r + SSL — 84 2967 bc 6.15 - -a Values are grams of cottonseed material required to make 1 OOg of wheat flour-cottonseed materi

al blend expressed on 14% moisture basis.k Mean values followed by the same alphabetical letter were not significantly different at the 5%

level.c Dry weight basis

b i l i t y c h a r a c t e r i s t i c s o f t h e p r o t e i n i n s o l u t i o n i n t h e s u p e r n a t a n t s w a s b e y o n d t h e s c o p e o f t h i s w o r k . T h e h i g h e s t p r o t e i n l e v e l t e s t e d a p p r o x i m a t e s t h e p r o t e i n

l e v e l o f m i l k . T h e p H c f t h e s u p e r n a t a n t s u s e d t o p r e p a r e t h e t e s t b e v e r a g e s w a s c a . 5 . 0 .

Whipping property evaluationsW h i p p i n g t e s t s r e s u l t s a r e g i v e n i n T a

b l e 6 . F i v e c o t t o n s e e d w h e y m a t e r i a l s w e r e e a c h w h i p p e d a t 1 2 % p r o d u c t c o n c e n t r a t i o n a n d a l s o a t t h e p r o d u c t c o n c e n t r a t i o n r e q u i r e d t o g i v e 6 % p r o t e i n i n

a s o l u t i o n - s u s p e n s i o n f r o m e a c h m a t e r i a l .

F o a m v i s c o s i t i e s f r o m t e s t s a t p H 4 . 5 w e r e a n a l y z e d s t a t i s t i c a l l y b y a n a n a l y s i s o f v a r i a n c e u s i n g a c o m p l e t e l y r a n d o m

i z e d d e s i g n . A s i n d i c a t e d i n T a b l e 6 t h e

t w o p r o d u c t s w h i p p e d i n a d d i t i o n t o t h e t h r e e U F p r o d u c t s a s s e s s e d t h r o u g h o u t t h e s t u d y w e r e ( 1 ) U F B p r o d u c t w i t h a d d i t i o n a l s a l t s a n d c a r b o h y d r a t e s r e

m o v e d b y e x h a u s t i v e d i a l y s i s a n d t h e n f r e e z e d r i e d , a n d ( 2 ) a p r o d u c t f r o m s p r a y d r y i n g P r o c e s s B w h e y w i t h o u t m e m b r a n e t r e a t m e n t ( i . e . , u n f r a c t i o n a t e d w h e y s o l i d s ) .

Table 5—Mean scores from organoleptic evaluation o f p ro te in-fortified noncarbonated "ade" and instant fru it drinks

Protein levels

Beverages tested 0% 1 % 2% 3%

Orange-flavored ''ade"drink 7.47 7.60 7.37 7.10

Orange-flavored instantfru it d rink 7.63 7.13 7.40 7.23

B y D u n c a n ’s r a n g e t e s t , w h e n t h e m a t e r i a l s w e r e w h i p p e d ( f i v e t r i a l s e a c h ) a t

1 2 % p r o d u c t c o n c e n t r a t i o n , U F B D i a l y z e d w a s s u p e r i o r t o o t h e r p r o d u c t s e x

c e p t U F B . U F B w a s n o t s u p e r i o r i n

f o a m v i s c o s i t y t o w h o l e w h e y s o l i d s o r U F C - N S P . U F C - S P w a s i n f e r i o r t o a l l o f t h e o t h e r p r o d u c t s .

W h e n t h e m a t e r i a l s w e r e t e s t e d ( a g a i n b y f i v e t r i a l s e a c h ) a t a c o m m o n % p r o

t e i n c o n c e n t r a t i o n , e a c h w a s s i g n i f i c a n t l y

d i f f e r e n t i n f o a m v i s c o s i t y . F r o m f o a m v i s c o s i t i e s o b t a i n e d ( T a b l e 6 ) i t i s a p p a r

e n t t h a t p r o t e i n c o n t e n t a l o n e d o e s n o t d e t e r m i n e t h e w h i p p a b i l i t y o f a p r o d u c t .

T h e f o a m s o b t a i n e d f r o m U F p r o d u c t s

B a n d C - N S P w e r e p l e a s a n t t a s t i n g a n d

v e r y s t a b l e . F o a m s f r o m s p r a y - d r i e d , u n

f r a c t i o n a t e d w h o l e w h e y s o l i d s h a d a

n o t i c e a b l y b i t t e r t a s t e a l t h o u g h t h e f o a m

v i s c o s i t y w a s d e s i r a b l y h i g h . T h i s b i t t e r n e s s w a s a t t r i b u t e d t o t h e h i g h a s h c o n

t e n t o f w h o l e w h e y s o l i d s . W h i p p i n g

p r o p e r t i e s o f p r o t e i n p r o d u c t s f r o m c o t

t o n s e e d w h e y s p r o v e d t o b e d e f i n i t e l y

s u p e r i o r t o t h o s e o f e i t h e r p r o t e i n i s o l a t e s o r w h i p p a b l e e x t r a c t s f r o m c o t t o n s e e d

f l o u r s p r e v i o u s l y t e s t e d a t t h e F P R D C . T o p r o v i d e a c o n t r o l f o r c o m p a r i s o n , a c o m m e r c i a l w h i p p i n g p r o d u c t , s p r a y -

d r i e d e g g w h i t e s o l i d s ( c o n t a i n i n g 1 % s o d i u m l a u r y l s u l f a t e a s a w h i p p i n g a i d ) ,

w a s w h i p p e d a t 6 % p r o t e i n c o n c e n t r a t i o n a n d m e a s u r e d b y t h e s a m e p r o c e d u r e . I t

y i e l d e d a f o a m w i t h a v i s c o s i t y o f 1 5 8 , 1 0 0 c p s . E g g w h i t e f o a m s w e r e l e s s

s t a b l e t h a n f o a m s f r o m U F B a n d U F C - N S P p r o d u c t s a t r o o m t e m p e r a t u r e .

H o w e v e r , f o a m s f r o m t h e w h e y p r o d u c t s w e r e n o n c o a g u l a b l e b y h e a t a s w e r e e g g

w h i t e f o a m s .

REFERENCESAACC. 1970. “ Approved M ethods,” American

Association of Cereal Chemists, St. Paul, Minn.

AOCS. 1971. “ Official and Tentative Methods,” 3rd ed. American Oil Chemists’ Society, Chicago.

Berardi, L.C., Martinez, W.H. and Fernandez, C.J. 1969. Cottonseed protein isolates: Two-step extraction procedure. Food Tech- nol. 23(10): 75.

Dubois, M., Gilles, K.A., Ham ilton, J.K., Rebers, P.A. and Smith, F. 1956. Colorim etric m ethod for determ ination of sugars and related substances. Anal. Chem. 28: 350.

Goldsmith, R.L., Stawiarski, M.M., Wilhelm,E.T. and Keeler, G. 1972. Treatm ent of soy whey by m embrane porcessing. Presented at the Third National Symposium on Food Processing Wastes, New Orleans, Louisiana (March).

Groves, F.W. and Graf, T.F. 1965. An economic analysis of whey utilization and disposal in Wisconsin. Agric. Econ. Bull. 44, Univ. of Wisconsin, Madison.

Kohler, G.O. and Palter, R. 1967. Studies on m ethods for amino acid analysis of wheat products. Cereal Chem. 44: 512.

Lawhon, J.T. and Cater, C.M. 1971. Effect of processing m ethod and pH of precipitation

Table 6—Whipping test data on products from cottonseed wheys

Foam viscosities w ith sugar added, cps% Volume increase with

sugar added (pH 4.5)Vo l liqu id separated

after 1 hr, mlMaterialswhipped

1 2 % product cone pH 4.5a pH 7.0

6% protein cone pH 4.5as>

1 2 % product cone

6% protein cone

1 2 % product cone pH 4.5 pH 7.0

UF B 107,900 ab 64,100 91,600 b 650 667 0 3UF C-NSP 71,900 b 57,600 53,700 d 630 500 0 3UF C-SP 13,700 c 2,000 2,400 e 280 233 0 10UF B-Dialyzed Process B

138,300 a 69,300 72,100 c 575 550 0 -

Whey solids 93,200 b 68,100 114,000 a 717 700 8.5 23.5

Means followed by same alphabetical letter were not significantly different at the 5% level

PROTEIN CO N CE NTR ATES FROM COTTONSEED W H E Y S - W l

on the yields and functional properties of protein isolates from glandless cottonseed. J. Food Sc i. 36(3): 372.

Lyman, C.M., Chang, W.Y. and Couch, J.R. 1953. Evaluation of protein quality in cottonseed meals by chick growth and by a chemical index method. J. Nutr. 49: 679.

Office of Research and Monitoring, Environmental Protection Agency. 1971. Membrane processing of cottage cheese whey for pollution abatem ent. Project No. 12060 DXF (July).

Schram, E., Moore, S. and Bigwood, E.J. 1954.

Chromatographic determ ination of cystine as cysteic acid. Biochem. J. 57: 33.

Spackman, D.H., Stein, W.H. and Moore, S. 1958. Automatic recording apparatus for use in the chromatography of amino acid. Anal. Chem. 30: 1190.

Sumner, J.B. 1944. A m ethod for the colorimetric determ ination of phosphorus. Science 100: 413.

Webb, N.B., Ivey, F.J., Craig, H.B., Jones, V.A. and Monroe, R.J. 1970. Measurement of emulsifying capacity by electrical resistance. J. Food Sci. 35(4): 401.

Ms received 6/12/73; revised 9 /27 /73 ; accepted 9 /30/73._________________________

Appreciation is expressed by the authors to M.N. Kahn for his work in the bread baking evaluations and to Gloria Quave, Jack Pirson, Claudia W iltrout, John Allen and Dwain Mul- sow for technical assistance in obtaining products and data.

This research was funded in part by USDA Research Agreement No. 12-14-100-11021 (72) and in part by the Natural Fibers & Food Protein Committee of Texas.

S. E. F L E M IN G , F. I/V. S O S U L S K I, A . K iL A R A a n d E. S. H U M B E R T

D e p a rtm e n ts o f C ro p S cience a n d D a iry & F o o d S cience

U n iv e rs ity o f S aska tchew an , S aska to on , S aska tch ew an , C anada S 7 N O W O

VISCOSITY AND WATER ABSORPTION CHARACTERISTICS OF SLU RRIES OF SUNFLOWER AND SOYBEAN FLOURS, CONCENTRATES AND ISOLATES

Table 1—Composition of the soybean and sunflower products (dry basis)

Protein product

Crudeprote in*

(%)

Crudefat(%)

Crudefiber(%)

Ash(%)

Soy flour 50.2 1.8 2.7 6.4Soy concentrate (Isopro) 63.7 0.3 3.0 3.5Soy isolate (Supro 610) 85.8 0.1 0.1 3.7Soy isolate (Promine D) 86.1 0.2 0.1 3.9Sunflower flour 53.0 1.8 3.6 8.2Sunflower concentrate — 60 69.9 1.7 5.2 5.4Sunflower concentrate — 80 68.6 1.0 5.1 6.9Sunflower isolate (60) 87.7 0.1 0.5 3.2* N X 5.7

INTRODUCTIONT H E U T I L I Z A T I O N o f s o y f l o u r s , c o n c e n t r a t e s a n d i s o l a t e s i n p r e p a r e d f o o d s h a s i n c r e a s e d r a p i d l y a n d e x c e e d s t h a t o f o t h e r c o n c e n t r a t e d s e e d p r o t e i n s . T h e f u n c t i o n a l a n d p h y s i c a l p r o p e r t i e s o f t h e s e p r o t e i n s h a v e d e f i n e d t h e i r r o l e i n

b a k e d g o o d s , m e a t p r o d u c t s a n d s o y b e v e r a g e s ( J o h n s o n , 1 9 7 0 ) .

T h e p r o p e r t i e s o f e m u l s i f i c a t i o n , v i s

c o s i t y a n d w a t e r - h o l d i n g c a p a c i t y h a v e r e n d e r e d s o y p r o t e i n f u n c t i o n a l i n m e a t p r o d u c t f o r m u l a t i o n s . T e x t u r e d v e g e t a b l e p r o t e i n h a s b e e n w i d e l y p r o d u c e d b y e i t h e r f i b e r s p i n n i n g o r t h e r m o p l a s t i c e x

t r u s i o n p r o c e s s e s ( R a k o s k y , 1 9 7 0 ) . I n m a n y c a s e s t h e p r o c e s s i n g c o n d i t i o n s h a v e b e e n a l t e r e d t o t a k e a d v a n t a g e o f

t h e p h y s i c a l a n d f u n c t i o n a l p r o p e r t i e s o f t h e r a w m a t e r i a l a n d t o p r o d u c e a w i d e v a r i e t y o f d e s i r a b l e e n d p r o d u c t s . F o r e x a m p l e , E l m q u i s t ( 1 9 6 5 ) v a r i e d t h e p H

a n d t e m p e r a t u r e o f s a f f l o w e r s e e d m e a l

d i s p e r s i o n s t o o b t a i n s p i n n i n g s o l u t i o n s

w i t h a w i d e r a n g e o f v i s c o s i t i e s . S z c z e s - n i a k a n d E n g e l ( 1 9 6 0 ) v a r i e d t h e p H o f s o y f l o u r - c a s e i n d i s p e r s i o n s t o p r o d u c e a s p i n n i n g s o l u t i o n w i t h t h e t a c k i n e s s a n d h i g h v i s c o s i t y r e q u i r e d f o r a f i l a m e n t w i t h s t r e t c h a b i l i t y e s s e n t i a l f o r r e s i s t a n c e t o m a s t i c a t i o n .

A c c o r d i n g t o B r i s k e y ( 1 9 7 0 ) a n d H e r m a n s s o n ( 1 9 7 0 ) t h e d e g r e e o f p r o t e i n

h y d r a t i o n i s r e l a t e d t o t h e v i s c o s i t y o f t h e s y s t e m . T h e s e a r e i n f l u e n c e d b y p H , i o n i c s t r e n g t h a n d t e m p e r a t u r e . H o w e v e r , i t i s n o t k n o w n t o w h a t e x t e n t w a t e r a b s o r p t i o n c a n b e t a k e n a s a m e a s u r e o f t h e d e g r e e o f s w e l l i n g o f p r o t e i n m o l e c u l e s . T h e r e f o r e , t h e d u a l p r o p e r t i e s o f w a t e r a b s o r p t i o n a n d v i s c o s i t y o v e r a r a n g e o f s l u r r y c o n c e n t r a t i o n s s h o u l d b e d e t e r m i n e d t o a s s e s s t h e p o t e n t i a l f o o d u s e s o f a n e w p r o t e i n s o u r c e .

D e f a t t e d s u n f l o w e r m e a l h a s p o t e n t i a l i n t h e f o o d i n d u s t r y b e c a u s e t h e f l o u r s c o n t a i n h i g h l e v e l s o f p r o t e i n a n d n o k n o w n t o x i c s u b s t a n c e s ( C l a n d i n i n ,1 9 5 8 ) . T h e m a j o r d i s a d v a n t a g e s o f s u n f l o w e r m e a l p r o d u c t s a r e t h e l o w l e v e l s o f l y s i n e a n d h i g h p r o p o r t i o n s o f s i m p l e s u g a r s a n d p h e n o l i c a c i d s w h i c h a r e r e a d i l y c o m p l e x e d o r o x i d i z e d i n t o d a r k b r o w n a n d g r e e n c o m p o u n d s . T h e s e l o w m o l e c u l a r w e i g h t c o m p o u n d s c a n b e d i f

f u s i o n e x t r a c t e d f r o m s u n f l o w e r k e r n e l s ( S o s u l s k i e t a l . , 1 9 7 3 ) , a n d , a f t e r o i l e x t r a c t i o n t h e r e s u l t i n g m e a l i s a p r o t e i n c o n c e n t r a t e w i t h s t a b l e c o l o r c h a r a c t e r i s t i c s .

T h e o b j e c t i v e s o f t h e p r e s e n t s t u d y w e r e t o c o m p a r e t h e w a t e r a b s o r p t i o n a n d v i s c o s i t y c h a r a c t e r i s t i c s o f 5 , 1 0 , 1 5 a n d 2 0 % s l u r r i e s o f u n t r e a t e d a n d p H - a c t i v a t e d s l u r r i e s o f s u n f l o w e r f l o u r , c o n c e n t r a t e s a n d i s o l a t e s w i t h s o y f l o u r , c o n c e n t r a t e s a n d i s o l a t e s a t r o o m t e m p e r a t u r e a n d d u r i n g a h e a t i n g a n d c o o l i n g c y c l e .

EXPERIMENTALTH E SO Y F L O U R , soy protein con cen trate (Iso p ro) and soy protein isolates (Su pro 6 1 0 and Prom ine D) were obtained from com m ercial sources. Dehulled sunflow er kernels (o b tained from Co-op V egetable Oil Mills L td ., A ltona, M anitoba) were ground, fat extracted with Skelly F and desolventized under vacuum at 4 5 °C to prepare sunflow er flour. T he phenolic com pounds were removed from the sunflow er kernels by the continuous procedure o f Sosulski et al. ( 1 9 7 3 ) in which the cut kernels were diffused at 6 0 °C for 4 hr or 8 0 °C for 1.5 hr with 0 .001 N HC1 at a solvent-to-kernel ratio o f 6 0 :1 . A fter air drying at 5 0 °C for 3 hr, the diffused kernels were aspirated to remove the testa, ground, extracted with Skelly F and desolventized to produce sunflow er con cen trate - 6 0 and sunflow er co n cen trate - 8 0 . The sunflow er protein isolate was prepared from the undenatured co n cen trate - 6 0 by two ex tractions with 0 .2% NaOH follow ed by isoelectric

precip itation at pH 4 .5 and cen trifugation (Sosulski and Bakal, 1 9 6 9 ). T he protein iso late was washed tw ice with acidified w ater (pH 4 .5 ) , centrifuged and stored at refrigeration tem peratures (w et iso late) or freeze dried (dry iso late).

All samples were ground and sifted to pass a 125 mesh T y ler screen. P rox im ate analyses for crude protein , fat, fiber and ash were d eterm ined by the AOAC (1 9 7 0 ) m ethods. T he factor, 5 .7 , for converting the K jeldahl nitrogen !N) value to protein percentage was used.

T he slurries were prepared by eith er adding the w ater to the protein product with stirring for 1 min (short m ix) or by grinding the protein product with enough w ater to form a th ick paste for 10 min (long m ix) then adding the rem aining water to m ake 5 , 10 , 15 and 20% slurries (g dry m atter/g total x 1 0 0 ). F u n ctional properties were measured im m ediately after slurry preparation. F or pH -activation, 1 .25N NaOH was added in 1 min with co n tin u ous stirring to reach pH 1 2 .2 and 6 .ON HC1 was added to return the pH to 6 .0 in 10 min. This slurry was designated as “ pH activ ated .”

T he viscosities o f the untreated and pH- activated soy and sunflow er slurries were d eterm ined with a B rookfield viscosim eter, m odel L V F , at room tem perature using the T-spindles and Helipath stand for slurry viscosities over 5 0 0 cps, and the standard rotating spindles for less viscous solutions.

Most o f the slurries were m easured at several speeds o f ro tation , but those recorded were taken at 12 rpm. T he H elipath perm its relative m easurem ents o f paste v iscosities by slowly low ering the spindle at low ro tation al speeds through the m atrix , m easuring undisturbed structure at all tim es. A Brabender viscoam ylo- graph was used to m easure viscosity during a


VISCOSITY A N D WATER A B SO RPTIO N PROPERTIES - 1 8 9

Table 2—Water absorption and apparent viscosity o f untreated and pH-activated slurries o f soy and sunflower flours, concentrates and isolates at room temperature

Brookfield apparent viscosity (cps)

Centrifuge water absorption Short m ix Long m ix

(g H2 O/g product) in water in 5% NaCl in water

in water in 5% NaCl Concentration o f product (w /w)

short m ix long m ix short m ix 15 15 5 10 15 20

Soybean: Flour 2.77 2.60 3.25

Untreated protein products

25 70 25 230 2,000Isopro 3.65 2.75 3.53 50 1,950 10 200 330 28,320Supro 610 6.25 6.25 3.85 >83,300 76,110 160 10,500 >83,300 >83,300Promine D 7.75 4.15 3.95 45,000 29,000 1,300 3,200 7,500 25,200

Sunflower: Flour 1.28 1.55 2.75 190 12,500 90 135 200 330Concentrate —60a 3.90 2.92 3.88 35,820 19,990 300 2,200 13,000 83,300Concentrate —803 3.90 2.65 4.08 12,650 14,810 50 400 10,800 83,300Isolate (Dry) 2.73 3.05 2.80 3,500 22,000 10 80 500 3,780Isolate (Wet) 4.05 2.68 3.75 800 830 - 20 130 680

Soybean: Flour 3.42 2.59 2.23pH-activated products

2,500 40 250 470 1,400 4,200Isopro 4.59 3.81 3.76 27,070 4,980 40 690 24,990 >83,300

Sunflower: Flour 3.41 2.51 2.54 2,700 8,500 120 660 2,800 22,000Concentrate —60 5.10 3.78 4.31 15,830 12,500 320 3,120 37,480 >83,300Concentrate —80 5.34 3.73 4.34 31,650 29,160 160 1,300 25,200 >83,300

a Sunflower concentrate —60 and concentrate —80 refer to diffusion extraction at 60°C and 80°C, respectively.

heating and coolin g cy cle according to the AACC (1 9 7 0 ) m ethod 2 2 -1 0 in which 65g (14% m oisture basis) o f product were com bined with 4 6 0 ml o f distilled w ater. T he instrum ent was programmed to increase from 30 to 9 7 .5 °C at 1 .5°C per m in, hold at 9 7 .5 °C for 15 m in, and decrease to 3 0 °C at 1 .5 °C per min. T h e viscosity data reported in this paper are the m eans o f 3 to 5 determ inations but only a single viscoamylograph curve was made on each product.

Water absorption was measured by a m od ification o f the centrifugation technique o f Jan ick i and W alczak (1 9 5 4 ) in which 10% dispersions (m oisture-free basis, w /w ) were prepared in w ater with eith er the short or long m ixing m ethods as previously described. O ther slurries were prepared in a m edium o f 5% NaCl. All slurries w ere centrifuged at 2 5 0 0 rpm for 15 min in a slant-tube type centrifuge and the volum e o f released w ater was measured. The w ater retained per gram o f protein product was calculated as w ater absorbed.


P r o x i m a t e c o m p o s i t i o n

T h e p r o x i m a t e a n a l y s i s o f s o y a n d s u n f l o w e r p r o d u c t s ( T a b l e 1 ) i n d i c a t e d t h a t t h e d e h u l l e d a n d d e f a t t e d s u n f l o w e r

f l o u r s , c o n c e n t r a t e s a n d i s o l a t e s w e r e g e n e r a l l y h i g h e r i n p r o t e i n t h a n t h e s o y

p r o t e i n p r o d u c t s w h i l e t h e s u n f l o w e r f l o u r s w e r e a l s o h i g h e r t h a n s o y i n c r u d e f i b e r a n d a s h . T h e d i f f u s i o n e x t r a c t i o n

p r o c e s s r e m o v e d c o n s t i t u e n t s w h i c h w e r e h i g h i n a s h b u t t h e c r u d e f i b e r w a s e n

r i c h e d i n t h e c o n c e n t r a t e s d u e t o t h e e x

t r a c t i o n o f t h e m o r e s o l u b l e c o n s t i t u e n t s . T h e r e f o r e , t h e s u n f l o w e r c o n c e n t r a t e s w e r e a b o u t 5 % h i g h e r i n p r o t e i n a n d 2 % h i g h e r i n c r u d e f i b e r a n d a s h t h a n t h e s o y

c o n c e n t r a t e . T h e s o y a n d s u n f l o w e r i s o

l a t e s w e r e s i m i l a r i n c o m p o s i t i o n , b e i n g p r i m a r i l y p r o t e i n .

E f f e c t o f s l u r r y p r e p a r a t i o n m e t h o d o n f u n c t i o n a l p r o p e r t i e s

T h e w a t e r a b s o r p t i o n a n d v i s c o s i t y c h a r a c t e r i s t i c s o f t h e u n t r e a t e d s l u r r i e s o f t h e s o y f l o u r a n d c o n c e n t r a t e ( I s o p r o ) w e r e l o w e r t h a n t h o s e o f t h e s o y i s o l a t e ,

r e g a r d l e s s o f t h e m i x i n g m e t h o d ( T a b l e 2 ) . H o w e v e r , t h e w a t e r a b s o r p t i o n s w e r e g e n e r a l l y l o w e r a n d t h e v i s c o s i t i e s h i g h e r i n s l u r r i e s s u b j e c t e d t o t h e l o n g t h a n t h e s h o r t m i x . W a t e r a b s o r p t i o n a n d v i s c o s i t i e s t e n d e d t o i n c r e a s e w i t h c o n c e n t r a t i o n o f p r o t e i n i n t h e p r o d u c t .

T h e s u n f l o w e r c o n c e n t r a t e s h a d t h e

h i g h e s t w a t e r a b s o r p t i o n s a n d v i s c o s i t i e s o f a l l t h e s u n f l o w e r p r o d u c t s , w i t h t h e e x c e p t i o n o f t h e w e t i s o l a t e ( s h o r t m i x ) a n d d r y i s o l a t e ( l o n g m i x ) w h i c h h a d h i g h

w a t e r a b s o r p t i o n b u t l o w v i s c o s i t i e s . T h e l o n g m i x p r o c e d u r e i m p r o v e d w a t e r a b s o r p t i o n o f t h e f l o u r a n d d r y i s o l a t e b u t t h e v i s c o s i t i e s o f t h e f l o u r s l u r r i e s r e m a i n e d c o n s t a n t w h i l e s l u r r i e s o f o t h e r s u n f l o w e r p r o d u c t s b e c a m e l e s s v i s c o u s .

E f f e c t o f s a l t o n f u n c t i o n a l

p r o p e r t i e s

L e m a n c i k a n d Z i e m b a ( 1 9 6 2 ) n o t e d a p o s i t i v e c o r r e l a t i o n b e t w e e n w a t e r a b s o r p t i o n a n d n i t r o g e n s o l u b i l i t y i n d e x o f

s o y f l o u r s . S o s u l s k i a n d B a k a l ( 1 9 6 9 ) d e m o n s t r a t e d t h a t o n l y 2 0 % o f t h e s u n f l o w e r f l o u r p r o t e i n s w e r e w a t e r s o l u b l e b u t n e a r l y 8 0 % c o u l d b e e x t r a c t e d w i t h

5 % N a C l . T h e r e f o r e w a t e r a b s o r p t i o n a n d v i s c o s i t y m e a s u r e m e n t s w e r e m a d e o n

1 5 % s l u r r i e s i n 5 % N a C l s o l u t i o n s . T h e w a t e r a b s o r p t i o n o f s o y f l o u r w a s h i g h e r i n 5 % N a C l t h a n i n w a t e r , b u t w a s l o w e r f o r a l l o t h e r s o y p r o d u c t s ( T a b l e 2 ) . T h e v i s c o s i t i e s , h o w e v e r , w e r e h i g h e r f o r b o t h s o y f l o u r a n d I s o p r o N a C l s l u r r i e s . S u n f l o w e r f l o u r h a d h i g h e r w a t e r a b s o r p t i o n i n 5 % N a C l t h a n i n w a t e r , b u t t h a t o f t h e w e t i s o l a t e d e c r e a s e d , w h i l e o t h e r p r o d u c t s r e m a i n e d r e l a t i v e l y c o n s t a n t . T h e v i s

c o s i t i e s o f a l l s u n f l o w e r p r o d u c t s o t h e r t h a n t h e c o n c e n t r a t e —6 0 i n c r e a s e d i n 5 %

N a C l . T h e f l o u r a n d w e t i s o l a t e s h o w e d l a r g e v i s c o s i t y i n c r e a s e s . T h e s e p r o t e i n s m a y b e h a v e i n a s i m i l a r m a n n e r i n g r o u n d m e a t p r o d u c t s w h e r e t h e a q u e o u s p h a s e c o n t a i n s a s i g n i f i c a n t p r o p o r t i o n o f s a l t .

I t a p p e a r e d t h a t p r o t e i n s o l u b i l i t y w a s a f a c t o r i n d e t e r m i n i n g t h e v i s c o s i t y b e

h a v i o r o f t h e s u n f l o w e r p r o d u c t s s i n c e t h e y a r e m o r e s o l u b l e i n s a l t s o l u t i o n s t h a n d i s t i l l e d w a t e r . A s i m i l a r p h e n o m e n o n h a s b e e n n o t e d f o r t h e s o y P r o m i n e

p r o d u c t s i n w h i c h 1 0 % ( w / w ) u n h e a t e d d i s p e r s i o n s o f P r o m i n e - F ( 6 5 % w a t e r - s o l u b l e p r o t e i n ) h a d a v i s c o s i t y o f 0 . 5

p o i s e w h e r e a s 1 0 % d i s p e r s i o n o f P r o - m i n e - D ( 7 5 % w a t e r - s o l u b l e p r o t e i n ) h a d a v i s c o s i t y o f 1 0 p o i s e ( A n o n y m o u s , 1 9 7 2 ) .

F u n c t i o n a l p r o p e r t i e s o f u n t r e a t e d s l u r r i e s

A s p r e v i o u s l y r e p o r t e d b y o t h e r w o r k e r s ( H e r m a n s s o n , 1 9 7 2 ) , t h e v i s c o s i t i e s o f t h e u n t r e a t e d s l u r r i e s i n c r e a s e d e x p o n e n t i a l l y w i t h i n c r e a s i n g c o n c e n t r a t i o n o f e a c h s o y p r o d u c t , w i t h t h e e x c e p t i o n o f I s o p r o ( T a b l e 2 ) . T h e v i s c o s i t y o f t h e s o y c o n c e n t r a t e w a s s i m i l a r t o t h e


Fig. 1—S ta b i l i ty o f th e u n tre a te d s lu rr ie s a f te r s ta n d in g fo r 4 h r a t ro o m te m p e ra tu re . F ro m le f t to r ig h t : 20% s u n f lo w e r f lo u r , 15% s u n f lo w e r c o n c e n tra te —6 0 , 15% o f s u n f lo w e r c o n c e n tra te —8 0 a n d 20% so y

f lo u r.

f l o u r a t l o w c o n c e n t r a t i o n s b u t w a s o v e r t e n f o l d g r e a t e r i n t h e 2 0 % s l u r r y . S u p r o 6 1 0 s h o w e d h i g h e r v a l u e s t h a n t h e f l o u r a n d c o n c e n t r a t e a t a l l c o n c e n t r a t i o n s w h i l e P r o m i n e D w a s h i g h l y v i s c o u s a t l o w c o n c e n t r a t i o n s , b u t l e s s v i s c o u s t h a n

I s o p r o a t 2 0 % . I n g e n e r a l , v i s c o s i t y i n c r e a s e d w i t h p r o t e i n c o n t e n t o f t h e s a m p l e .

T h e v i s c o s i t i e s o f t h e s u n f l o w e r p r o d u c t s a l s o i n c r e a s e d e x p o n e n t i a l l y w i t h

c o n c e n t r a t i o n , b u t s u n f l o w e r f l o u r r e m a i n e d f a i r l y s t a b l e ( T a b l e 2 ) . T h e c o n c e n t r a t e —6 0 s h o w e d t h e h i g h e s t v i s

c o s i t i e s a t a l l c o n c e n t r a t i o n s w h i l e a t 2 0 % b o t h c o n c e n t r a t e s w e r e e x c e e d i n g l y v i s

c o u s . A l t h o u g h t h e s u n f l o w e r i s o l a t e s w e r e h i g h i n p r o t e i n a n d l o w i n c a r b o h y d r a t e , t h e y e x h i b i t e d l o w v i s c o s i t y c h a r a c t e r i s t i c s .

M o s t o f t h e 5 % s l u r r i e s t e s t e d h a d l o w v i s c o s i t i e s b u t t h e s o y i s o l a t e , P r o m i n e D ,

w a s r e l a t i v e l y v i s c o u s ( T a b l e 2 ) . T h e s u n f l o w e r f l o u r s l u r r y w a s m o r e v i s c o u s t h a n t h e c o r r e s p o n d i n g s o y f l o u r s l u r r y a t 1 0 %

b u t a t h i g h e r c o n c e n t r a t i o n s w a s l e s s v i s

c o u s . T h e s u n f l o w e r c o n c e n t r a t e s h a d c o n s i d e r a b l y h i g h e r v i s c o s i t i e s a t a l l c o n c e n t r a t i o n s t h a n t h e s o y c o n c e n t r a t e b u t t h e i s o l a t e s d i d n o t p a r a l l e l t h e s o y i s o l a t e s i n v i s c o s i t y c h a r a c t e r i s t i c s .

T h e s t a b i l i t i e s o f t h e u n t r e a t e d s l u r r i e s w e r e e v a l u a t e d a f t e r s t o r a g e o n t h e l a b o r a t o r y b e n c h f o r 4 h r . A s s h o w n i n F i g u r e 1, t h e s u n f l o w e r f l o u r s e p a r a t e d f r o m t h e a q u e o u s p h a s e q u i t e r a p i d l y , c o m m e n c i n g s e p a r a t i o n a f t e r 5 m i n . T h i s f l o u r s l u r r y a l s o e x h i b i t e d t h e c h a r a c t e r i s t i c d a r k g r e e n c o l o r w h i c h d e v e l o p e d w h e n t h e c h l o r o g e n i c a c i d i n t h e s u n f l o w e r f l o u r w a s e x p o s e d t o a q u e o u s m e d i u m . A t t h e

e n d o f t h e 4 - h r p e r i o d , t h e w a t e r h a d a l s o b e g u n t o s e p a r a t e f r o m t h e 2 0 % s o y f l o u r s l u r r y . H o w e v e r , t h e l i g h t - c o l o r e d c o n c e n

t r a t e — 6 0 s l u r r y r e m a i n e d s t a b l e u n t i l t h e n e x t d a y ( 1 8 h r ) w h i l e t h e 1 5 % c o n c e n

t r a t e — 8 0 w a s s t i l l h o m o g e n e o u s a f t e r a n a d d i t i o n a l 2 4 h r o f s t o r a g e i n t h e r e f r i g

e r a t o r . T h e s u n f l o w e r c o n c e n t r a t e s w e r e a

s t a b l e w h i t e c o l o r , w h e r e a s t h e s o y f l o u r w a s y e l l o w - b r o w n a n d t h e s u n f l o w e r

f l o u r w a s d a r k g r e e n d u e t o c h l o r o g e n i c a c i d o x i d a t i o n . T h e s t a b l e h i g h v i s c o s i t i e s

a n d w h i t e c o l o r s o f t h e s e c o n c e n t r a t e s m a y o f f e r s o m e a d v a n t a g e o v e r s o y b e a n

p r o d u c t s i n f o o d a p p l i c a t i o n s s u c h a s

s a u c e s , p u d d i n g s a n d m e a t p r o d u c t s .

pH-activation processT h e s o y i s o l a t e s h o w e d v e r y h i g h v i s

c o s i t i e s , e s p e c i a l l y i n t h e m o r e c o n c e n t r a t e d s l u r r i e s . T h i s i s n o r m a l l y c o n

s i d e r e d t o b e d u e t o t h e h i g h e r p r o t e i n c o n c e n t r a t i o n a n d t h e e l i m i n a t i o n o f i n t e r f e r i n g c o m p o u n d s s u c h a s f i b e r . H o w e v e r , t h e v i s c o s i t y o f s o y f l o u r s i n c r e a s e s m a r k e d l y d u r i n g a l k a l i n e p r o t e i n i s o l a t i o n a n d t h e p H t r e a t m e n t m a y c o n t r i b u t e t o t h e h i g h v i s c o s i t y o f t h e f i n a l i s o l a t e . E x

p e r i m e n t s w e r e c o n d u c t e d t o a s s e s s t h e i n f l u e n c e o f t h e p H c y c l e o n t h e w a t e r

a b s o r p t i o n s a n d v i s c o s i t i e s o f t h e s o y f l o u r a n d c o n c e n t r a t e s l u r r i e s b y a d j u s t i n g t h e p H t o o v e r 1 2 a n d b a c k t o p H 6 .

I n i t i a l l y , s l u r r i e s o f 2 0 % s u n f l o w e r f l o u r i n d i s t i l l e d w a t e r w e r e c y c l e d t h r o u g h t h e p H t r e a t m e n t n o r m a l l y a p p l i e d t o i s o l a t e s , a n d v i s c o s i t i e s o f o v e r1 0 , 0 0 0 c p s w e r e r e a d i l y o b t a i n e d . I t w a s f o u n d t h a t r a p i d p H a d j u s t m e n t t o p H1 2 . 2 a n d b a c k t o p H 6 . 0 w i t h c o n c e n t r a t e d N a O H a n d H C 1 r e s u l t e d i n h i g h e r v i s c o s i t i e s . W h i l e 2 . 5 N N a O H a n d 6 . O N

H C 1 w e r e u s e d i n t h e p r e s e n t r e p o r t , h i g h e r c o n c e n t r a t i o n s o f H C 1 w e r e e f f e c t i v e i n a c h i e v i n g m o r e v i s c o u s s l u r r i e s d u e , i n p a r t , t o l e s s d i l u t i o n . T h e u s e o f

1 2 . O N H C 1 w a s d i s c o n t i n u e d d u e t o t h e

f o r m a t i o n o f a n o n h o m o g e n e o u s s l u r r y , e x c e s s i v e p r o t e i n d e n a t u r a t i o n a n d i t s d i s a d v a n t a g e s f o r a p p l i c a t i o n i n t h e f o o d i n d u s t r y . T h e 1 0 m i n u s e d t o r e d u c e t h e p H

f r o m 1 2 . 2 t o 6 . 0 w a s r e q u i r e d b e c a u s e o f t h e m e c h a n i c a l p r o b l e m s w i t h s t i r r i n g t h e

m o r e v i s c o u s s l u r r i e s . H i g h e r v i s c o s i t i e s w e r e a c h i e v e d w h e n t h e p H w a s a d j u s t e d

t c 6 . 0 w i t h i n 2 — 3 m i n .T h e p H - a c t i v a t i o n p r o c e s s i m p r o v e d

t h e w a t e r a b s o r p t i o n p r o p e r t i e s o f m o s t o f t h e s o y a n d s u n f l o w e r p r o d u c t s r e g a r d l e s s o f t h e m i x i n g m e t h o d ( T a b l e 2 ) . T h i s p r o c e s s a l s o i m p r o v e d t h e v i s c o s i t i e s o f t h e s o y f l o u r a n d I s o p r o s l u r r i e s . T h e v i s c o s i t y o f t h e 1 5 % I s o p r o s l u r r y w a s m u c h l o w e r t h a n t h e v i s c o s i t y o f S u p r o 6 1 0 ,

b u t m u c h h i g h e r t h a n P r o m i n e D .S l u r r i e s o f s u n f l o w e r f l o u r a n d c o n c e n

t r a t e s w e r e m o r e v i s c o u s a f t e r p H - a c t i v a - t i o n , w i t h t h e c o n c e n t r a t e s s h o w i n g c o n s i d e r a b l y h i g h e r v i s c o s i t i e s a t e a c h

c o n c e n t r a t i o n t h a n t h e f l o u r . T h e c o n c e n t r a t e — 6 0 w a s m o r e v i s c o u s t h a n t h e c o n

c e n t r a t e — 8 0 a t a l l c o n c e n t r a t i o n s . T h e s u n f l o w e r w e t i s o l a t e w a s l e s s v i s c o u s

t h a n t h e o t h e r p r o t e i n p r o d u c t s .

T h e s u n f l o w e r f l o u r a n d c o n c e n t r a t e s

w e r e g e n e r a l l y m o r e v i s c o u s t h a n t h e c o r r e s p o n d i n g s o y b e a n s l u r r i e s . T h e p H - a c t i v a t e d 5 % s l u r r i e s o f s u n f l o w e r c o n c e n

t r a t e s h a s v i s c o s i t i e s e q u i v a l e n t t o o r

g r e a t e r t h a n t h e 5 % s l u r r y o f S u p r o 6 1 0 ,

b u t l e s s t h a n P r o m i n e D a t 5 % . T h i s m i g h t h a v e s o m e s i g n i f i c a n c e i n f o o d a p

p l i c a t i o n s w h e r e t h e s e p r o d u c t s a r e u s e d i n d i l u t e c o n c e n t r a t i o n s s u c h a s i m i t a t i o n

m i l k a n d g r a v i e s . T h e s u n f l o w e r p r o d u c t s s h o w e d a p a r t i c u l a r l y h i g h r e s p o n s e t o p H - a c t i v a t i o n . W h i l e f u r t h e r i n v e s t i g a t i o n s a r e r e q u i r e d , i t a p p e a r e d t h a t p H - a c t i v a t i o n m a y b e a p o t e n t i a l t e c h n i q u e

f o r e x t e n d i n g t h e u t i l i z a t i o n o f f l o u r s a n d c o n c e n t r a t e s i n t o f o o d a n d i n d u s t r i a l a p p l i c a t i o n s w h i c h p r e s e n t l y r e q u i r e p r o d

u c t s w i t h h i g h v i s c o s i t y c h a r a c t e r i s t i c s .

Viscoamylograph analysisT h e v i s c o a m y l o g r a p h c u r v e s d e m o n

s t r a t e d t h a t o n l y s o y i s o l a t e d e v e l o p e d a

p e a k v i s c o s i t y u n d e r t h e t e m p e r a t u r e

p r o g r a m ( T a b l e 3 ) . T h i s p r o d u c t p e a k e d a t 6 8 ° C ( 7 6 0 B U ) a n d d e c r e a s e d t o o n l y 7 5 B U a t 9 7 . 5 ° C w i t h t h e c o l d v i s c o s i t y

r e a c h i n g 3 2 5 B U . A l t h o u g h n o p e a k v i s c o s i t y w a s o b t a i n e d f o r s o y f l o u r i n t h i s s t u d y , P a u l s e n a n d H o r a n ( 1 9 6 5 ) h a d o b t a i n e d a p e a k v i s c o s i t y w h e n u s i n g a 2 0 % s l u r r y . T h e u n t r e a t e d s u n f l o w e r p r o d u c t s h a d l o w v i s c o s i t i e s d u r i n g t h e h e a t i n g

p e r i o d b u t t h e c o l d v i s c o s i t i e s o f t h e c o n c e n t r a t e s r o s e t o a b o u t 2 5 0 B U a t 3 0 ° C . T h e p H - a c t i v a t e d s o y f l o u r a n d c o n c e n t r a t e a n d t h e s u n f l o w e r f l o u r a n d i s o l a t e s

d i d n o t s h o w h i g h v i s c o s i t i e s d u r i n g t h e t e m p e r a t u r e p r o g r a m . H o w e v e r , t h e s u n f l o w e r c o n c e n t r a t e s , e s p e c i a l l y t h e c o n c e n t r a t e —6 0 d e v e l o p e d r e l a t i v e l y h i g h v i s c o s i t i e s d u r i n g t h e 9 7 . 5 ° C t r e a t m e n t

a n d t h e s e i n c r e a s e d t o c o l d v i s c o s i t i e s w e l l a b o v e t h a t o f s o y i s o l a t e . L o w p e a k v i s c o s i t i e s w i t h h i g h c o l d v i s c o s i t i e s a r e d e s i r a b l e p r o p e r t i e s i n f o o d a p p l i c a t i o n s w h e r e g e l a t i o n m u s t o c c u r a f t e r t h e c o o k -

VISCOSITY A N D WATER AB SO RPTIO N PROPERTIES - 1 9 1

Table 3—Viscosity and gelation data from viscoamylograph curves o f untreated and pH-activated slurries o f soy and sunflower flours, concentrates and isolates (Brabender units)

Untreated slurry pH-activated slurry

Peak 97.5° C fo r Cold Peak 97.5°C ColdProtein product 70° C 15 min 30° C 70° C fo r 15 min 30° C

Soybean: Flour 0 5 20 20 50 150Isopro 0 0 5 35 50 1 1 0Supro 610 750 75 325 - - -

Sunflower: Flour 3 92 125 0 60 115Concentrate —60 45 92 240 45 375 750Concentrate —80 35 140 270 75 175 425Isolate (Dry) 0 0 8 — — —

i n g p r o c e s s . T h i s i s e s p e c i a l l y i m p o r t a n t i n t h e c a n n i n g i n d u s t r y a n d t h e p r e p a r a t i o n o f m e a t p r o d u c t s s u c h a s s a u s a g e , w i e n e r s a n d l u n c h e o n m e a t s .

General discussionT h e s o y p r o t e i n i s o l a t e , S u p r o 6 1 0 , e x

h i b i t e d i t s c h a r a c t e r i s t i c a l l y h i g h v i s c o s i t y w h i l e P r o m i n e D s h o w e d h i g h v i s c o s i t i e s o n l y a t l o w c o n c e n t r a t i o n s . T h e s u n f l o w e r c o n c e n t r a t e - 6 0 h a d t h e h i g h e s t

v i s c o s i t y o f a l l s u n f l o w e r p r o d u c t s a n d w a s c o n s i d e r a b l y m o r e v i s c o u s t h a n t h e c o r r e s p o n d i n g s o y p r o d u c t s . T h e s l u r r i e s o f s u n f l o w e r a n d s o y c o n c e n t r a t e s w h i c h w e r e c y c l e d t h r o u g h t h e p H t r e a t m e n t

h a d v i s c o s i t i e s b e t w e e n t h o s e o f t h e s o y

i s o l a t e s . T h e v i s c o a m y l o g r a p h c u r v e d e m

o n s t r a t e d t h a t t h e s o y i s o l a t e h a d a h i g h p e a k a n d c o l d v i s c o s i t y w h i l e t h e s u n

f l o w e r c o n c e n t r a t e s h o w e d n o p e a k b u t v e r y h i g h c o l d v i s c o s i t i e s . T h e s u n f l o w e r c o n c e n t r a t e s a p p e a r e d t o b e i d e a l b a s i c p r o d u c t s f o r g e n e r a l f o o d u s e .

B y v a r y i n g t h e t e m p e r a t u r e , m i x i n g

r e g i m e , s l u r r y m e d i u m , a n d b y p H - a c t i v a -

t i o n , a w i d e r a n g e o f v i s c o s i t i e s w e r e o b t a i n e d i n t h e p r e s e n t s t u d y . W i t h t h i s i n f o r m a t i o n , t h e f o o d p r o c e s s o r c o u l d s e l e c t t h e p r o d u c t w i t h t h e v i s c o s i t y c h a r a c t e r i s t i c s m o s t s u i t a b l e t o t h e p r o c e s s i n g c o n d i t i o n s , e q u i p m e n t a n d e n d p r o d u c t c h a r a c t e r i s t i c s . F u r t h e r r e s e a r c h w i l l b e c o n d u c t e d t o r e l a t e t h e m o d e l s y s t e m t o t h e a c t u a l f o o d p r o d u c t .

REFERENCESAACC. 1968. “ AACC Approved M ethods”

(formerly “ Cereal Laboratory M ethods,”

7th ed.) American Association of Cereal Chemists, St. Paul, Minn.

Anonymous. 1972. “ Technical Service Manual. Promine-F. Isolated Soy Protein.” Chem- urgy Div., Central Soya, 111.

AOAC. 1970. “ Official Methods of Analysis,” 11th ed. Association of Official Analytical Chemists, Washington, D.C.

Briskey, E.J. 1970. Functional evaluation of protein in food systems. In “ Evaluation of Novel Protein Products,” Ed. Bender, A.E., Kihlberg, R., Lofqvist, B. and Munck, L. Pergamon Press. Toronto.

Clandinin, D.R. 1958. Sunflower seed oil meal. In “ Processed Plant Protein Foodstuffs,” Ed. Altschul, A.M. p. 557. Academic Press, Inc., New York.

Elmquist, L.F. 1965. Safflower seed meal nonisolated protein. U.S. Patent 3,175,909.

Hermansson, A.M. 1972. Functional properties of proteins for foods-swelling. Lebensm. Wiss. u. Technol. 5(1): 24.

Janicki, N.A. and Walczak, J. 1954. Wateriness of meat and m ethods of its determination. In Hamm, R. 1960. Biochemistry of meat hydration. Adv. Food Res. 10: 355.

Johnson, D.W. 1970. Functional properties of oilseed proteins. J. Am. Oil Chem. Soc. 47: 402.

Lemancik, J.F . and Ziemba, J.V. 1962. Versatile soy flours. Food Eng. 34(7): 90.

Paulsen, T.M. and Horan, F.E. 1965. Functional characteristics of edible soya flours. Cer. Sci. Today. 10(1): 14.

Rakosky, J. 1970. Soy products for the meat industry. J. Agr. Food Chem. 18: 1005.

Szczesniak, A.S. and Engel, E. 1960. Soy f l o u r /casein combination. U.S. Patent 2,952,543.

Sosulski, F.W. and Bakal, A. 1969. Isolated proteins from rapeseed, flax and sunflower meals. Can. Inst. Food Sci. Tech. J. 2(1): 28.

Sosulski, F.W., Sabir, M.A. and Fleming, S.E. 1973. Continuous diffusion of chlorogenic acid from sunflower kernels. J. Food Sci. 38: 468.

Ms received 7 /2 /73; revised 9 /1 /73 ; accepted 9 /6 /7 3._______________________________

The research was supported by grants from the Hantleman Agriculture Research Fund and the National Research Council of Canada. The authors are indebted to Miss J.J. Soroka and Mrs. D. Knapp for technical assistance.

N. E. H A R R IS , 5. J. B IS H O V, A . R. R A H M A N , M . M . R O B E R T S O N a n d A . F . M A B R O U K

F o o d L a b o ra to ry , U.S. A r m y N a t ic k L a b o ra to r ie s , N a t ic k , M A 0 1 7 6 0

S O L U B L E C O F F E E : S H E L F L I F E S T U D IE S

INTRODUCTION

S O L U B L E C O F F E E is t h e d r ie d w a te r e x t r a c t o f r o a s t e d g r o u n d c o f f e e . T h e p r o d u c t c o n s i s t s o f b r o w n c o l o r e d f re e - f lo w in g p a r t i c l e s o f u n i f o r m s ize ( M o o r e s a n d S t e f a n u c c i , 1 9 6 4 ) . S o l u b l e c o f f e e c o m p r is e s t w o d i s t i n c t p r o d u c t t y p e s , v iz . , s p r a y d r ie d a n d f r e e z e d r ie d . S o l u b le c o f f e e p r o d u c e r s ad d n a t u r a l c o f f e e a r o m a in c o f f e e o i l t o t h e p o w d e r . A c c o r d i n g t o F e d e r a l S p e c i f i c a t i o n C o f f e e , In s t a n t ( 1 9 6 4 A m e n d . 1 9 6 7 ) t h e r e q u i r e m e n t s f o r s p r a y -d r ie d c o f f e e a r e : M o i s t u r e c o n t e n t 3 % m a x i m u m , a n d c a f f e i n e 3 . 2 % m i n i m u m . T h e d is t in c t iv e f e a t u r e s o f f r e e z e - d r ie d c o f f e e as c i t e d in t h e m i l i t a r y I n t e r i m P u r c h a s e D e s c r i p t i o n S - l l - 2( 1 9 7 2 ) a r e : m o i s t u r e c o n t e n t 2 . 6 % m a x i m u m ; c a f f e i n e 2 . 2 % m i n i m u m ; a n d c a r b o h y d r a t e s 3 5 % m a x i m u m o n d ry bas is . In g e n e r a l t h e s a m e c o m p o u n d s f o u n d in f r e s h c o f f e e a r e c o n t a i n e d i n s o l u b l e c o f f e e ; h o w e v e r , t h e r a t i o s a n d a m o u n t s a re c o m p l e t e l y d i f f e r e n t a n d a re d e p e n d e n t u p o n t h e p r o c e s s o f r e m o v i n g w a t e r ( G i a n t u r c o , 1 9 6 7 ) . A s m o r e h ig h ly v o la t i le a n d c h e m i c a l l y u n s t a b l e f la v o r c o n s t i t u e n t s a re r e t a in e d o r a d d e d t o s o lu b le c o f f e e , t h e p r o b l e m o f s t a b i l i t y , i . e . , s t a l in g , in c r e a s e s .

D e t e r i o r a t i o n o f in i t ia l q u a l i t y o r s t a l ing o f t h e s e c o f f e e s h a s l o n g b e e n a t t r i b u t e d t o r e a c t i o n s w i t h h e a d s p a c e o x y g e n . S iv e t z a n d F o o t e ( 1 9 6 3 ) c la im e d t h a t f la v o r c h a n g e s in p r o p e r l y p a c k e d s o lu b le c o f f e e w o u ld b e n o m i n a l a f t e r a p e r io d o f 6 m o n t h s t o sev e ra l y e a r s i f i t w e r e sea led a i r - t ig h t a n d s t o r e d at less t h a n 2 1 ° C . F u r t h e r m o r e t h e y s t a t e d t h a t in s t a n t c o f f e e s t a b i l i t y in a ir s t o r a g e w h e n n o t a r o m a t i z e d ( c o f f e e o i l a d d - b a c k ) w a s v e r y g o o d . R e c e n t s t u d ie s b y B i s h o v e t al .( 1 9 7 1 ) o n a v a r ie t y o f f r e e z e - d r ie d f o o d s s t o r e d u n d e r “ z e r o ” o x y g e n i n d ic a t e d a p r e f e r e n c e f o r t h e s e f o o d s . P u b l i s h e d s h e l f s t u d ie s d e a l in g w i t n s o l u b l e c o f f e e s s t o r e d u n d e r “ z e r o ” o x y g e n h e a d s p a c e c o n d i t i o n s w e r e v i r t u a l ly n o n e x i s t a n t . In f a c t W i lh e lm ( 1 9 5 8 a , b ) in t w o a r t i c l e s r e p o r t e d a b o u t p a c k a g i n g r e q u i r e m e n t s o f i n s t a n t c o f f e e b u t h e m a in ly d is cu ss e d e a r ly w o r k w i t h c o m m e r c i a l e q u i p m e n t a n d c o n t a in e r s . W h i t e ( 1 9 5 9 ) r e c o m m e n d e d u s in g a ir t ig h t c o n t a i n e r s t o p r o t e c t t h e q u a l i t y o f s o l u b l e c o f f e e b u t a lso d id n o t o f f e r s u p p o r t i v e d a ta b y p a n e l s t u d ie s a s t o w h y l o w o x y g e n in t h e h e a d - s p a c e o f t h e p a c k a g e d p r o d u c t w a s n e c e s

sa r y . T h e q u a l i t y o f s o l u b l e c o f f e e is m a in l y d u e t o t h e v o la t i le a r o m a o f o v e r 3 0 0 c o m p o u n d s w i t h b o i l i n g p o in t s v a r y in g b e t w e e n 1 6 0 ° C a n d o v e r 2 5 0 ° C ( T h i j s s e n 1 9 6 9 ) . T h e i n s t a b i l i t y o f t h e a r o m a f la v o r o f s o l u b l e c o f f e e d u r in g s t o r a g e is t h e m a jo r p r o b l e m f a c i n g t h e c o f f e e i n d u s t r y . L o s s o f f la v o r d u e t o a g in g h a s b e e n r e d u c e d s o m e w h a t b y b e t t e r p a c k a g in g , b y m o r e e f f i c i e n t d is t r i b u t i o n , a n d ra p id d e l iv e r y a n d b y m o r e c a r e in s t o c k r o t a t i o n a n d t u r n o v e r ( L e e ,1 9 6 5 ) .

E ld e r ( 1 9 4 0 ) r e p o r t e d t h a t r o a s t in g c o f f e e b e a n s r e s u l t e d in in c r e a s in g s t a b i l i t y o f t h e c o f f e e o i l d u e t o f o r m a t i o n o f h e t e r o c y c l i c i m i n o c o m p o u n d s , s o m e o f w h i c h s h o w e d a n t i o x i d a n t p r o p e r t i e s . T h e o x y g e n o f a ir is p o t e n t e n o u g h t o o x i d i z e i m p o r t a n t c o n s t i t u e n t s o f f la v o r less c o m p o u n d s ( L e e , 1 9 6 5 ) .

T h i s s t u d y w a s u n d e r t a k e n b e c a u s e t h e m i l i t a r y is p r o c u r in g f r e e z e - d r ie d c o f f e e in l im i t e d q u a n t i t i e s a n d w o u ld l ik e t o d e s ig n a s y s t e m t h a t o f f e r s a d e q u a t e p r o t e c t i o n a g a in s t d e t e r i o r a t i o n . A s t u d y o f t h i s n a t u r e c o u ld e n t a i l a m u l t i t u d e o f r e s e a r c h ; h o w e v e r , it w a s l im i t e d in s c o p e t o a n s w e r p r a c t i c a l q u e s t i o n s s u c h as : d o e s a lo w o x y g e n h e a d s p a c e p r o t e c t t h e s e c o f f e e s ? H o w d o e s a f r e e z e - d r ie d c o f f e e c o m p a r e w i t h a s p r a y -d r ie d c o f f e e as t o k e e p in g q u a l i t y s t o r e d in v a r io u s p a c k s , a n d is a d e f i n i t e m a x i m u m t o l e r a n c e o n h e a d s p a c e o x y g e n n e e d e d in t h e p u r c h a s e d o c u m e n t o r s p e c i f i c a t i o n ? C u r r e n t l y a re s id u a l o x y g e n o f n o t g r e a t e r t h a n 2 % is a c u s t o m a r y s p e c i f i c a t i o n s t a n d a r d f o r s t o r in g f r e e z e - d r ie d f o o d s ( U . S . A r m y N a t i c k L a b o r a t o r i e s , 1 9 6 7 ) . A c c o r d i n g t o P a le s e ( 1 9 7 2 ) , a l o w o x y g e n a t m o s p h e r e p a c k o f th is n a t u r e is c o s t ly ( a b o u t 2 4 p e r 8 - o z p a c k ) .

EXPERIMENTALM aterials

Spray-dried so luble coffee. A fresh batch o f spray-dried agglom erated soluble coffee was p rocured from a coffee p ro d u cer in their com m ercial 10-oz jars. The m oistu re co n ten t was 4.5%.

Freeze-dried soluble coffee. A fresh batch o f freeze-dried soluble coffee was p ro cu red from a coffee p roducer in their com m ercial 8-oz jars. The m oistu re co n ten t was 2.0%.Gases

O ne gas was a m ix tu re o f analy tical grade 98% n itrogen /2% oxygen v/v. T he o th e r gas

used was 5% hydrogen v/v in n itrogen . C a ta lys t w as palladium pow dered m eta l, 0 .5% w /w on kaolin as pellets.Packaging

8 oz each o f b o th spray-dried in s tan t and freeze-dried in s tan t soluble coffees w ere repacked in herm etically-sealed 401 X 411 cans in a con tro lled hum id ity ro o m regu la ted at 21°C and 20% relative h u m id ity .

F o r the air pack no gas was added to the headspace. To achieve 2% oxygen , the c o n ten ts o f the can were purged four tim es in a closed cham ber by pulling a 30-in. vacuum hold ing 30 sec to rem ove any a tm osphe ric gases and then back flushing from a tan k the desired 2% o x y gen m ix tu re gas. F o r the “ zero ” oxygen packs the same p roced u re as above was fo llow ed excep t th a t the cans con ta in ed 0.5g o f a palladium catalyst (H oudry Processing & D evelopm en t C o.) w rapped in “ K im w ipes” tissue paper and fastened to the can lid w ith “ S co tch ” transp aren t tape and the cans purged w ith the gas con ta in ing 5% hydrogen /95% n itrogen . T he cans afte r closure w ere d ipped in m o lten para ffin w ax, a tre a tm e n t found necessary to p reven t gas leakage through the can lid d o u b le seam seal closure. All cans were sto red at 37.8°C and w ithdraw n a f te r 1 w k, 1 m o n th , 2 m o n th s, 3 m on ths, 4 m o n th s, 6 m o n th s, 9 m o n th s and 12 m o n th s fo r headspace gas analysis and sensory evaluation . T he sam ples w ere run in d u p lica te . H eadspace gas analysis

Residual oxygen, carbon d iox ide an d residual hydrogen c o n te n ts w ere d e te rm in ed by the gas ch rom atograph ic m e th o d o f Bishov and H enick (1966). Sam pling was achieved w ith a can-lid piercing needle on a gas-tight syringe. T he sam pling area on the can lid w as firs t covered w ith a self-adhesive tape o f closed-cell p o lyu re thane foam . RTV sealant w as used to cover the ex ternal pa tch to p reven t leakage before sensory evaluation .Panel testing

Tw o d iffe ren t panels were used to evaluate the stored sam ples.

A technolog ical panel o f 12 se lected judges w ho rep o rted judg em en ts on co lor, flavor, od o r and appearance on a 9 -po in t scale (Peryam and Shapiro , 1955). 40g o f spray-dried so luble coffee were dissolved in 1 gal o f boiling w ater and served as black coffee in 2-fl oz porcelain cups a t a tem p era tu re o f 63°C . 36g o f freeze-dried soluble coffee w ere reco n stitu ted in a gallon o f boiling w ater and served in th e sam e m anner as the spray-dried soluble coffees. Spray-dried or freeze-dried coffee trea tm en ts o f air pack, 2% oxygen pack and “ zero ” oxygen pack w ere served to th e techn ical panel in separa te sessions as a 3 o f 3 com plete b lo ck design.

A consum er panel o f 30 ju dges random ly selected at each session re p o rte d prefe rences on a 9 -po in t scale (Pilgrim and P ery am , 1958). T he panel was served coffee tre a tm e n ts initially and afte r 12 m onths.


SOLUBLE COFFEE: SHELF STUDIES- 1 9 3


H e a d s p a c e g as s t u d i e s —S o l u b l e c o f f e e s

D a t a ( T a b l e 1 ) i n d ic a t e d t h a t as t h e s o l u b l e c o f f e e s a g e d , t h e r e w a s a g e n e r a l d e c r e a s e in t h e a m o u n t o f o x y g e n in t h e h e a d s p a c e gas o f b o t h a ir a n d 2 % o x y g e n p a c k s o f s p r a y -d r ie d s o l u b l e ( S D S ) a n d f r e e z e - d r ie d s o l u b l e c o f f e e s ( F D S ) . In t h e “ z e r o ” o x y g e n p a c k s t h e r e w a s n o a p p r e c i a b l e c h a n g e in t h e o x y g e n c o n t e n t o v e r t h e e n t i r e s t o r a g e p e r io d . T h i s su g g e s te d t h a t t h e c a n s w e r e n o t l e a k in g a n d t h a t

t h e c o f f e e w a s n o t a b s o r b i n g o x y g e n . T o c h e c k t h i s , t h e a m o u n t o f r e s id u a l h y d r o g e n t h a t r e m a i n e d in t h e “ z e r o ” o x y g e n p a c k s w a s a n a l y z e d . T h e h e a d s p a c e gas a n a ly s i s i n d ic a t e d t h e p r e s e n c e o f h y d r o g e n in t h e gas p h a s e , t h u s c o n f i r m i n g t h e a b s e n c e o f a gas l e a k a n d t h a t all o f t h e o x y g e n a b s o r b e d d u r in g p r o c e s s in g o n t h e c o f f e e p a r t i c l e s r e a c t e d w i t h t h e h y d r o g e n r e s u l t in g in i ts c o m p l e t e a b s e n c e in t h e gas p h a s e .

S a m p l e s p a c k e d in “ z e r o ” o x y g e n a t m o s p h e r e s h o w e d n o s i g n i f i c a n t d i f f e r e n c e in o x y g e n c o n t e n t y e t s o l u b l e c o f f e e

p a c k e d in e i t h e r a ir o r 2 % o x y g e n a t m o s p h e r e s h o w e d a g r a d u a l d e c r e a s e in o x y g e n h e a d s p a c e c o n t e n t d u r in g s to r a g e . F o r e x a m p l e , t h e S D S c o f f e e a b s o r b e d m o r e o x y g e n t h a n t h e F D S c o f f e e . C o m p a rin g t h e t w o c o f f e e s as t o c a r b o n d i o x id e e v o l u t i o n , i t w as f o u n d t h a t S D S c o f f e e g av e o f f s i g n i f i c a n t l y m o r e c a r b o n d i o x i d e ( a ir a n d 2 % o x y g e n p a c k s ) t h a n F D S c o f f e e s t o r e d u n d e r s im i la r c o n d i t io n s . C o n v e r s e l y , a t “ z e r o ” o x y g e n t h e r e w a s n o d i f f e r e n c e in c a r b o n d i o x i d e e v o l u t i o n b e t w e e n t h e s e t w o c o f f e e s .

G e n e r a l l y s p e a k in g , S D S i n s t a n t c o f f e e

Table 1—Headspace oxygen and C 0 2 fo r soluble coffees3 packed in "ze ro " oxygen, 2% oxygen and air in hermetically-sealed containers stored at 37.8°C

0//o

SampleIn itia l

O, o o

1 weeko 2 c o 2

1 montho 2 c o 2

3 monthso 2 c o 2

4 monthso 2 c o 2

6 monthso 2 c o 2

9 monthso 2 c o 2

1 2 monthso 2 c o 2 o 2

sig.c o 2

SDS air 20 1.0 17.5 1.9 13.6 6.7 7.8 14.7 6.5 15.5 4.1 18.5 - - - - NS *F DS air 20 1.0 14.2 1.2 17.3 3.0 9.7 6.3 7.9 7.4 4.4 8.5 2.8 8.9 - -SDS 2% 02 2 1.1 1.8 1.1 1.6 3.9 0.5 8.2 0.3 10 .2 0.3 10.7 0.3 14.2 0.0 16.3 * *FDS 2% 02 2 0.7 1.8 0.7 1.4 1.9 0.4 3.8 0.2 4.6 0.2 5.5 0.3 6.5 0.0 8.2SDS 0% 0 1.0 0 0.6 0.2 1.7 0.0 9.0 0.0 6.0 0.0 11.9 0.4 13.8 0.1 17.8 NS NSFDS 0% 0 1.2 0 1.4 0.1 4.8 0.0 3.6 0.0 4.1 0.0 5.5 0.3 6.4 0.0 7.7

* S i g n i f i c a n t d i f f e r e n c e a t 5 % l e v e l

a S D S = s p r a y - d r i e d s o l u b l e ; F D S = f r e e z e - d r i e d s o l u b l e ; d a t a m e a n o f t w o s a m p l e s

Table 2—Soluble coffees-Comparison of headspace gas treatments as judged by a technical panel using mean sensory ratings at each withdrawal from storage at 37.8°Ca

Treatment I n it. sig. 1 wk sig. 1 mo sig. 2 mos sig. 3 mos sig. 4 mos sig. 6 mos sig. 9 mos sig. 12 mos sig.

'Ze ro " oxygen Color 7.0 6.8 7.4Odor 6.3 6.7 ' 6.7Flavor 5.5 5.8 6.5Appearance 6.9 6.8 7.3

2% Oxygen Color 7.0 6.9 7.1Odor 6.3 6.1 6.3Flavor 5.5 NS 5.4 NS 6.0

Appearance 6.9 6.8 6.9

Airpack Color 7.0 6.8 7.3Odor 6.3 6.2 6.4Flavor 5.5 5.5 5.8Appearance 6.9 6.8 7.2

'Zero" oxygen Color 7.0 6.9 7.2Odor 6.3 6.3 6.3Flavor 5.8 6.0 6.3Appearance 6.9 6.8 6.9

2% oxygen Color 7.0 6.8 7.3Odor 6.3 NS2 6.2 NS 6.4

Flavor 5.8 5.5 6.7


Airpack Color 7.0 6.9 7.4Odor 6.3 6.3 6.5Flavor 5.8 5.9 6.5


a 5 . 0 = f a i r ; 7 . 0 = g o o d

* S i g n i f i c a n t 5 % l e v e l ; N S = n o t s i g n i f i c a n t

Spray-dried soluble

7.1 7.1 7.3 6.9 6.8 7.26.9 6.5 6.0 6.2 6.0 6.1

6.9 5.9 6.1 6.4 6.0 5.57.1 7.0 6.8 6.9 6.7 6.9

7.1 7.2 7.3 6.8 6.9 7.16.4 6.5 5.8 6.1 6.3 6.2

6.1 NS 6.3 JNS 5.3 NS 6.3 NS 6 .4 NS 5.7 NS

6.9 6.9 6.8 6.7 6.8 6.8

7.0 7.2 7.2 6.9 6.8 7.06.4 6.4 5.7 6.0 6.2 6.0

6.4 6.3 5.3 5.8 5.3 ' 5.3

6.8 7.1 6.8 6.6 6.4 6.6Freeze-dried soluble

7.0 7.2 7.4 7.2 7.0 7.36.3 5.5 5.7 6.3 6.3 6.0

5.9 6.3 6.3 6.2 6.0 5.3

6.8 7.2 7.3 7.1 7.0 7.1

7.1 7.2 7.4 7.2 7.0 7.3

5.9 NS 5.7 NS 6.2

00LO

fi NS 6.5 NS 6.5 NS

6.0 5.4 6.3 5.8 6.3 5.7

6.8 7.3 7.4 7.3 7.0 7.1

7.0 7.3 7.4 7.2 6.9 7.3

5.9 5.3 6.3 5.6 6.1 6.0

5.7 5.4 6.4 5.3 6.1 5.5

6.8 7.3 7.4 7.1 6.9 7.0


p r o d u c e d g r e a t e r q u a n t i t i e s o f c a r b o n d i o x i d e t h a n F D S . C h a r t in g p e a k lev e ls v e r s u s s t o r a g e t i m e , it w a s f o u n d t h a t t h e t w o c o f f e e s v a r ie d as t o w h e n t h e h ig h e s t lev e l o c c u r r e d b u t i t w a s a f t e r 6 m o n t h s s t o r a g e a t 3 7 . 8 ° C f o r t h e a i r p a c k o f S D S , a f t e r 1 2 m o n t h s f o r S D S 2 % o x y g e n p a c k , a n d a f t e r 1 2 m o n t h s f o r t h e S D S “ z e r o ” o x y g e n p a c k . F o r F D S t h e p e a k c a r b o n d i o x i d e lev e l o c c u r r e d a t 9 m o n t h s f o r t h e a i r p a c k , 1 2 m o n t h s f o r t h e 2 % o x y g e n p a c k a n d a t 1 2 m o n t h s at t h e “ z e r o ” o x y g e n p a c k . I t s h o u ld b e e m p h a s iz e d t h a t t h e r e w e r e n o s a m p le s a v a i la b le t o a n a l y z e f o r h e a d s p a c e g ases f o r t h e 9 m o n t h S D S air w i t h d r a w a l or S D S 1 2 m o n t h s o f F D S s a m p l e s in air p a c k . F u r t h e r m o r e t h e d a ta in T a b l e 1 i n d ic a t e t h a t as c o f f e e a g e s a t 3 7 . 8 C, h e a d s p a c e o x y g e n c o n t e n t d e c r e a s e d g r a d u a l ly a n d c a r b o n d io x i d e c o n t e n t in c r e a s e d . I n a i r p a c k s w h e r e m o r e o x y g e n w a s a v a i la b le a b s o r p t i o n o f o x y g e n an d e v o l u t i o n o f c a r b o n d io x i d e b y s o l u b l e c o f f e e s d o n o t f o l l o w a d i r e c t 1 :1 re la t i o n s h i p . T h i s in c r e a s e d level o f c a r b o n d i o x i d e m a y o r ig i n a t e f r o m t h e c a r b o x y l g r o u p s o f t h e a m in o a c id s b y n o n e n z y - m a t i c b r o w n i n g ( R e y n o l d s , 1 9 6 3 ) . L o c k h a r t ( 1 9 6 9 ) r e p o r t e d t h a t r o a s t e d c o f f e e c o n t a i n s 1 1 - 1 4 % p r o t e in , 1 2 - 1 4 % f a t an d 0 . 4 —2 . 0 % su g ar . In a n a q u e o u s e x t r a c t o f r o a s t e d c o f f e e t h e f o l l o w in g a m in o a c id s w e r e i d e n t i f i e d a n d q u a n t i f ie d ( m g / l O O g ) d r y w t : a l a n i n e ( 1 5 4 ) , a s p a r t i c ( 1 7 2 ) , g l u t a m i c ( 1 3 8 ) , g ly c in e ( 9 2 ) , l e u c i n e ( 5 1 ) , p h e n y l a l a n i n e ( 1 0 3 ) a n d v a l in e ( 1 5 4 ) ( U n d e r w o o d a n d D e a t h e r a g e , 1 9 5 2 ) . T h e p r e s e n c e o f su l f u r - c o n t a i n i n g a m i n o a c id s , c y s t i n e an d m e t h i o n i n e in a q u e o u s e x t r a c t s o f r o a s t e d c o f f e e h a d b e e n r e p o r t e d b y B a r b e r a ( 1 9 5 6 ) .

S e n s o r y p a n e l t e s t s

In T a b l e 2 s o l u b l e c o f f e e s w e r e e v a l u a t e d b y a t e c h n i c a l p a n e l a t t h e s a m e

w i t h d r a w a l t im e as w h e n h e a d s p a c e gas a n a ly s i s w a s p e r f o r m e d . D a t a i n d ic a t e d t h a t t h e o d o r o f t h e s p r a y -d r ie d s o l u b l e c o f f e e ( “ a r o m a t i z e d ” ) w a s a f f e c t e d t o s o m e e x t e n t b y o x y g e n in t h e h e a d s p a c e . T h e “ z e r o ” o x y g e n c o f f e e s a m p l e s w e r e ju d g e d t o b e s ig n i f i c a n t l y b e t t e r t h a n e i t h e r t h e 2 % o x y g e n o r a i r - p a c k t r e a t m e n t s a f t e r 1 w k a n d a f t e r 2 m o n t h s s t o r age.

N o n e o f t h e o t h e r c o f f e e t r e a t m e n t s w a s ju d g e d s i g n i f i c a n t l y d i f f e r e n t as t o o d o r , c o l o r , f la v o r o r a p p e a r a n c e w h e n c o m p a r i n g l ik e p a r a m e t e r s at e a c h w i t h d r a w a l p e r io d u n t i l a f t e r 9 m o n t h s at 3 7 . 8 ° C . A t t h a t t i m e t h e f la v o r o f t h e s p r a y -d r ie d c o f f e e in t h e a ir p a c k w as s i g n i f i c a n t l y d i f f e r e n t f r o m t h e o t h e r p a c k s . A f t e r 2 m o n t h s a t 3 7 . 8 ° C b o t h S D S a n d F D S c o f f e e d e v e lo p e d a n u n u s u al s h e l la c - l ik e o d o r s t o r e d u n d e r “ z e r o ” o x y g e n . T h i s o d o r w a s d e t e c t a b l e b y “ s m e l l ” o n o p e n i n g t h e c a n . F u r t h e r m o r e , t h i s o d o r p e r s is t e d in t h e “ z e r o ” o x y g e n p a c k s in s u b s e q u e n t w i th d r a w a l s a m p l e s o f t h e s e d r ie d c o f f e e s . T h i s o d o r w a s n o t a p p a r e n t in a n y o t h e r t r e a t m e n t s . A ls o , it d id n o t c a r r y - o v e r i n t o t h e r e c o n s t i t u t e d p r o d u c t s o f “ z e r o ” o x y g e n s a m p le s o f b l a c k c o f f e e an d a p p a r e n t l y had l i t t l e o r n o e f f e c t o n p a n e l ra t in g s . T h is a r o m a t r a n s f o r m a t i o n m a y b e d u e t o t h e d e v e l o p m e n t o f c a r b o n y l c o m p o u n d s ( F o r s s e t a l . , 1 9 6 7 ) .

F o r f r e e z e - d r ie d s o l u b l e c o f f e e , t h e r e w e r e n o s ig n i f i c a n t d i f f e r e n c e s f o r c o l o r , o d o r , f la v o r o r a p p e a r a n c e at a n y w i t h d ra w a l p e r io d u p t o 1 y r e x c e p t a f t e r 3 m o n t h s a t 3 7 . 8 ° C . A t t h i s w i t h d r a w a l , t h e c o f f e e s t o r e d u n d e r “ z e r o ” o x y g e n h e a d s p a c e w a s ju d g e d to b e s ig n i f i c a n t ly b e t t e r t h a n t h e 2 % a n d a ir p a c k s . S i n c e a t e c h n i c a l p a n e l is m a in l y c o n c e r n e d w i th q u a n t i t a t i v e d i f f e r e n c e s in c o f f e e q u a l i t y b u t n o t i ts a c c e p t a n c e , t h e c o f f e e s a m p le s w e r e e v a lu a t e d b y a c o n s u m e r p a n e l . In T a b l e 3 t h e d a ta s h o w t h a t t h e r e w e r e n o

d i f f e r e n c e s b e t w e e n t h e s p r a y -d r ie d s o l u b le c o f f e e in i t ia l ly b u t a f t e r 1 2 m o n t h s at 3 7 . 8 ° C , t h e s p r a y -d r ie d s o l u b l e c o f f e e s t o r e d in 2 % o x y g e n p a c k s w as ju d g e d b y t h e p a n e l t o b e s i g n i f i c a n t l y b e t t e r in a c c e p t a n c e t h a n t h e air p a c k , b u t n o t b e t t e r t h a n t h e “ z e r o ” o x y g e n o r u n s t o r e d n e w s a m p le . A n e w s a m p l e w a s i n t r o d u c e d t o s e e w h a t e f f e c t a g ed c o f f e e h a d o n c o n s u m e r a c c e p t a n c e . P a r a d o x i c a l l y , t h e 2 % o x y g e n s a m p l e t a s t e d as g o o d as t h e f r e s h s a m p l e ( c o n t r o l ) . F o r t h e f r e e z e - d r ie d c o f f e e t r e a t m e n t s t h e p a n e l d id n o t f in d a s i g n i f i c a n t d i f f e r e n c e in a c c e p t a n c e f o r a n y o f t h e h e a d s p a c e t r e a t m e n t s . T h e s e d a ta su g g e s te d t h a t f r e e z e - d r ie d s o l u b l e c o f f e e h a s a lo n g e r s h e l f l i f e t h a n s p r a y -d r ie d s o l u b l e c o f f e e u s e d in t h i s s t u d y b u t t h e r e a s o n f o r th is d i f f e r e n c e w a s n o t c le a r . It w a s e s t a b l is h e d ( S i v e t z , 1 9 6 3 ) t h a t m o i s t u r e c o n t e n t o f s o l u b l e c o f f e e is a c r i t i c a l f a c t o r f o r k e e p in g q u a l i t y . H e s t a t e d t h a t t h e f la v o r o f t h e p r o d u c t is r e la t iv e ly s t a b l e at 3 % m o i s t u r e c o n t e n t . M o r e o v e r , L e e ( 1 9 6 5 ) r e p o r t e d t h a t m o i s t u r e in a ir o n e v e n a fa i r ly d r y d a y c a n h y d r o l y z e s o m e o f t h e e s t e r s , a c e t a l s , a n d k e t a l s in c o f f e e a r o m a t o less f l a v o r s o m e c o n s t i t u e n t s . It w as f o u n d t h a t t h e c o m m e r c i a l s p r a y - dried c o f f e e u s e d in t h i s s t u d y h a d a m o i s t u r e c o n t e n t o f 4 . 5 % w h i le t h e f r e e z e - d r ie d c o f f e e h a d o n l y 2 . 0 % m o i s t u r e . T h e t e n t a t i v e c o n c l u s i o n w e d r a w is t h a t m o i s t u r e c o n t e n t a p p e a r s t o b e m o r e c r i t i c a l t o t h e k e e p i n g q u a l i t y o f s o l u b l e c o f f e e t h a n h e a d s p a c e gas t r e a t m e n t . T h i s is in a g r e e m e n t w i t h a s t a t e m e n t b y S iv e t z a n d F o o t e ( 1 9 6 3 ) . F o r s p r a y -d r ie d s o lu b le c o f f e e it s e e m s p r u d e n t , t h a t w h e n t h e p r o d u c t m o i s t u r e c o n t e n t e x c e e d s a c e r t a i n le v e l , it s h o u ld b e p a c k e d u n d e r l o w e r o x y g e n c o n d i t i o n s . T h e p ic - cu re o f w h a t h a p p e n s t o s o l u b l e c o f f e e o f d i f f e r e n t m o i s t u r e c o n t e n t s a n d s t o r e d in d i f f e r e n t a t m o s p h e r e s is b a s e d o n d e d u c - : i o n s f r o m p u b l i s h e d r e s u l t s . T h e s u b j e c t o f t h e s e i n t e r - r e l a t io n s h i p s w ill r e q u i r e m o r e e x t e n s i v e r e s e a r c h .

REFERENCESBarbera, C.E. 1956. Identification of cystine,

m ethionine, and proline in raw and roasted coffee. Coffee and Tea Inds. 79: 12.

Bishov, S.J. and Henick, A.S. 1966. A gas chrom atographic m ethod for continuous accelerated study of 0 2 uptake in fats. J. Am. Oil Chem. Soc. 43: 477.

Bishov, S.J., Henick, A.S., Giffee, J.W., Nil,I.T., Prell, P.A. and Wolf, M. 1971. Quality and stability of some freeze-dried foods in “ zero” oxygen headspace. J. Food Sci. 36: 532.

Elder, L.W. 1940. Staling vs. rancidity in roasted coffee. Ind. Eng. Chem. 32: 798.

F e d e ra l S p e c if ic a t io n C offee, Instant, HHH-C-575b, May 4, 1964, Am endm ent-2, July 12, 1967.

Forss, D.A., Angelini, P., Basinet, M.L. and M erritt, C. Jr. 1967. Volatile com pounds produced by copper-catalyzed oxidation of bu tterfat. J. Am. Oil Chem. Soc. 44: 141.

Gianturco, M.A. 1967. Coffee Flavor. In “ The Chemistry and Physiology of Flavors,” Ed. Schultz, H.W., Day, E.A. and Libbey, L.M., p. 431. The Avi Publishing Co., Inc., West- port, Conn.

Table 3—Consumer panel testing of soluble coffees served black- hedonic ratinga

Headspace treatment In itia l12 months

37.8°C

AirSpray-dried soluble

5.7b 4.92% Oxygen 5.7b 5.8

NSC0% Oxygen 5.7b 5.3Control (new sample) NAd 5.4

A irFreeze-dried soluble

6 .0b 5.82% Oxygen 6 .0b 5.3 (\ISC0% Oxygen 6 .0b 5.8Control (new sample) NAd 5.7

e ; 6 = l i k e s l i g h t l ya 5 = n e i t h e r l i k e n o r d i s l i k

b S i n g l e d e t e r m i n a t i o n

c N S = n o t s i g n i f i c a n t

^ N A = n o t a p p l i c a b l e

* S i g n i f i c a n t c i f f e r e n c e a t 5 % l e v e l ; L S D = 0 . 6

SOLUBLE COFFEE: SHELF LIFE STUDIES- 1 9 5

Lee, S. 1965. Stabilization of coffee flavor by m icroencapsulation. Second International Colloquium on the Chemistry of Coffee, 3—7 May, Paris, France.

Lockhart, E.E. 1969. Chemistry of coffee. The Coffee Brewing Institute Publication No. 25: 4.

Moores, R.G. and Stefanucci, A. 1964. “ Coffee Encyclopedia of Chemical Technology,” p. 748. John Wiley & Sons, Inc., New York, New York.

Palese, D.J. 1972. Personal Communication.U.S. Army Natick Laboratories General Equipment & Packaging Laboratory.

Peryam, D.R. and Shapiro, R. 1955. Perception, preference, judgment—Clues to food quality. Ind. Quality Control 11(7): 15.

Pilgrim, F.J. and Peryam, D.R. 1958. “ Sensory Testing Methods—A Manual,” p. 4. Technical Report 25-58, QMF&CI, Chicago, 111.

Reynolds, T.M. 1963. Chemistry of nonenzymatic browning. 1. The reaction between aldoses and amines. In “ Advances in Food Research,” Vol 12. Academic Press, New York, N.Y.

Sivetz, M. 1963. “ Coffee Processing Technol

ogy,” Vol 2, p. 102. The Avi Publishing Co., Inc., W estport, Conn.

Sivetz, M. and Foote, H. 1963. “ Coffee Processing Technology,” Vol 1, p. 519. The Avi Publishing Co., Inc., Westport, Conn.

Thijssen, H.A.C. 1969. The effect of process variables on aroma retention in drying coffee extract. Fourth International Colloquium on the Chemistry of Coffee, 2—6 June, Amsterdam, Holland.

Underwood, G.E. and Deatherage, F.E. 1952. A study of the amino acids of green and roasted coffee including a new m ethod of protein hydrolysis. Food Res. 17: 425.

U.S. Army Natick Laboratories Interim Purchase Description for Food Packet Long Range Patrol. IP/DES S-17-7, 4 May 1967.

U.S. Army Natick Laboratories Interim Purchase Description for Coffee, Soluble, Freeze-dried. IP/DES S -ll-2 , 1 December1972.

White, F.J. 1959. Air tight container protects quality of Savarin instant coffee. Tea & Coffee Trade J. 116(3): 44.

Wilhelm, R.B. 1958a. Inert gas packaging of

instant coffee. Coffee & Tea Ind. 81(1): 105.

Wilhelm, R.B. 1958b. Packaging requirements of instant coffee. Tea & Coffee Trade J. 115 ( 2 ).

Ms received 3 /30/73; revised 8 /31 /73 ; accepted 9 /6/73.


This paper reports research undertaken at the U.S. Army Natick (Massachusetts) Laboratories and has been assigned No. T. P. 1344 in the series of papers approved for publication. The findings in this report are no t to be construed as an official Dept, of the Army position. Citation of trade names in this report does no t constitute an official endorsem ent or approval of the use of such items.

We are grateful to Houdry Processing & Development Co. for the palladium catalyst. The authors particularly thank Mr. R obert Kluter, Food Acceptance Laboratory, NLABS, for performing the acceptance tests and to PFC John R. Troy, NLABS, who perform ed the analysis of variance for Table 1.

R O B E R T H A G E N M A IE R , K A R L F. M A T T IL a n d C A R L M . C A T E R

F o o d P ro te in R esearch & D e v e lo p m e n t C enter, Texas A & M U n iv e rs ity , C o llege S ta t io n , T X 7 7 8 4 3

D E H Y D R A T E D C O C O N U T S K IM M I L K A S A F O O D P R O D U C T ;

C O M P O S IT IO N A N D F U N C T I O N A L I T Y

INTRODUCTION

“ C O C O N U T M I L K ” is t h e n a m e c o m m o n l y g iv en t o t h e l iq u id p r e p a r e d by a q u e o u s e x t r a c t i o n o f g r o u n d - u p c o c o n u t m e a t s . T h e o i l c o n t e n t o f c o c o n u t m ilk d i f f e r s m a r k e d l y f r o m t h a t o f c o w ’s m i l k : w h i le c o w ’s m i l k h a s a b o u t e q u a l a m o u n t s o f o i l a n d p r o t e i n , c o c o n u t m i l k h as a b o u t t e n t i m e s as m u c h o i l as p r o t e in . R e m o v a l o f t h e e x c e s s o i l b y c e n t r i f u g a t i o n y ie ld s a p r o d u c t g e n e r a l ly ca l le d c o c o n u t s k im m i lk .

C o c o n u t s k im m i l k h a s b e e n p r e v io u s ly c o n s i d e r e d as a h ig h p r o t e in f o o d p r o d u c t ( R a j a s e k h a r a n an d S r e e n iv a s a n , 1 9 6 7 ; S a l o n an d M a n iq u is , 1 9 6 9 ) . H o w ev er , t h e p r o d u c t is s t i ll n o t a v a i la b le b e c a u s e o f p r o b l e m s e n c o u n t e r e d in large- s c a le p r e p a r a t io n . T h e p r o b l e m s c e n t e r a r o u n d d i f f i c u l t i e s w i t h c e r t a i n u n i t o p e r a t i o n s , e s p e c ia l ly t h e e f f i c i e n t s e p a r a t i o n o f o i l f r o m o t h e r c o m p o n e n t s ( H a g e n m a i e r e t a l . , 1 9 7 2 a ) . O i l s e p a r a t io n is i m p o r t a n t b e c a u s e t h e e f f i c i e n c y o f oi l r e c o v e r y d e t e r m i n e s t h e c o s t o f t h e c o c o n u t s k im m i l k . R e c e n t l y , a p r o c e s s h a s b e e n d e s c r ib e d w h i c h a c h ie v e s ca . 9 1 % r e c o v e r y o f o i l a n d p r o d u c e s a d r ie d , e c o n o m i c a l c o c o n u t s k im m i l k ( H a g e n m a ie r e t a l . , 1 9 7 3 ) .

T h e p u r p o s e o f t h i s w o r k is t o p r e s e n t a c h e m i c a l d e s c r i p t i o n o f c o c o n u t s k im m i l k , d e s c r i b e s o m e p h y s i c a l - c h e m i c a l p r o p e r t i e s , a n d p r e s e n t s o m e t a s t e p a n e l e v a l u a t i o n s o f r e c o n s t i t u t e d c o c o n u t s k im m i l k as a b e v e r a g e . It is p r e s u m e d t h a t t h e a n a ly s e s p r e s e n t e d w ill p r o v id e a c h e m i c a l b a s i s f o r i n t e r p r e t a t i o n o f t h e p h y s ic a l , c h e m i c a l and n u t r i t i o n a l c h a r a c t e r i s t i c s o f c o c o n u t s k im m i lk .

EXPERIMENTALAMINO ACID analyses were perfo rm ed by standard ion exchange separa tion o f p ro te in h ydro lysa tes , w ith cysteine analyzed separately as cysteic acid. No co rrec tions w ere m ade for d es tru c tio n o f am ino acids during hydrolysis. Each rep o rted value is the average o f a t least three results. M eth ion ine was n o t analyzed for separa te ly ; how ever, a N 2 flush was used during hydro lysis to preven t ox ida tio n .

P ro te in so lubility was m easured by m ixing p ro d u c t w ith distilled w ater, ag itating for ca. 30 m in at room tem p era tu re , m easuring pH, then centrifuging 10 m in a t 25 ,000 x G. T he supern a ta n t liquid was filtered th rough W hatm an no. 41 f ilte r paper and analyzed fo r dissolved p ro te in by K jeldahl analysis, o r by the Low ry tech n ique ca lib ra ted against K jeldahl.

T he am o u n t o f p ro te in p rec ip ita ted by heat

coagulation was determ ined by heating an aqueous suspension o f p ro d u c t for 20 m in, then cooling to 25°C. Any w ater evapora ted o ff during heating was added back, and the p ro tein so lubility then m easured afte r centrifuging and filtering.

Oil co n ten t was d e term ined a fte r weighing the lipid ex trac t ob ta in ed by the ch loroform - m ethano l ex trac tio n m eth o d o f Bligh and D yer(1959). T he ex trac ted oil was analyzed by standard m ethods.

R educing sugars were m easured by the M unson and W alker m ethod as described by T riebo ld and A urand (1963). R educing sugars a f te r inversion were m easured afte r heating the w ater ex trac t fo r 1 hr a t 60°C in 0.7M HC1. Sucrose id en tifica tion was m ade by m atching gas ch rom atography p a tte rn s w ith standards (trim ethy lsily l derivatives o f the samples) before and after inversion.

E quilibrium m oisture co n ten ts w ere m eas

ured after exposure o f sam ples to air o f contro lled relative hum id ity un til ap p aren t equ ilib rium was a tta ined (no fu rth e r w eight change). V iscosity was m easured w ith a B rookfield v iscom eter, w ith the spindle ro ta tin g a t 12 rpm .

The low m olecular w eight n itrogen is tak en as the n itrogen th a t passes th ro u g h an u ltra filtra tio n m em brane w ith a ra ted m olecu lar w eight cu t-o ff o f 1,000. M em branes w ere checked fo r passage o f larger m olecu les by u ltra filtra tio n o f bovine serum album in solutions.

M ineral co n ten ts were d e te rm in ed by a tom ic ab so rp tio n sp ec tro m etry , e x cep t fo r chloride and phosphorus. C hloride was d e te rm ined by vo lum etric analysis, and p h o sphorus from w eight o f a phosph o -m o ly b d a te com plex .

Sam ples o f d eh y d ra ted c o c o n u t skim m ilk were prepared w ith p ilo t p lan t eq u ip m en t. T he p repara tio n was as described by H agenm aier e t al. (1973). The p repara tio n consists o f w ater

Table 1—Chemical analysis of spray-dried coconut skim m ilk

% Composition15

Coconut water Tap waterused in used in Standard

processing processing dev

Protein Crude Protein

(IM X 6.25) 25 30 0 .8%Low Molecular Wt.

N (as % of N) 8 7 0.5

FatCrude Fat 5 7 1Free Fatty Acids

(as % o f Oil) 3.2 1.4 0.3Non-saponfiables

(as % of Oil) 3.2 0.5Iodine Value

(as % of Oil ) 6.3 _ 0.7Carbohydrates

Reducing Sugars 2.8 2.0 0.2Reducing Sugars 45 37 1.5

after inversion Sucrose 33 1.5Crude Fiber 0.03 0.03 0.02

MineralsPhosphorus 0.5 0.5 0.05Calcium 0.17 0.06 0.01Magnesium 0.26 0.36 0.03Potassium 3.6 3.3 0.2Sodium 0.9 1.4 0.3Chloride 1.6 1.6 0.2Ash accounted fo ra 8.2 9.3 0.4Ash (by analysis) 8.8 9.2 0.5

a B y c a l c u l a t i o n f r o m s t a t e d m i n e r a l c o n t e n t s b A t l e v e l o f 3 % m o i s t u r e


DEHYDRATED COCONUT SKIM MILK-IQT

ex trac tio n o f g round co co n u t m eats , rem oval o f solids by pressure f iltra tio n , and separa tion o f aqueous phase from oil phase w ith a centrifuge. The aqueous phase was dried by spray drying at air o u tle t tem pera tu res o f 8 5 -9 3 ° C , excep t w here no ted .

T he w ater used in p ilo t p lan t p repara tions was tap w ater w hich w as very soft; a typical com position : 390 ppm b icarbonate , 130 ppm Na, 54 ppm Cl, 1.5 ppm Ca and 0.5 ppm Mg. D istilled w ater was used in lab o ra to ry w ork.

All resu lts rep o rted in tables are the averaged resu lts o f analyses o f a t least tw o independently prepared sam ples.


T A B L E 1 s h o w s t h e c h e m i c a l c o n t e n t o f dried c o c o n u t s k im m i lk . T h e v a lu e s f o r p r o t e in , f a t a n d a sh a re s im ila r t o t h e v a lu e s r e p o r t e d b y R a o e t a l . ( 1 9 6 7 ) f o r c o c o n u t s k im m i lk p r e p a r e d b y t h e K r a u s s - M a f f e i p r o c e s s . In a d d i t i o n t o t h e r e s u l t s s h o w n in t h e t a b l e , t h e c o c o n u t s k im m i l k m a d e w i t h u s e o f c o c o n u t w a t e r h a d t h e f o l l o w in g t r a c e m in e r a l c o n t e n t s : 17 ± 4 p p m o f io d in e , 7 0 ± 3 0 p p m o f i r o n , 5 0 ± 2 0 p p m o f c o p p e r , 15 ± 5 p p m o f m a n g a n e s e , 3 ± 2 p p m o f c h r o m i u m a n d le ss t h a n 2 p p m o f c o b a l t .

S o m e o f t h e m in e r a l s in t h e p r o d u c t s w e r e c o n t r i b u t e d b y t h e t a p w a t e r u sed in p r o c e s s in g . T h e p r o d u c t m a d e w i t h c o c o n u t w a t e r as t h e e x t r a c t i n g l iq u id w a s

Fig. 1—S o lu b i l i t y as fu n c t io n o f p H , fo r c o c o

n u t s k im m i lk (a t 2 5 ° C, 10% so lid s ).

m a d e w i t h o n l y a b o u t 0 . 5 g ta p w a t e r p er g c o c o n u t m e a t s . T h e p r o d u c t m a d e w i t h o u t u s e o f c o c o n u t w a t e r w a s m a d e w i th 4 . 5 g t a p w a t e r p er g c o c o n u t m e a ts . In t h e l a t t e r c a s e , t h e io n s in t h e t a p w a t e r w o u ld c o n t r i b u t e t h e f o l l o w in g c a l c u la t e d

Fig . 2 —S o lu b i l i t y as fu n c t io n o f c o n c e n tra t io n

fo r c o c o n u t s k im m i lk (a t 2 5 ° C, p H 7 .0 ).

i o n c o n t e n t s t o t h e d r ie d p r o d u c t : s o d i u m , 0 . 6 % ; c h l o r i d e , 0 . 2 % ; c a l c i u m ,0 . 0 0 7 % ; m a g n e s i u m , 0 . 0 0 2 % . B a s e d o n t h e s e d a t a , it is e v id e n t t h a t t h e in c r e a s e d s o d iu m c o n t e n t o f p r o d u c t m a d e w i t h o u t c o c o n u t w a t e r is d u e t o s o d i u m in t h e t a p w a te r .

F r o m t h e d a ta in T a b l e 1, it is s e e n t h a t c r u d e p r o t e i n , p lus a s h , p lu s m o i s t u r e , p lu s c r u d e f a t , p lu s r e d u c in g su gars a f t e r in v e r s io n e q u a ls o n l y 8 6 % f o r p r o d u c t p r e p a r e d w i t h o r w i t h o u t c o c o n u t w a t e r . T h e 1 4 % u n a c c o u n t e d f o r is c u r r e n t l y u n i d e n t i f i e d a n d u n e x p l a i n e d .

T h e a m i n o a c id c o n t e n t o f c o c o n u t s k im m i l k w a s n o t o b s e r v a b l y d e p e n d e n t o n t h e a m o u n t o f c o c o n u t w a t e r u s e d in t h e p r o c e s s in g . ( N o t su rp r is in g , s in c e t h e c o c o n u t w a t e r c o n t a i n s o n l y 3 % o f t h e t o t a l p r o t e i n . ) T h e r e s u l t s in T a b l e 2 are a v e ra g e d a m in o a c id c o n t e n t s o f s a m p le s p r e p a r e d w i t h a n d w i t h o u t c o c o n u t w a t e r . T h e a m i n o a c id c o n t e n t s in T a b l e 2 a re s im ila r t o v a lu e s r e p o r t e d f o r a s im ila r p r o d u c t p r e p a r e d b y S r in iv a s a n et al . ( 1 9 6 4 ) .

T h e s o l u b i l i t y o f s p r a y -d r ie d c o c o n u t s k im m i l k as a f u n c t i o n o f p H is s h o w n in F i g u r e 1. T h e r e s u l t s a re a v erag ed f r o m e x p e r i m e n t s w i t h t w o in d e p e n d e n t l y p r e p a r e d s a m p le s , e a c h p r e p a r e d w i t h use o f c o c o n u t w a t e r . T h e p r o d u c t is s h o w n t o b e h ig h ly s o l u b l e e x c e p t in t h e pH r a n g e 3 . 0 —6 . 0 . T h e s e d a t a su g ge st t h a t c o c o n u t s k im m i l k m ig h t b e s u i ta b le f o r a n e u t r a l pH b e v e r a g e .

T h e e f f e c t o f c o n c e n t r a t i o n o n s o l u b i l i t y , f o r t h e s a m e p r e p a r a t io n s , is s h o w n in F ig u r e 2 . T h e a m o u n t d is so lv e d is s h o w n t o b e n e a r ly a c o n s t a n t f r a c t i o n o f t o t a l so l id s o v e r t h e e n t i r e c o n c e n t r a t i o n ra n g e i n v e s t ig a t e d . A p p r o x i m a t e l y 9 0 % o f t h e p r o t e in a n d t o t a l so l id s w e re o b s e r v e d to b e s o l u b l e a t pH 7 . 0 a n d 2 5 ° C . A d d i t io n -

Table 2—Am ino acid composition o f spray-dried coconut skim m ilk

Am ino acid g/16g NaRatio to

egg values13Ratio to

FAO pattern13

EssentialIsoleucine 2.6 39% 60%Leucine 5,4 61% 1 1 0 %Lysine 4.6C 71% 106%(Total Aromatic) (6 .1 ) (61%) (106%)Phenylalanine 3.8 65% 132%Tyrosine 2.3 55% 80%(Total Sulfur Containing) (3.0) (54%) (70%)Cysteine 1.7 71% 84%Methionine 1.3 41% 56%Threonine 2.4 47% 83%Tryptophan 0.9 56% 62%Valine 4.0 55% 93%

NonessentialHistidine 2.2Arginine 15.5Aspartic Acid 7.1Glutamic Acid 22.0Serine 3.7Proline 3.5Alanine 4.1Glycine 3.8

TO TAL 90.9a S t a n d a r d d e v i a t i o n i s c a . 0 . 1 g / 1 6 g Nb E a c h a m i n o a c i d c o n t e n t w a s d i v i d e d b y a m i n o a c i d c o n t e n t o f e g g o r

1 9 5 7 F A O p r o v i s i o n a l p a t t e r n , f o r s a m e a m o u n t o f n i t r o g e n . E g g a n d

F A O v a l u e s u s e d w e r e a s r e p o r t e d i n F A O / W H O , 1 9 6 5 , P r o t e i n

R e q u i r e m e n t s .c A v a i l a b l e l y s i n e w a s 4 . 3 g / 1 6 g N f o r s a m p l e s f r e e z e d r i e d o r s p r a y

d r i e d a t a i r o u t l e t t e m p e r a t u r e s o f 8 8 — 1 0 5 ° C , d e c r e a s i n g t o 3 . 8 g / 1 6 g

N a t a i r o u t l e t t e m p e r a t u r e s o f 1 0 7 — 1 1 6 ° C . A v a i l a b l e l y s i n e w a s

d e t e r m i n e d b y t h e m e t h o d o f C a r p e n t e r ( 1 9 6 0 ) .


u

Fig . 3 —P ro te in s o lu b i l i t y a t 2 5 ° C a f te r 9 0 ° C h e a t c o a g u la tio n , f o r c o c o n u t s k im m i lk ( a tp H

7 .0 ).

T O T A L % S O L I D S

F ig . 4 — V is c o s ity as fu n c t io n o f c o n c e n tra t io n , f o r c o c o n u t s k im m i lk (p H 7 .5 , 3 0 0 C).

s u c r o s e i n t h e s a m p l e s ( s e e T a b l e 1 ) a re r e s p o n s i b l e f o r m o s t o f t h e w a t e r b in d in g . T h e w a t e r b i n d i n g o f i s o la t e d c o c o n u t p r o t e i n h a s b e e n p r e v io u s ly r e p o r t e d ( H a g e n m a i e r , 1 9 7 2 ) .

T h e d a t a in F i g u r e 4 s h o w t h e v i s c o s i ty o f s o l u t i o n s o f c o c o n u t s k i m m i l k . T h e v i s c o s i t i e s a r e s im i la r t o d a t a f o r s u c r o s e s o l u t i o n s a t t h e s a m e c o n c e n t r a t i o n s o v e r t h e r a n g e 0 —6 5 % so l id s , w h i c h is p r o b a b l y d u e t o t h e h ig h s u c r o s e c o n t e n t o f t h e s a m p le s . T h e s o l u t i o n s u s e d f o r v i s c o s i t y m e a s u r e m e n t s w e r e m a d e in t h e l a b o r a t o r y w i t h c o c o n u t w a t e r u s e d as a n e x t r a c t i n g l iq u id . T h e v i s c o s i t y d a ta w ill b e o f in t e r e s t f o r p r o d u c t a p p l i c a - : i o n s a n d in p r o c e s s in g w o r k w h e r e v i s c o u s s o l u t i o n s a r e h a n d le d .

T h e s p e c i f i c g r a v i t y w a s m e a s u r e d f o r s o l u t i o n s o f c o c o n u t s k im m i l k p r e p a r e d i n t h e l a b o r a t o r y . D a t a f o r s p e c i f i c g ra v i t y a t 3 0 ° C w a s u s e d t o d e v e lo p e q u a t i o n ( 1 ) , w h e r e f s is t a k e n as t h e w e ig h t f r a c t i o n o f so l id s :

S p g r = 1 . 0 0 + 0 . 4 1 f s + 0 . 1 2 ( f s ) 2 ( 1 )

E q ( 1 ) c a n b e u s e d t o g e t a ra p id e s t i m a t e o f s o l id s c o n t e n t f r o m m e a s u r e m e n t o f s p e c i f i c g r a v i t y o v e r t h e c o n c e n t r a t i o n r a n g e 0 —8 0 % s o l id s .

F o r t a s t e p a n e l e v a l u a t i o n s s a m p l e s o f s p r a y -d r ie d c o c o n u t s k i m m i l k w e r e m i x e d w i t h t a p w a t e r a n d s e r v e d a t r o o m t e m p e r a t u r e . P r e f e r e n c e t e s t s w i t h s ix - m e m b e r p a n e ls w e r e u s e d t o e s t a b l i s h b e s t c o n c e n t r a t i o n . T h e r e w a s n o s t a t i s t i c a l ly s i g n i f i c a n t p r e f e r e n c e o v e r t h e c o n c e n t r a t i o n ra n g e 5 —1 5 % so l id s . T h e r e f o r e , c o n c e n t r a t i o n w as s e l e c t e d m o r e f r o m n u t r i t i o n a l c o n s i d e r a t i o n s . S e l e c t e d c o n c e n t r a t i o n w a s 1 2 . 5 % so l id s , w h i c h w o u ld g ive 3 . 7 5 % c r u d e p r o t e i n w h e n f o r m u l a t e d w i t h a d r y s a m p l e c o n t a i n i n g 3 0 % c r u d e p r o t e in .

F o r a c o m p a r i s o n w i t h d a ir y s k i m m i lk e a c h p a n e l i s t w a s s i m u l t a n e o u s l y p r e s e n t ed t w o u n i d e n t i f i e d s a m p l e s : o n e o f r e h y d r a t e d c o c o n u t s k i m m i l k ( 1 2 . 5 % s o l id s ) a n d o n e o f r e h y d r a t e d N F D M ( 9 . 4 % s o l id s ) . W i th 2 2 p a n e l i s t s a n d a h e d o n i c s c o r e o f 1 —9 , t h e c o c o n u t s k i m m i lk s c o r e d 6 . 2 a n d t h e r e h y d r a t e d N F D M

Table 4—Equilibrium moisture content of coconut skim m ilk, at 25°C

\Aateractivity

% Moisture after equ ilib ra tion3

Prepared w ith coconut water

Prepared w ith o u t coconut water

0.91 51 480.84 36 330.81 36 330.75 24 220A3 8 10

al d a t a i n d ic a t e d t h a t s o l u b i l i t y c o n t i n u e d t o b e h ig h a t 6 7 % s o l id s a n d 3 3 % w a te r .

T h e h e a t c o a g u l a t i o n o f t h e p r o t e in s in c o c o n u t s k i m m i l k w a s a lso o b s e r v e d . F i g u r e 3 s h o w s t h e s o l u b i l i t y a t 2 5 ° C a n d p H 7 . 0 , a f t e r h e a t i n g t h e s a m p le s t o 9 0 ° C f o r 2 0 m in . S o l u b i l i t y d e c r e a s e s w i t h in c r e a s in g c o n c e n t r a t i o n . H o w e v e r , i n c r e a s e d c o n c e n t r a t i o n w i t h n o h e a t i n g did n o t r e d u c e p r o t e i n s o l u b i l i t y ( s e e F ig . 2 ) . T h e r e f o r e , t h e d a t a o f F i g u r e 3 i n d ic a t e t h a t s t a b i l i t y t o h e a t c o a g u l a t i o n is b e t t e r a t lo w c o n c e n t r a t i o n s ( 2 —3 % so l id s ) . A l t h o u g h 2 —3 % s o l id s is b e l o w t h e u s e fu l c o n c e n t r a t i o n r a n g e f o r e c o n o m i c p r o c e ss in g , t h e h e a t s t a b i l i t y a t t h e s e lo w c o n c e n t r a t i o n s m a y b e a n a d v a n t a g e in s o m e p r o d u c t a p p l i c a t io n s .

T h e d a ta in T a b l e 3 s h o w t h e c o m b i n e d e f f e c t s o f p H a n d t e m p e r a t u r e o n t h e h e a t c o a g u l a t i o n o f t h e c o c o n u t s k im m i l k p r o t e in s . T h e r e s u l t s a re a v e ra g e d f r o m t w o e x p e r i m e n t s w i t h i n d e p e n d e n t ly p r e p a r e d s p r a y -d r ie d s a m p le s (p r e p a r e d w i t h c o c o n u t w a t e r ) . T h e d a ta i n d ic a t e t h a t a n u p w a r d a d ju s t m e n t o f p H ( f r o m

t h e u n a d j u s t e d v a lu e o f 6 . 0 —6 . 5 ) will p r e v e n t h e a t c o a g u l a t i o n . T h e s e r e s u l t s a re in a g r e e m e n t w i t h d a ta f o r p u r i f ie d c o c o n u t p r o t e in s ( H a g e n m a i e r e t a l . , 1 9 7 2 b ) . T h e d a t a in T a b l e 3 su g g e st t h a t c o c o n u t s k i m m i l k c a n w i t h s t a n d t h e h e a t t r e a t m e n t u s e d t o s t e r i l i z e e v a p o r a t e d m i l k , w i t h o u t m u c h p r o t e i n c o a g u l a t i o n .

T h e m o i s t u r e c o n t e n t s o f s a m p le s o f c o c o n u t s k im m i l k s o l id s a t r e g u la t e d v a lu e s o f r e la t iv e h u m i d i t y a r e s h o w n in T a b l e 4 . T h e s e d a ta s h o w t h a t c o c o n u t s k im m i l k is v e r y h y g r o s c o p i c . T h e t e n d e n c y f o r c o c o n u t s k i m m i l k t o b in d w a t e r is r e s p o n s i b l e f o r t h e d i f f i c u l t y e x p e r i e n c e d i n d r y in g t h i s p r o d u c t . T h i s d i f f i c u l t y h a s a ls o b e e n n o t i c e d b y o t h e r s ( R a j a s e k h a r a n a n d S r e e n iv a s a n , 1 9 6 7 ) . T h e s a m p l e s l o s t t h e i r p o w d e r y c h a r a c t e r a n d b e c a m e g lassy o r l iq u id o v e r t h e e n t i r e r a n g e o f w a t e r a c t i v i t y in v e s t ig a t e d , 0 . 4 3 —0 . 9 1 . T h e h y g r o s c o p i c n a t u r e o f t h i s p r o d u c t p r e s e n t s p a r t i c u l a r p r o b l e m s b e c a u s e t r o p i c a l c o c o n u t p r o d u c in g r e g i o n s a r e i n v a r ia b ly q u i t e h u m id . It is a s s u m e d t h a t t h e p o t a s s i u m , s o d i u m a n d

Table 3—Effect o f pH on irreversible heat dénaturation of the prote in in coconut skim m ilk. Protein so lub ility at 25°C after heating fo r 20 min at specified temperature

pH

% o f Protein dissolved3

25° C 55° C 70° C 80° C 90° C 95° C 125°Cb

8.0 84 82 83 83 80 80 927.5 84 83 85 81 80 76 797.0 83 83 83 79 75 70 756.5 82 81 80 75 49 44 43a E a c h s a m p l e w a s 5 % s o l i d s a n d 9 5 % w a t e r . S t a n d a r d d e v i a t i o n i s c a .

4 % .b -------- . --------

T h e s a m p l e s a t 1 2 5 C w e r e a u t o c l a v e d . S t a n d a r d d e v i a t i o n i s 2 . 5 % .

DEHYDRATED COCONUT SKIM MILK- 1 9 9

s c o r e d 3 . 5 . T h e p r o b a b i l i t y ( P ) t h a t th is d i f f e r e n c e is d u e t o c h o i c e w a s c a l c u l a t e d w i t h s t u d e n t s ’ t t o b e 5 % < P < 1 0 % .

S a m p l e s o f d e h y d r a t e d c o c o n u t s k im m i lk s h o w e d n o e v id e n t d e t e r i o r a t i o n w i th 6 m o n t h s s t o r a g e a t 2 0 —3 0 ° C as a d ry p o w d e r in glass c o n t a i n e r s . W et s a m p les w e r e s t a b l e t o m i c r o b i o l o g i c a l g r o w t h a t m o i s t u r e c o n t e n t s o f 3 2 % o r less.

T h e d a ta p r e s e n t e d in t h i s p a p e r ( c h e m i c a l a n a ly s is , f u n c t i o n a l p r o p e r t i e s a n d o r g a n o l e p t i c e v a l u a t i o n ) su g ge st t h a t c o c o n u t s k im m i l k m ig h t b e u sed in t h e f o r m u l a t i o n o f a n a c c e p t a b l e b e v e r a g e o r o t h e r f o o d p r o d u c t .

T h e n e x t s ta g e o f o u r r e s e a r c h in v o lv e s a n u t r i t i o n a l e v a l u a t i o n o f c o c o n u t s k im m i lk an d a m o r e d e ta i l e d l o o k a t f o o d u ses . C a r e fu l c o n s i d e r a t i o n is a l s o b e in g

g iv en t o t h e c o n s t r u c t i o n o f a f ie ld p i lo t p la n t f o r p r o d u c t i o n o f a p p r o x i m a t e l y 5 0 k g p e r d a y o f d e h y d r a t e d c o c o n u t s k im m i l k . I t is a s s u m e d t h a t w i t h s u c h a p i lo t f a c i l i t y a r e a s o n a b l y a c c u r a t e e s t i m a t e m a y b e m a d e o f p r o d u c t i o n c o s t s .

REFERENCES

Bligh, E.C. and Dyer, W.J. 1959. A rapid m ethod of total lipid extraction and purification. Can. J. Biochem. & Physiol. 37: 911.

Carpenter, K.J. 1960. The estim ation of available lysine in animal-protein foods. Biochem. J. 77: 604.

Hagenmaier, R. 1972. Water binding of some purified oilseed proteins. J. Food Sci. 37: 965.

Hagenmaier, R., Cater, C.M. and Mattil, K.F. 1972a. Critical unit operations of the aqueous processing of fresh coconuts. J. Amer.Oil. Chem. Soc. 49: 178.

Hagenmaier, R., Cater, C.M. and Mattil, K.F. 1972b. A characterization of two chrom ato

graphically separated fractions of coconut protein. J. Food Sci. 37: 4.

Hagenmaier, R., Cater, C.M. and Mattil, K.F.1973. Aqueous processing of fresh coconuts for recovery of oil and coconut skim milk.J. Food Sci. 38: 516.

Rajesekharan, N. and Sreenivasan, A. 1967. The use of coconut preparations as a protein supplement in child feeding. J. Food Sci. & Technol. 4: 59.

Rao, G.R., Ramanatham, G., Indira, K., Rao,U.S.B., Chandrasekhara, M.R., Carpenter,K. J. and Bhatia, D.S. 1967. Nutritive value of coconut protein concentrates obtained by wet processing. Indian J. Exp. Biol. 5: 114.

Salon, D.T. and Maniquis, P.L. 1969. Developm ent of a village process of preparing coconut skim m ilk beverage for feeding children. Phil. J. Nutr. 22: 164.

Srinivasan, K.S., Indira, K. and Chandrasekhara, M.R. 1964. Amino acid composition and electrophoretic behaviour of coconut proteins. Indian J. Biochem. 1: 146.

Triebold, H.O. and Aurand, L.W. 1963. “ Food Composition and Analysis.” D. Van Nostrum Co.

Ms received 6/23/73; revised 9 /4 /73; accepted9/6/73.

HOWARD R. MOSKOWITZ, RONALD A. SEGARS, JOHN G. KAPSAL/S and ROBERT A. KLUTERU.S. Army Natick Laboratories, Natick, MA 01760

S E N S O R Y R A T I O S C A L E S R E L A T I N G H A R D N E S S A N D C R U N C H IN E S S

T O M E C H A N I C A L P R O P E R T IE S O F S P A C E C U B E S

INTRODUCTIONR E C E N T D E V E L O P M E N T S o f p r o c e d u res f o r d i r e c t s e n s o r y s c a l in g in p s y c h o p h y s ic s a n d p s y c h o m e t r i c s hav e d e m o n s t r a t e d t h a t e q u a t i o n s m a y b e d e v e lo p e d t o r e la t e t e x t u r e p r o p e r t i e s assessed in- s t r u m e n t a l l y t o t e x t u r e p r o p e r t i e s ju d g e d b y t h e h u m a n o b s e r v e r ( M o s k o w i t z e t a l . ,1 9 7 2 ) . O n e p a r t i c u l a r m e t h o d , ra tio scaling , h a s p r o v id e d p o w e r f u n c t i o n s o f th e f o r m S = K I m , w h i c h r e la t e s th e p r o p o r t io n a l c h a n g e in m e a s u r e d m e c h a n i c a l p r o p e r t i e s ( I ) t o t h e r e s u l t a n t p r o p o r t io n a l c h a n g e o f s u b j e c t i v e l y p e r c e iv e d m a g n i t u d e ( S ) . T h e e x p o n e n t m g o v e r n s t h e r a t e a t w h i c h p e r c e iv e d i n t e n s i t y i n c re a se s w i t h p h y s ic a l m a g n i t u d e . V a lu e s o f m a r o u n d 1 .5 d e s c r ib e h o w a p p a r e n t r o u g h n e s s c h a n g e s w i th t h e m e a s u r e d grit s ize o f s a n d p a p e r ( S t e v e n s a n d H arris , 1 9 6 2 ) a n d su g ge st t h a t s in c e t h e e x p o n e n t e x c e e d s 1 . 0 s u b je c t i v e r o u g h n e s s a c c e l e r a t e s w i t h grit s ize . S m a l l i n c r e a s e s in p h y s ic a l r o u g h n e s s are a c c e n t u a t e d p e r c e p t u a l l y . S u b j e c t i v e l y e s t i m a t e d h a r d n e ss , in c o n t r a s t , o b e y s a p o w e r f u n c t i o n w h o s e e x p o n e n t ra n g e s f r o m 0 . 6 —0 . 8 ( H a r p e r a n d S t e v e n s , 1 9 6 4 ) , w h e n th e p h y s ic a l c o r r e l a t e is t h e f o r c e / i n d e n t a t i o n r a t io . S u b j e c t i v e h a r d n e s s d e c e le r a t e s w i t h p h y s ic a l m a g n i t u d e , so t h a t t h e per- c e iv e r c o n t r a c t s t h e ra n g e o f i n s t r u m e n t a l h a r d n e s s . S u b j e c t i v e l y e s t i m a t e d v i s c o s i ty a n d f lu i d i t y are g o v e r n e d b y p o w e r f u n c t io n s w i t h r e la t iv e ly lo w e x p o n e n t s ( 0 . 5 ; M o s k o w i t z , 1 9 7 2 ; S t e v e n s a n d G u i r a o , 1 9 6 4 ) . In o r d e r t o i n c r e a s e t h e a p p a r e n t s u b je c t i v e v i s c o s i t y o f g u m o r o i l l iq u id s b y a f a c t o r o f 1 0 , t h e p h y s ic a l v i s c o s i ty m u s t b e in c r e a s e d b y 1 0 0 t im e s .

P o w e r f u n c t i o n s p ro v id e a u s e fu l o r g a n iz in g p r in c ip le t o r e p r e s e n t s u b je c t i v e - i n s t r u m e n t a l r e l a t i o n s q u a n t i t a t i v e l y . A large arr a y o f t e x t u r e d e s c r i p t o r s h a s b e e n r e p o r t e d ( S z c z e s n i a k a n d K l e y n , 1 9 6 3 ; Y o s h i k a w a e t a l . , 1 9 7 0 ) , a n d c o r r e s p o n d in g ly large n u m b e r s o f m e c h a n i c a l p r o p e r t ies c a n be o b t a i n e d f r o m in s t r u m e n t s t h a t m e a s u r e e i t h e r o n e p r o p e r t y s p e c i f i c a l ly , o r i n t e g r a t e a n u m b e r o f m e c h a n i cal p r o p e r t i e s . T h e n u m b e r o f c o r r e l a t io n s b e t w e e n a p e r c e p t u a l a t t r i b u t e f o r t e x t u r e a n d a m e c h a n i c a l p r o p e r t y is la rg e , a n d w h e n s e v e ra l m e c h a n i c a l p r o p e r t i e s a re c o n s i d e r e d in c o n c e r t t h e n u m b e r o f p o t e n t i a l c o r r e l a t i o n s in c r e a s e s e x p o n e n t i a l l y .

B y a j u d i c i o u s r e p r e s e n t a t i o n o f th e s u b j e c t i v e - i n s t r u m e n t a l r e l a t i o n in t e r m s

o f a s in g le f u n c t i o n a l f o r m ( p o w e r f u n c t io n s , o r a n y o t h e r a r b i t r a r y f u n c t i o n ) o n e m a y d e t e r m i n e th e f o l l o w in g : ( a ) w h a t s e t o f p o w e r f u n c t i o n s ( o r a n y o t h e r c lass o f f u n c t i o n ) d e s c r ib e t h e s u b je c t iv e - i n s t r u m e n t a l r e l a t i o n ; ( b ) w h e t h e r s u b s t i t u t io n o f o n e m e c h a n i c a l p r o p e r t y f o r a n o t h e r a l te r s t h e e x p o n e n t o f p o w e r f u n c t i o n s w h e n a s in g le t e x t u r e a t t r i b u t e is p r e d ic t e d f r o m a n u m b e r o f d i f f e r e n t m e c h a n i c a l v a r ia b le s ; a n d ( c ) w h e t h e r w ell d e f in e d c o m b i n a t i o n s o f m e c h a n i c a l p r o p e r t i e s t o y ie ld d er iv ed m e c h a n i c a l p r o p e r t i e s t r a n s f e r t o t h e s u b je c t i v e r e a lm . P o s s ib i l i t y ( c ) la y s t h e g r o u n d w o r k f o r a t ru e s u b j e c t i v e p s y c h o l o g y o f t e x t u r e , e x i s t i n g s id e -b y -s id e w i th t h e p h y s ic s o f t e x t u r e . C o m b i n a t i o n s o f m e c h a n i ca l p r o p e r t i e s , in th is s y s t e m , w o u ld be r e f l e c t e d by a p p r o p r i a t e c o m b i n a t i o n s o f t h e i r s u b je c t i v e c o r r e l a t e s . O n ly r a t i o - s c a l ing p r o c e d u r e s , h o w e v e r , a l lo w th is p o s s i b i l i t y . In te r v a l ( c a t e g o r y ) s c a le s d o n o t , s in c e r a t i o s o f m e c h a n i c a l p r o p e r t i e s c a n n o t be p a ra l le le d b y r a t i o s o f in te rv a l - t y p e s e n s o r y m e a s u r e m e n t s .

T h i s s t u d y d e v e lo p s o n e p a r t o f a p s y c h o p h y s i c s o f t e x t u r e t h a t is ba se d u p o n fu n c tio n a l re lations b e t w e e n s u b j e c t ive a t t r i b u t e s a n d m e c h a n i c a l p r o p e r t i e s . T h e w o r k c o n c e r n s t w o a t t r i b u t e s r e la te d t o h a r d n e s s : p e r c e iv e d h a r d n e s s i t s e l f , and c r u n c h i n e s s . C r u n c h i n e s s , d e f in e d o p e r a t io n a l l y f o r t h e o b s e r v e r s , is th e p e r c e iv e d h a r d n e s s o f a f o o d a f t e r i t is c r u s h e d an d c h e w e d in t h e m o u t h 2 —3 t im e s . A l t h o u g h t h e s e l e c t i o n o f th e b e s t p r e d i c t o r e q u a t i o n c a n b e m a d e by s t a t i s t i c a l c r i te r ia o n l y a f t e r a f u n c t i o n f a m i ly ( l in e a r , p o w e r , e x p o n e n t i a l , l o g a r i t h m i c , e t c . ) has b e e n s e l e c t e d t o d e f in e t h e s u b je c t i v e - i n s t r u m e n t a l r e l a t i o n , we a r b i t r a r i ly s e l e c t e d th e p o w e r f u n c t i o n in v iew o f p r e v io u s r e s u l t s w i t h t h e d ir e c t s c a l in g o f o t h e r t e x t u r e c o n t i n u a . T h e e x p o n e n t o f a p o w e r f u n c t i o n is i n d e p e n d e n t b o t h o f t h e m u l t i p l i c a t iv e c h a n g e o f u n i t s f o r a m e c h a n i c a l p r o p e r t y a n d a m u l t ip l i c a t iv e c h a n g e in t h e s ize o f n u m b e r s s e l e c t e d by t h e o b s e r v e r ( c h a n g e in m o d u l u s f o r th e ju d g m e n t s ) .

In r e c e n t y e a r s c o n s i d e r a b l e w o r k has b e e n d o n e w i th s p a c e c u b e s as p a r t o f th e N A S A p r o g r a m . S p a c e c u b e s a re f o o d s w h o s e s ize a n d c o m p o s i t i o n m a k e t h e m e x c e l l e n t m o d e l s y s t e m s f o r th e s t u d y o f h a r d n e s s . H o l le n d e r ( 1 9 6 5 ) an d K l i c k a e t al. ( 1 9 6 7 ) d is cu ss e d t h e c o m p o s i t i o n , q u a l i t y a n d p e r f o r m a n c e o f t h e s e f o o d s in

d e ta i l . T h e s p a c e c u b e s a re s u f f i c i e n t l y sm a l l an d h a r d s o t h a t t h e y c a n b e e v a l u a te d f o r h a r d n e s s b y c r u s h in g in t h e m o u t h , w h i le s i m u l t a n e o u s l y b e in g t r a c t a b le f o r i n s t r u m e n t a l m e a s u r e m e n t o f m e c h a n i c a l p r o p e r t i e s .

EXPERIMENTAL

THE STIM ULI w ere four fo rm ula tions o f space cubes (s traw berry , graham cracker, sugar cookie and cheese cracker). In the tw o experim en ts, the space cubes w ere w ithdraw n from storage after 12 m o n th s at 4 .5°C , and after 16 m o n th s a t 4 .5°C . P roperties varied suffic ien tly w ith fo rm ulation so th a t the variously flavored space cubes had d ifferen t hardnesses and crunchiness, sensorially, and w ere governed by d ifferen t fo rce /d e fo rm atio n curves. T he space cubes were un ifo rm , m easuring 1.77 cm on each side. They w ere thus bite-sized, and easily evaluated by the panelists, as well as ideally sized for in stru m ental assessm ent o f tex tu re p roperties .

M echanical tex tu re p roperties w ere m easured w ith the In stro n U niversal T esting Machine (F loor Model TT-DM ), equ ipped w ith a load-strain con tro l un it. The cubes w ere com pressed un:-axially u nder parallel plate con d itions until the applied force being d isplayed on the recorder show ed a decrease o f 5 10% ind icating th a t ru p tu re had occurred . C om pression rates o f 0 .05 , 0 .20 , 1.00, 2 .00 and 5 .00 cm /m in were used in o rder to stu d y the effec ts o f this aspect o f the test p rocedure . A com pression rate o f 5 cm /m in was the m axim um possible rate th a t allow ed the Instron V4-sec reco rder to respond accurately to the rapid ly increasing force. The p late used for com pression had a surface area greater than th a t o f the sam ples. Ideally, this m eans th a t all parts o f the cube were subjected to the same strain . In ac tu a lity , how ever, rough areas, uneven surfaces and n o n parallel faces all p roduced deviations from the ideal com pression , thus causing sca tter in the ob ta ined m easurem ents. Ten rep lica tions were m ade a t each in strum en ta l tes t cond ition as a m inim um for sta tistical analysis.

F rom the fo rce-deform ation curve the fo llow ing properties were calculated:( 1 ) A p p a r e n t m o d u l u s o f e l a s t i c i t y (the ratio

o f stress to strain along the linear p o rtio n o f the loading curve; it m ay be a useful m easure o f the stiffness or rig id ity o f the sam ple).

(2) U l t i m a t e s t r e n g t h - ( t h e stress at ru p tu re ; a possible corre la te o f hardness).

S u b j e c t i v e a s s e s s m e n t o f t e x t u r e p r o p e r t i e s

Tw o separa te experim en ts w ere co n d u c ted in o rder to evaluate the m echanical p roperties related to subjective hardness. In E x p erim en t I, 48 panelists evaluated b o th the hardness and the crunchiness o f three flavors o f space cubes (straw berry , graham cracker and sugar cookie)


S E N S O R Y R A T I O S C A L E S - 201

Table 1—Sensory-Instrumental Functions

(1) log Hardness = 0.41 log Modulus of elasticity + 0.61 (r = 0.80)(2) log Hardness = 0.61 log U ltim ate strength + 1.02 (r = 0.75)(3) log Crunchiness = 0.55 log Mod. of elasticity - 0.05 (r = 0.89)(4) log Crunchiness = 0.72 log U ltim ate strength + 0.61 (r = 0.74)<5)a log Crunchiness = 1.22 log Hardness - 0.62 (r = 0.93)(6 )a log U ltim ate strength = 0.65 log Mod. elasticity - 0.58 (r = 0.93)

a Represents th e average regression fu n c tio n w hen i t is com puted w ith each variab le serving as th e c r ite r io n

after a storage period o f 1 2 m onths. In E xp erim ent II, 30 panelists (1 8 from E xp erim ent I. 12 new panelists) estim ated the ‘first hardness’ (corresponding to hardness in E xp erim ent I) and the ‘second hardness’ (corresponding to crunchiness) o f four flavors o f space cubes withdrawn from storage after 16 m onths.

The sealed cans containing the space cubes were withdrawn from storage and allowed to equilibrate at room tem perature for about 24 hr prior to the test. The sealed cans were opened on the test day, and im m ediately thereafter the cubes were transferred to quart-sized jars, (wide m outhed, Mason) with d esiccant packets at the b o tto m . The d esiccant m aintained the dryness o f the space cubes, and prevented the cubes from picking up m oisture that could alter their m echanical properties.

F o r purposes o f standardizing both the te x ture descriptors and the sampling m eth od , the panelists were presented w ith the follow ing d efin itions and procedures:(1 ) H ard n ess-th e am ount o f force exerted by

the m olar teeth needed to crack the cube in the m outh (first b ite ) ; and

(2) C ru n ch in ess-th e am ount o f force necessary to crush and grind the cube during the second and subsequent chews.

T he panelists were provided with the follow ing instructions, and told to perform exactly according to the procedure outlined.(1 ) Put the largest granule (soy protein chip)

into your m outh betw een your molar teeth (you may use either side o f your m outh ).

(2 ) B ite down gradually until the granule b r e a k s -th e force required to do this is hardness.

(3 ) Continue to chew two or three tim es to get an idea o f the am ount o f force needed to crush and grind the m aterial. This overall force is crunchiness.The panelists were instructed in the use o f

ratio scaling procedures [specifically the m eth od o f m agnitude estim ation (M oskow itz, 1 9 7 0 ; M oskow itz and Sidel, 1 9 7 1 ) ] . Their two num erical estim ates were to reflect ratios o f hardness or crunchiness, respectively. In both experim ents irregularly shaped soy protein chips were used as standards to anchor the judgm ents. In both experim ents care was taken to m ake the m agnitude estim ates o f bo th hardness and crunchiness com parable, bo th across stim uli, and com parable to each oth er. F or exam ple, a rating o f 3 0 0 on hardness and 10 0 on cru nch iness indicates that the im pression o f hardness is three times as great as the im pression o f crunch-

relating sensory judgm ent S to m echanical property I. The geom etric mean is the preferred measure o f central tendency for magnitude estim ates since they d istribute log-norm ally, and a power fu n ction is the preferred fun ctional form for subjective-instrum ental relations derived from m agnitude estim ation .

RESULTST A B L E 1 p r e s e n t s t h e f u n c t i o n s t h a t r e l a t e b o t h h a r d n e s s a n d c r u n c h i n e s s t o t h e m o d u l u s o f e l a s t i c i t y a t 5 c m / m i n c o m p r e s s i o n r a t e , a n d t o t h e u l t i m a t e s t r e n g t h a t 5 c m / m i n . N o t e t h a t t h e d a t a f r o m t h e t w o e x p e r i m e n t s h a v e b e e n p o o l e d t o y i e l d e s t i m a t e s f o r s e v e n d i f f e r e n t c u b e s . F i g u r e 1 a l s o s h o w s t h e b e s t - f i t t i n g p o w e r f u n c t i o n s r e l a t i n g t h e m e a n m a g n i t u d e e s t i m a t e t o t h e t w o p h y s i c a l c o n t i n u a .

T h e p r e s e n t r e s u l t s s u g g e s t t h a t b o t h h a r d n e s s a n d c r u n c h i n e s s a r e g o v e r n e d , a s a f i r s t a p p r o x i m a t i o n , b y s i m p l e p o w e r f u n c t i o n s , a l t h o u g h s o m e d e p a r t u r e s f r o m

t h e f u n c t i o n y i e l d s c a t t e r a b o u t t h e b e s t f i t t i n g r e g r e s s i o n l i n e . T h e e x p o n e n t f o r

h a r d n e s s i s a p p r o x i m a t e l y 0 . 4 w h e n t h e i n d e p e n d e n t p h y s i c a l c o n t i n u u m i s t h e

m o d u l u s o f e l a s t i c i t y , a n d 0 . 6 w h e n h a r d n e s s i s a f u n c t i o n o f t h e u l t i m a t e

s t r e n g t h . T h u s , h a r d n e s s g r o w s l e s s r a p i d l y t h a n t h e m e c h a n i c a l l y m e a s u r e d t e x t u r e a t t r i b u t e a g a i n s t w h i c h i t i s c o r r e

l a t e d . C r u n c h i n e s s s h o w s a s i m i l a r d e c e l e r a t i n g f u n c t i o n , a n d i t s e x p o n e n t i s 0 . 5 a s a f u n c t i o n o f t h e m o d u l u s o f e l a s t i c i t y , a n d 0 . 7 a s a f u n c t i o n o f t h e u l t i m a t e s t r e n g t h ( T a b l e 1 ) . S u b j e c t i v e l y , a

t e n f o l d i n c r e a s e i n t h e m e a s u r e d m e c h a n

i c a l p r o p e r t y i s p e r c e i v e d o n l y a s a t h r e e - t o f i v e f o l d i n c r e a s e i n h a r d n e s s a n d i n c r u n c h i n e s s . T h e t a c t i l e a n d k i n e s t h e t i c

s y s t e m s t h a t r e s p o n d t o m e c h a n i c a l p r o p e r t i e s a n d t r a n s f o r m t h e s e t o i n f o r m a t i o n a b o u t t e x t u r e t e n d t o c o m p r e s s t h e r a n g e o f p h y s i c a l v a r i a t i o n . T h e r a n g e o f h a r d n e s s i m p r e s s i o n s o n t h e s u b j e c t i v e s i d e i s c o n s i d e r a b l y s m a l l e r t h a n w h a t w o u l d b e e x p e c t e d w e r e t h e s e n s o r y s y s t e m t o m a p v e r i d i c a l l y t h e p h y s i c a l p r o p e r t i e s i n t o t h e s u b j e c t i v e r e a l m . L a r g e c h a n g e s i n t h e f o r c e - d e f o r m a t i o n c u r v e m a y y i e l d o n l y m i n o r p e r c e p t u a l c h a n g e s .

F r o m s i m p l e p s y c h o p h y s i c a l m e a s u r e m e n t o n e m a y d e d u c e o n l y t h e f o r m o f t h e i n t e n s i t y f u n c t i o n f o r t e x t u r e , b u t

n o t n e c e s s a r i l y c o n c l u d e w h i c h o f m a n y p o t e n t i a l p h y s i c a l c o n t i n u a i s m o s t r e s p o n s i b l e f o r p r o d u c i n g t h a t t e x t u r e a t t r i b u t e . H e r e , t w o c o n t i n u a c o r r e l a t e w i t h i m p r e s s i o n s o f h a r d n e s s a n d c r u n c h i n e s s . T h e y a r e c o r r e l a t e d w i t h e a c h o t h e r

M E C H A N I C A L PROPERTY

Fig. 1—Relation between the geometric mean o f subjective magnitude estimation (both o f hardness and o f crunchiness) and the mechanical properties o f the modulus o f elasticity and the ultim ate strength. Both mechanical properties were measured by the Instron Universal Testing Machine at a compression speed o f 5 cm /m in. The subjective-instrum ental functions are p lo tted in log-log coordinates, in which power

mess.T h e geom etric mean o f the m agnitude esti

m ates was com puted for each space cu be, and for each attribute. A regression analysis was used in order to find the b est-fittin g param eters (k , m ) o f a simple power fu n ctio n S = k lm ,

functions (S = k lm, S = sensory property, / = mechanical property) show up as straight lines. The exponents o f best-fitting power functions are always less than 1.0. The dimensions o f both mechanical properties are kg /cm 1, and the abscissa numbers for the modulus o f elasticity and for the ultim ate strength must be m ultiplied by 200 and 5, respectively.

2 0 2 - J O U R N A L O F F O O D S C I E N C E - V o l u m e 3 9 (1 9 7 4 )

( T a b l e 1 ) , a n d t h e r e g r e s s i o n f u n c t i o n r e l a t i n g t h e m a l l o w s t h e i n v e s t i g a t o r t o p r e d i c t t h e m o d u l u s o f e l a s t i c i t y f r o m t h e u l t i m a t e s t r e n g t h a n d v i c e v e r s a . T h e r e a r e a l s o t w o p o s s i b i l i t i e s f o r d e t e r m i n i n g w h i c h p h y s i c a l c o r r e l a t e i s m a x i m a l l y r e s p o n s i b l e f o r t h e s u b j e c t i v e t e x t u r e i m p r e s s i o n . O n e i s t h e d e g r e e o f c o r r e l a t i o n , o r t h e s t a t i s t i c a l c r i t e r i o n o f b e s t - f i t . B y t h a t c r i t e r i o n t h e m o d u l u s o f e l a s t i c i t y a t 5 c m / m i n s h o w s t h e h i g h e r c o r r e l a t i o n c o e f f i c i e n t , a n d i s t h u s m o r e s t r o n g l y r e

l a t e d b o t h t o h a r d n e s s a n d t o c r u n c h i n e s s . T h e o t h e r p o s s i b i l i t y i s t h e s i z e o f

t h e e x p o n e n t s . O n e c a n a r g u e t h a t e x p o n e n t s c l o s e r t o 1 . 0 r e p r e s e n t a v e r i d i c a l

s u b j e c t i v e - i n s t r u m e n t a l f u n c t i o n . A s t h e e x p o n e n t e i t h e r i n c r e a s e s o r d e c r e a s e s f r o m 1 . 0 s u b j e c t i v e r a t i o s o f t e x t u r e m a g

n i t u d e s g r a d u a l l y d i v e r g e f r o m i n s t r u - m e n t a l l y m e a s u r e d r a t i o s . I d e a l l y , t h e r e f o r e , i n a s t u d y o f h u m a n t e x t u r e p e r c e p t i o n t h e i n v e s t i g a t o r s h o u l d d e t e r

m i n e w h i c h o f h i s s u b j e c t i v e - i n s t r u m e n t a l

f u n c t i o n s a r e m o s t v e r i d i c a l . A c c o r d i n g t o

t h i s c r i t e r i o n o f v e r i d i c a l i t y t h e m e c h a n i c a l p r o p e r t y o f u l t i m a t e s t r e n g t h i s m o r e

a p p r o p r i a t e , s i n c e i t s e x p o n e n t s f o r b o t h h a r d n e s s a n d c r u n c h i n e s s e x c e e d e d t h o s e o b t a i n e d w i t h t h e m o d u l u s o f e l a s t i c i t y .

DISCUSSIONE Q U A T I O N S r e l a t i n g s u b j e c t i v e t o i n s t r u m e n t a l t e x t u r e m e a s u r e s h a v e a t l e a s t t w o i m p o r t a n t a p p l i c a t i o n s . O n o n e h a n d , t h e y i l l u s t r a t e h o w r a t i o s o f m e c h a n i c a l p r o p e r t i e s m a y b e t r a n s f o r m e d t o r a t i o s o f p e r c e i v e d t e x t u r e m a g n i t u d e . A

p s y c h o p h y s i c s o f t e x t u r e m a y b e d e v e l

o p e d , w i t h p r e d i c t o r e q u a t i o n s t h a t p a r a l l e l f o r m u l a e i n p h y s i c s b y a c o l l e c t i o n o f a r e p r e s e n t a t i v e s e t o f e x p o n e n t s r e l a t i n g

m e c h a n i c a l t o s u b j e c t i v e a t t r i b u t e s . T h i s

a p p l i c a t i o n c o n s t i t u t e s a n a p p r o a c h t o t e x t u r e b a s e d u p o n f u n c t i o n a l r e l a t i o n s b e t w e e n t w o r e c e p t o r s y s t e m s , t h e p h y s i c a l i n s t r u m e n t a n d t h e e v a l u a t i n g o b

s e r v e r .T h e s e c o n d a p p l i c a t i o n i s t h a t o f q u a l

i t y c o n t r o l . C o n t i n u a l i n s t r u m e n t a l m o n i

t o r i n g o f p r o c e s s e s w h o s e e n d p r o d u c t s a r e f o o d s w i t h d e s i r e d t e x t u r e c a n y i e l d

o n l y m e a s u r e s o f p h y s i c a l m a g n i t u d e s r e a d b y a n i n s t r u m e n t . T h e s e p h y s i c a l

m e a s u r e s r e f l e c t s h i f t s f r o m t h e i d e a l v a l

u e s o f m e c h a n i c a l p r o p e r t i e s d e s i r e d i n t h e p r o d u c t , b u t d o n o t i n d i c a t e w h e t h e r

l a r g e s h i f t s i n m e c h a n i c a l p r o p e r t i e s c o r r e s p o n d t o i m p o r t a n t , t o m o d e r a t e o r p e r h a p s e v e n t o o n l y m i n o r c h a n g e s i n

t h e s u b j e c t i v e t e x t u r e a t t r i b u t e c o r r e l a t e d w i t h t h e m e a s u r e . B y m e a n s o f a p p r o p r i a t e i n s t r u m e n t a l - s u b j e c t i v e e q u a t i o n s o n e

m a y e f f e c t i v e l y r e s c a l e t h e p r o c e s s m o n i

t o r i n g i n s t r u m e n t t o r e a d d i r e c t l y t h e t e x t u r e m a g n i t u d e t h a t w o u l d b e o b t a i n e d f r o m s u b j e c t i v e e v a l u a t i o n . I n t h e e s t i m a t i o n o f h a r d n e s s , f o r e x a m p l e , t h e o u t p u t o f a c o n t i n u a l m o n i t o r o f t h e m o d u l u s o f e l a s t i c i t y c o u l d b e t r a n s f o r m e d t o r e a d d i r e c t l y i n t e r m s o f s u b j e c t i v e h a r d n e s s ( i . e . , b y r a i s i n g t h e i n s t r u m e n t a l r e a d i n g t o t h e 0 . 4 p o w e r ) . A p p r o p r i a t e i m p l e

m e n t a t i o n o f q u a l i t y c o n t r o l , t h e r e f o r e , c o u l d b e e f f e c t e d w i t h r e s p e c t t o s u b j e c

t i v e l i m i t s , a n d t h e p r o c e s s m o n i t o r w o u l d r e s p o n d as i f i t w e r e a p a n e l o f h u m a n j u d g e s .

REFERENCESHarper, R. and Stevens, S.S. 1964. Subjective

hardness of compliant materials. Quarterly J. Exp. Phychol. 16: 204.

Hollender, H.A. 1965. Technology of space foods, p. 17. Activities Report, Research & Development Associates, Inc., U.S. Army Natick Laboratories, Natick, Mass.

Kapsalis, J.G., Drake, B. and Johansson, B. 1970. Texture properties of dehydrated foods: Relationships with the therm odynamics of water vapor sorption. J. Text. Stud. 1: 285.

Klicka, M., Hollender, H.A. and LaChance, P.A. 1967. Foods for astronauts. J. Amer. Dietet. Assoc. 51: 238.

Moskowitz, H.R. 1970. Ratio scales of sugar sweetness. Percept. & Psychophys. 7: 315.

Moskowitz. H.R. 1972. Scales of subjective viscosity and fluidity of gum solutions. J. Text. Stud. 3: 89.

Moskowitz. H.R. and Sidel, J.L. 1971. Magnitude and hedonic scales of food acceptability . J. Food Sci. 36: 677.

Moskowitz, H.R., Drake, B. and Akesson, C.A.1972. Psychophysical measures of texture. J. Text. Stud. 3: 135.

Stevens, S.S. and Guirao, M. 1964. Scaling of apparent viscosity. Science 144: 1157.

Stevens, S.S. and Harris, J.R . 1962. The scaling of subjective roughness and sm oothness. J. Exp. Psychol. 64: 489.

Szczesniak. A.S. and Kleyn, H. 1963. Consumer awareness of tex ture and of other food a ttributes. Food Technol. 17: 74.

Yoshikawa, S., Nishimaru, S., Tashiro, S. and Yoshida, M. 1970. Collection and classification of words for the description of food texture. 1, 2, 3. J. Text. Stud. 1: 427.

Ms received 6/28/73; revised 9 /17 /73 ; accepted9/19/73._____________________________________

The authors acknowledge and thank the members of the Sensory Evaluation Unit, Food Acceptance Group (Natick Laboratories) for their assistance in the study, and especially acknowledge Day Waterman for aid in the design of the study. H.R. Moskowitz and R.A. Kluter are members of the Pioneering Research Laboratory of the U.S. Army Natick Laboratories. J.G. Kapsalis and R.A. Segars are members of the Food Laboratory of the Natick Laboratories. A preliminary version of this report was presented at the meeting of the Institute of Food Technologists, May, 1972, in Minneapolis, Minnesota.

H O W A R D G. S C H Ü T Z and JOSEPH D. D A M RE LL Dept, o f Consumer Sciences, University o f California, Davis, CA 95616

PREDICTION OF HEDONIC RATINGS OF RICE BY SENSORY ANALYSIS

INTRODUCTIONT H E A B I L I T Y t o r e l a t e t h e s e n s o r y c h a r

a c t e r i s t i c s o f c o o k e d r i c e t o t h e i r p a l a t a - b i l i t y o r h e d o n i c v a l u e w o u l d b e u s e f u l i n

t h e d e v e l o p m e n t o f n e w v a r i e t i e s o f r i c e , i m p r o v e m e n t o f p r e s e n t r i c e s , d e v e l o p m e n t o f i m p r o v e d p r e p a r a t i o n i n s t r u c

t i o n s , a n d m o r e m e a n i n g f u l q u a l i t y c o n t r o l p r o c e d u r e s . N o r m a l l y , t h e p a y a b i l i t y o f r i c e i s a f f e c t e d b y a v a r i e t y o f

f a c t o r s , a m o n g w h i c h a r e : c o o k i n g t i m e ; a m o u n t o f w a t e r u s e d i n c o o k i n g ; a m o u n t o f s t a r c h i n t h e r i c e ; t h e c o n s i s t e n c y o f r i c e a f t e r i t i s c o o k e d . I n a d d i t i o n , t h e v a r i e t y o f r i c e , w h e t h e r i t i s s h o r t , m e d i u m , o r l o n g g r a i n , i s c r i t i c a l i n t h e w a y i n w h i c h t h e p r e v i o u s f a c t o r s o p e r a t e . A l

t h o u g h t h e r e h a v e b e e n s t u d i e s ( S i m p s o n e t a l . , 1 9 6 5 ; B a t c h e r e t a l . , 1 9 5 6 , 1 9 5 7 ) w h i c h e v a l u a t e t h e c o o k i n g q u a l i t y o f r i c e

w i t h r e g a r d t o s u c h v a r i a b l e s a s v a r i e t i e s , t h e s e w e r e j u d g e m e n t s m a d e b y a n e x p e r t - t y p e p a n e l a n d d i d n o t i n v o l v e c o n

s u m e r e v a l u a t i o n o f t h e r i c e p r o d u c t s . I n a d d i t i o n , t h e s e s t u d i e s d i d n o t q u a n t i t a t i v e l y r e l a t e t h e r e l a t i v e i m p o r t a n c e o f

s e n s o r y c h a r a c t e r i s t i c s t o q u a l i t y , b u t r a t h e r u s e d p h y s i c a l a n d c h e m i c a l m e a s u r e m e n t s a n d r e l a t e d t h e m t o s e n s o r y

c h a r a c t e r i s t i c s o f r i c e . A l s o , t h e r e h a v e b e e n s t u d i e s ( K a i t z , 1 9 7 1 ; P r a t t , 1 9 6 0 )

o n t h e p r e f e r e n c e f o r v a r i o u s t y p e s o f r i c e s u s i n g q u e s t i o n n a i r e t e c h n i q u e s i n w h i c h c o n s u m e r s s t a t e d t h e i r p r e f e r e n c e s

a n d d e s c r i b e d t h e c h a r a c t e r i s t i c s t h e y l i k e d a n d d i s l i k e d a b o u t r i c e . H e r e a g a i n t h e r e w a s n o q u a n t i t a t i v e r e l a t i o n s h i p d e t e r m i n e d b e t w e e n t h e r i c e c h a r a c t e r i s

t i c s a n d a c c e p t a n c e . T o o u r k n o w l e d g e a s t u d y h a s n o t b e e n r e p o r t e d i n w h i c h t h e

s e n s o r y c h a r a c t e r i s t i c s o f c o o k e d r i c e w e r e r e l a t e d t o c o n s u m e r p a l a t a b i l i t y . T h u s , t h e o b j e c t i v e o f o u r s t u d y w a s t o d i s c o v e r w h i c h s e n s o r y c h a r a c t e r i s t i c s o f c o o k e d r i c e a r e i m p o r t a n t , a n d t o w h a t d e g r e e , i n d e t e r m i n i n g c o n s u m e r p a l a t a b i l i t y . O u r t a s k t h e n w a s t o s e l e c t a v a r i e t y o f r i c e s a v a i l a b l e t o c o n s u m e r s a n d d e v i s e a m e t h o d w h i c h w o u l d e n a b l e u s t o d e t e r m i n e t h e i r s e n s o r y c h a r a c t e r i s t i c s b y u s e o f a t r a i n e d p a n e l a s a s e n s o r y

i n s t r u m e n t . W i t h t h e s a m e r i c e s w e w o u l d m e a s u r e h e d o n i c v a l u e f o r a g r o u p o f c o n

s u m e r s . T h e n , u s i n g t h e s e n s o r y a n a l y s i s d a t a a s p r e d i c t o r s , w e h o p e d t o d e t e r m i n e t h e q u a n t i t a t i v e r e l a t i o n s h i p b e t w e e n t h e s e s e n s o r y c h a r a c t e r i s t i c s a n d p a l a t a b i l i t y a n d t o e s t i m a t e t h e i r r e l a t i v e i m

p o r t a n c e .

EXPERIMENTALR ice samples

R ice products were selected from those available and sold for table use in grocery m arkets. Many varieties o f rices were experim ented with from the standpoint o f developing a set o f experim ental samples which covered a wide range o f sensory ch aracteristics. In order to obtain this range o f sensory ch aracteristics, it was necessary to prepare som e o f the rices in a m anner n ot suggested by the m anufacturer. In general, the samples were boiled in as little w ater as possible in order to m inim ize variation in tex ture during the tim e interval betw een preparation and serving. A specific e ffo rt was made to utilize rices o f sh ort, m edium and long grain as well as those that had been parboiled or precooked. Eventually , 2 0 rice sam ples were developed resulting from a com bin ation o f variety o f rice and cond ition s under which they were prepared. T he rice samples are listed in Table I.

Sensory panel training and analysis

S ix fem ale students from the College o f A gricultural & Environm ental Scien ces at the University o f C alifornia, Davis were trained in three 2 -hr sessions to evaluate the sensory characteristics o f cooked rice. T he first training session consisted o f the exposure o f the subjects to a sample o f the 2 0 cooked rices. During this session, which was o f a group discussion nature, sensory a ttrib u te term s were developed by having the su b jects w rite down and discuss as many o f the words that they could think o f that de-

Table 1 —Description o f rice samples

P ofrice/water

(g/cc)

Cooktime(Min)

1 . Short grain 0.30 192 . Short grain 0.44 32a3. Short grain 0.30 30a4. Short grain 0.34 195. Medium grain 0.34 14a6 . Medium grain 0.55 15a7. Medium grain 0.55 158 . Medium grain 0.30 259. Medium grain 0.21 15

10. Parboiled medium grain 0.48 251 1 . Parboiled medium grain 0.34 20a12. Parboiled medium grain 0.50 2013. Parboiled medium grain 0.37 2514. Parboiled medium grain 0.75 2715. Long grain 0.47 2016. Long grain 0.34 2517. Long grain 0.28 2018. Precooked long grain 0.43 1019. Parboiled long grain 0.30 2720 . Precooked long grain 0 .10 15

a Washed be fo re p rep ara tion

scribed sensory characteristics o f the rice samples. From the term s developed from the first session, the experim en ters selected 15 which in their judgem ent represented the broader sensory ch aracteristics o f tex tu re , appearance and taste. T he 15 sensory characteristics w ere: (1 ) size o f grain (length and width o f kern el); (2 ) com pactness (apparent density o f rice sam ple);(3 ) yellow ness (yellow co lo r) ; (4 ) flu ffiness (discreteness o f rice kernels in m o u th ); (5 ) stickiness (stickin g to m outh su rfaces); (6 ) m oistness (w etness in m ou th ); (7 ) dryness (lack o f w etness in m ou th ); (8 ) chalkiness (graininess in m ou th ); (9 ) starchiness (coating surfaces in m ou th ); ( 1 0 ) rubberiness (resistance to chew ing); ( 1 1 ) firm ness tfo rce to b ite through kern els); ( 1 2 ) tenderness (lack o f force to bite through kern els); (1 3 ) doneness (com pleteness o f co o k in g ); (1 4 ) rice flavor (ch aracteristic rice flavor); (1 5 ) o th er flavor (any nonrice flavor). In selecting the term s, tw o pairs o f opposite attribu tes were included in order to minimize the problem that these characteristics m ight be nonlinearly related to hedonic value. These were “ m oistness-dryness” and “ firm ness-tenderness.” T he statistical analyses to be conducted would take in to accou n t the ex ten t to which these term s are redundant.

During the second training session, the subje c ts were again exposed to a variety o f cooked rice sam ples and the 15 term s selected were discussed in term s o f developing a com m on understanding o f w hat each o f the sensory ch aracteristics m eant with regard to each o f the rice samples. In the third training session, the subje c ts gained exp erience in utilizing a sensory analysis rating scale for each o f the 15 characteristics in the evaluation o f a variety o f cooked rices. T he scale was a 7-point bipolar rating scale, with 1 representing a low am ount o f a characteristic and 7 representing a high am ount o f that ch aracteristic.

T he sensory analysis o f the 2 0 experim ental rice sam ples was carried ou t over a 2 -day period in a taste testing laboratory located on the Davis cam pus o f the University o f C alifornia. The tem perature in the testing booth s was held con stan t on both days at 2 1 °C ± 1°. Illum ination was provided by incandescent bulbs in each testing b o o th . All testing to o k place between 10 and 12 a.m . R ice sam ples were m aintained at serving tem perature through this period in a 7 3 °C w ater bath . On each day, 11 samples o f cooked rice were presented to each o f the six trained panelists. T h e samples were presented in a random order and as a ch eck on the reliability o f the jud gem en ts, tw o samples were rep licated but w ith d ifferen t identifying num bers on each o f the tw o test days. F or each sample, the judges were instructed to rate the degree o f each o f the 15 sensory characteristics on the 7-point scale previously described. The reliability co effic ien ts (correlation co effic ien t betw een rep licated sam ples) were 0 .9 0 and0 .8 6 , respectively, which indicates a high degree o f internal reliability .

V o lu m e 3 9 ( 1 9 7 4 ) - J O U R N A L OF F O O D SC IE N C E - 2 0 3


Fig. 1— Average sensory and hedonic scores for four best liked and for four least liked rice samples.

H e d o n i c r a t i n g b y c o n s u m e r p a n e l

F o r the jud gem en t o f hedonic value o f the 2 0 cooked rices it was considered desirable to use a representative group o f housewives. This type o f population was m ost conveniently available in Sacram en to , Calif. Since the testing facilities to be used did not allow for adequate preparation co n tro l, it was necessary to develop a m eans o f transporting the rice from the U niversity o f C alifornia, Davis, to Sacram ento with m inim um tem perature loss. It was determ ined by exp erim en tation that i f a 1 -gal polyurethane contain er was utilized, the tem peratures o f the rice in the, contain er would remain at a reasonable serving tem perature for a 1 0 -hr period (actual holding tim e was from V/i to 4 hr). This was true even in the case where the containers were opened and closed and rice samples removed at regular intervals. Tem perature measurem ents made during the testing per se indicated that there was about a 17°C tem perature change from the beginning to the end o f testing; how ever, at the conclusion o f the test day the low est tem perature was still warm enough to be considered an appropriate serving tem perature (5 8 °C ). On each o f the tw o testing days 5 0 volunteer housewives (a donation was made to organizations to which the women belonged) evaluated the 2 0 rice samples that were previously analyzed by sensory analysis using a 7-point rating scale anchored only at the ends w ith 1 being “ dislike very m uch” and 7 being “ like very m u ch .” T he tests were organized on both days so th at four sets o f five sam ples each were presented, in a small paper cup, in a random order to each o f five groups o f 1 0

women who arrived at the testing location at prearranged tim es. W ater for m outh rinsing was provided to each su b ject. The experim en ts were cond ucted betw een 1 :0 0 and 3 :3 0 p.m . A short background questionnaire on age, incom e and

rice usage was com pleted by all su b jects before the testing. T he testing cond ition s for both days were supervised by one o f the experim enters so as to m inim ize com m u n ication among subjects.

Table 2—Average sensory scale judgements and hedonic ratings fo r 20 rice samples

SizeYellow

nessCompact

nessM oistness

D ryness

S tick iness

F lu ff iness

Chalkiness

Starchiness

Rubberi-ness

Firm ness

Tenderness

Doneness

RiceFlavor

OtherFlavor

HedonicValue

1. Short grain 3.5a 2.7 5.5 5.5 2.0 5.3 3.2 2.3 5.3 2.3 2.5 5.0 5.0 3.2 1.3 2.82. Short grain 1.0 5.8 1.0 1.3 4.5 1.7 3.0 3.7 2.2 2.8 4.8 2.5 4.7 3.5 2.3 3.33. Short grain 1.5 4.8 2.2 4.8 2.3 1.7 3.3 3.7 2.2 1.2 2.3 4.7 4.7 2.8 2.3 3.14. Short grain 2.3 1.7 3.3 3.3 2.8 3.8 2.7 1.2 3.2 2.2 1.7 4.7 4.3 2.8 1.2 3.75. Medium grain 3.5 1.5 4.8 3.5 2.3 4.3 2.3 2.3 4.5 1.7 2.0 4.8 3.3 2.3 1.5 2.56. Medium grain 1.8 1.5 5.0 3.0 3.7 4.2 2.2 1.7 4.3 2.7 2.8 3.8 5.3 2.2 1.0 3.97. Medium grain 2.0 2.3 3.3 1.2 4.5 3.0 1.7 2.5 2.7 4.0 4.3 2.8 5.5 2.5 1.5 3.68 . Medium grain 4.5 3.3 4.2 4.2 2.0 2.2 3.5 1.7 4.0 1.3 1.7 4.8 3.3 2.3 2.5 2.49. Medium grain 4.3 3.2 5.8 6.3 1.2 5.8 2.3 2.5 5.5 2.3 2.7 5.2 5.5 4.7 2.5 1.4

10. Parboiledmedium grain 3.9 1.4 4.6 2.3 3.8 4.1 1.8 2.3 4.0 2.6 2.4 4.7 4.9 2.4 1.3 4.3





15. Long grain 6.7 2.1 3.3 3.8 4.0 2.3 5.2 2.3 3.8 3.3 3.2 5.3 5.2 2.5 1.2 2.716. Long grain 6.3 2.6 3.6 2.8 5.2 2.0 4.8 1.6 2.6 4.4 4.7 4.1 5.8 3.3 1.4 3.917. Long grain 4.8 1.5 2.8 3.5 2.2 1.8 3.7 2.0 2.3 1.7 2.7 3.7 5.2 2.2 1.5 3.518. Precooked

long grain 4.7 4.7 1.8 1.8 4.2 1.0 4.0 1.8 1.3 4.0 4.2 2.3 5.7 1.7 2.3 5.619. Parboiled

long grain 4.7 4.7 1.8 1.8 4.2 1.0 4.0 1.8 1.3 4.0 4.2 2.3 5.7 1.7 2.3 5.220. Precooked

long grain 6.7 3.8 1.5 4.3 2.8 1.2 5.7 1.8 1.7 1.8 3.3 5.3 6.3 2.5 1.5 4.9

a 1 = lo w degree o f charac te ris tic : 7 = high degree o f charac te ris tic : (N = 6 fo r Sensory Scale Judgem erts ; N = 100 fo r H edonic Ratings)

P R E D IC T IO N O F R IC E R A T I N G S - 2 0 5

Table 3—Correlations between fifteen sensory variables and hedonic judgements

SizeYellow

nessCompact

nessMoistness

D ryness

S tick iness

F lu ffiness

Chalkiness

Starchiness

Rubberiness

F irm ness

Tenderness

Doneness

RiceFlavor

OtherFlavor

HedonicValue

Size 1.00 - 0.10 0.03 0.25 -0 .06 -0 .16 0.71b -0 .4 7 a 0.00 0.14 0.00 0.33 0.40 - 0 .11 0.01 0.10Yellowness 1.00 -0 .5 8 b -0 .33 0.39 —0.49a 0.24 0.42 -0.41 0.32 0.58b -0 .5 1 a 0.07 0.27 0.73b 0.27Compactness 1.00 0.56b -0.31 o CO CO cr -0 .4 5 a -0.17 0.83b -0 .04 -0 .2 8 0.49a -0 .14 0.35 -0 .14 -0 .6 5 bMoistness 1.00 —0.81b 0.59b 0.06 -0 .05 0 .66b -0 .5 7 b -0 .5 8 b 0.80b - 0.02 0.40 -0 .26 - 0 .66bDryness 1.00 -0 .4 3 a 0.07 0 .12 -0 .4 6 3 0.85b 0.83b -0 .6 7 b 0.21 -0 .07 0.41 0.51aStickiness 1.00 -0 .5 9 b 0.02 0.91b -0 .18 -0 .3 4 0.48a -0 .15 0.51a - 0.21 -0 .6 7 bFluffiness 1.00 -0 .3 4 -0 .4 5 a 0.01 0.10 0.16 0.42 -0 .16 -0 .05 0.31Chalkiness 1.00 0.04 0.07 0.40 -0 .30 -0 .1 5 0.34 0.37 -0 .24Starchiness 1.00 -0 .24 -0 .40 0.59b -0.31 0.49a - 0 .12 —0.76bRubberiness 1.00 0 .86b -0 .6 3 b 0.35 0.04 0.55b 0.34Firmness 1.00 -0 .7 4 b 0.40 0.21 0.60b 0.32Tenderness 1.00 -0 .1 5 0.22 -0 .4 5 a -0 .5 4 aDoneness 1.00 0.07 - 0 .12 0.39Rice Flavor 1.00 0.26 -0 .6 0 bOther Flavor 1.00 0.02Hedonic Value 1.00

1 S ig n ific a n t a t 5% level (0 .4 3 ) * S ig n ific a n t a t 1% level (0 .5 5 )

RESULTS & DISCUSSIONA V E R A G E S w e r e c o m p u t e d f o r e a c h o f t h e 1 5 s e n s o r y s c a l e s a n d f o r t h e h e d o n i c

j u d g e m e n t s f o r t h e 2 0 r i c e s . T h e r e s u l t s a r e s h o w n i n T a b l e 2 . i t c a n b e s e e n b y e x a m i n a t i o n o f t h i s t a b l e , t h a t t h e r e i s w i d e v a r i a t i o n i n t h e i n t e n s i t y o f t h e v a r

i o u s s e n s o r y d i m e n s i o n s , a s w e l l a s i n t h e v a r i a t i o n o f h e d o n i c v a l u e . T h u s , t h e o b

j e c t i v e o f h a v i n g a w i d e r a n g e o f c h a r a c t e r i s t i c s a s w e l l a s p a l a t a b i l i t y w a s

a c h i e v e d . W i t h o u t t h i s r a n g e o f c h a r a c t e r i s t i c s i t w o u l d n o t b e a p p r o p r i a t e t o u t i l i z e q u a n t i t a t i v e t e c h n i q u e s f o r p r e d i c t i n g

h e d o n i c v a l u e f r o m s e n s o r y a n a l y s i s , s i n c e

a r e s t r i c t e d r a n g e o f d a t a w o u l d n e i t h e r b e s t a t i s t i c a l l y m e a n i n g f u l n o r r e s u l t i n g e n e r a l i z a b l e i n f o r m a t i o n .

F i g u r e 1 i s a n i l l u s t r a t i o n o f t h e d i f f e r e n c e s i n t h e s e n s o r y c h a r a c t e r i s t i c s o f t h e

f o u r b e s t l i k e d a n d f o u r l e a s t l i k e d s a m

p l e s . T h e f i g u r e s h o w s t h e a v e r a g e s o n t h e

1 5 d i m e n s i o n s f o r t h e s e s a m p l e s , a s w e l l a s t h e i r a v e r a g e h e d o n i c r a t i n g . E x a m i n a t i o n o f t h i s f i g u r e r e v e a l s t h a t t h e s e n s o r y c h a r a c t e r i s t i c s a p p e a r t o p l a y d i f f e r e n t

r o l e s w i t h r e g a r d t o p a l a t a b i l i t y . F o r e x a m p l e , “ s i z e , ” “ f l u f f i n e s s ” a n d “ o t h e r f l a v o r ” s h o w v e r y l i t t l e d i f f e r e n c e i n a v e r

a g e r a t i n g f o r t h e s e t w o g r o u p s o f r i c e s , w h e r e a s “ c o m p a c t n e s s , ” “ m o i s t n e s s , ” “ d r y n e s s , ” “ s t i c k i n e s s , ” “ c h a l k i n e s s ” a n d “ s t a r c h i n e s s , ” s h o w v e r y l a r g e d i f f e r e n c e s i n a v e r a g e r a t i n g s f o r t h e s e t w o g r o u p s o f

r i c e s .I n o r d e r t o q u a n t i f y t h e r e l a t i o n s h i p

b e t w e e n t h e s e n s o r y d i m e n s i o n s a n d p a l a t a b i l i t y , a m u l t i p l e r e g r e s s i o n a n a l y s i s w a s c o n d u c t e d . F i r s t , e x a m i n a t i o n o f t h e p l o t s o f h e d o n i c v a l u e s v s . s e n s o r y d i m e n s i o n s i n d i c a t e d s u f f i c i e n t l i n e a r i t y t o j u s

t i f y s u c h a n a n a l y s i s . T h e n , u s i n g t i r e

B i o - M e d s t e p w i s e r e g r e s s i o n c o m p u t e r p r o g r a m ( D i x o n , 1 9 7 0 ) , t h e m u l t i p l e r e

g r e s s i o n w a s c o m p u t e d . T h e c o r r e l a t i o n s a m o n g a l l v a r i a b l e s a r e g i v e n i n T a b l e 3 .

E x a m i n a t i o n o f t h i s t a b l e r e v e a l s t h a t t h e r e a r e m a n y h i g h l y s i g n i f i c a n t r e l a t i o n

s h i p s a m o n g s e n s o r y c h a r a c t e r i s t i c s . T h e s e r e l a t i o n s h i p s c o u l d b e d u e t o t h r e e

f a c t o r s : ( 1 ) t h e c h a r a c t e r i s t i c s h a p p e n t o c o v a r y i n t h e r i c e s a m p l e s ; ( 2 ) t h e c h a r a c t e r i s t i c s a r e m e a s u r i n g t h e s a m e p r o p e r t y ;

a n d ( 3 ) t h e c h a r a c t e r i s t i c s a r e m e a s u r e s o f p r o p e r t i e s o p p o s i t e o f o n e a n o t h e r . E x

a m p l e s o f f a c t o r ( 1 ) a r e “ c o m p a c t n e s s ” a n d “ s t i c k i n e s s ” ( 0 . 8 8 ) a n d “ m o i s t n e s s ” a n d “ t e n d e r n e s s ” ( 0 . 8 0 ) . E x a m p l e s o f

f a c t o r ( 2 ) a r e “ s i z e ” a n d “ f l u f f i n e s s ” ( 0 . 7 1 ) a n d “ r u b b e r i n e s s ” a n d “ f i r m n e s s ” ( 0 . 8 6 ) . E x a m p l e s o f f a c t o r ( 3 ) a r e “ m o i s t n e s s ” a n d “ d r y n e s s ” ( —0 . 8 1 ) a n d “ f i r m

n e s s ” a n d “ t e n d e r n e s s ” ( —0 . 7 4 ) . T h e r e a r e a l s o s e v e r a l h i g h l y s i g n i f i c a n t r e l a t i o n

s h i p s b e t w e e n t h e s e n s o r y c h a r a c t e r i s t i c s a n d h e d o n i c v a l u e ( “ c o m p a c t n e s s ” — 0 . 6 5 , “ m o i s t n e s s ” —0 . 6 6 , “ s t i c k i n e s s ” — 0 . 6 7 , “ s t a r c h i n e s s ” —0 . 7 6 a n d “ r i c e f l a

v o r ” - 0 . 6 0 ) . T h e d i r e c t i o n o f t h e r e l a t i o n s h i p s b e t w e e n t h e s e n s o r y v a r i a b l e s a n d h e d o n i c v a l u e i n g e n e r a l a r e e a s y t o u n d e r s t a n d ; h o w e v e r , t h e r e a r e a f e w c a s e s w h i c h d e s e r v e s o m e p o s s i b l e e x p l a n a t i o n . T h e n e g a t i v e c o r r e l a t i o n ( - 0 . 6 0 )

Table 4 —M ultip le regression analysis fo r prediction of hedonic ratings3

Variable DescriptionRegressioncoefficient

Betavalue % Contribution

Y Hedonic Value(Constant 4.284)

x , Size 0.277 0.4846 5.49x 2 Yellowness 0.282 0.4588 5.19x 3 Compactness -0.468 -0.7480 8.47x . Moistness 0.523 0.7944 8.99X s Dryness 1.165 1.4641 16.57X 6 Stickiness 0.799 1.2070 13.67X. Fluffiness -0 .482 -0 .5202 5.89X, Chalkiness -0.503 -0.3899 4.41X , Starchiness -0.581 -0 .7545 8.54X ,„ Rubberiness -0.703 -0.8423 9.53X , , Tenderness -0.251 -0.2691 3.05X 1 3 Doneness 0.225 0.1666 1.89X , 4 Rice Flavor -0.941 -0.7349 8.32

a M u ltip le R = 0.97 0 ; A d justed R = 0.95 8 ; R 2= 0 .9 4 2 ; A d jus ted R 2= 0.917 ; S .E . o f E s tim a te= 0 .3 94 .


b e t w e e n “ r i c e f l a v o r ” a n d h e d o n i c v a l u e p e r h a p s r e f l e c t s t h e f a c t t h a t t h e s e c o n

s u m e r s f i n d a n y t h i n g o t h e r t h a n a b l a n d f l a v o r l e s s a c c e p t a b l e . “ F i r m n e s s w i t h i t s

p o s i t i v e c o r r e l a t i o n ( 0 . 3 2 ) a n d “ t e n d e r

n e s s ” w i t h i t s n e g a t i v e c o r r e l a t i o n ( — 0 . 5 4 ) w i t h h e d o n i c v a l u e a p p e a r t o i n d i c a t e t h a t

“ t e n d e r n e s s ” i s a s s o c i a t e d w i t h t h e n e g a t i v e p r o p e r t i e s o f s o f t n e s s w h i l e “ f i r m

n e s s ” i s a s s o c i a t e d w i t h p o s i t i v e p r o p e r t y

o f d r y n e s s .

T h e m u l t i p l e r e g r e s s i o n r e s u l t s i n c l u d

i n g t h e r e l a t i v e c o n t r i b u t i o n o f e a c h o f t h e v a r i a b l e s , w h i c h a r e i n c l u d e d i n t h i s r e g r e s s i o n ( E z e k i a l , 1 9 4 1 ) , t o h e d o n i c

v a l u e i s g i v e n i n T a b l e 4 . T h e f a c t t h a t “ f i r m n e s s ” a n d “ o t h e r f l a v o r ” d o n o a p p e a r i n t h e e q u a t i o n o b v i o u s l y d o e s n o t m e a n t h a t t h e y h a v e n o r e l e v a n c e t o h e d o n i c v a l u e , b u t t h a t w i t h t h e s t e p w i s e r e g r e s s i o n p r o g r a m u t i l i z e d t h e y d o n o t

a d d s i g n i f i c a n t l y t o t h e a b i l i t y t o p r e d i c t h e d o n i c v a l u e . T h e r e l a t i v e c o n t r i b u t i o n i n f o r m a t i o n a s s h o w n i n T a b l e 4 m a k e s

c l e a r t h e f a c t t h a t s o m e v a r i a b l e s c o n t r i b u t e m o r e i m p o r t a n t l y t o h e d o n i c v a l u e

t h a n o t h e r s ; f o r e x a m p l e , “ d r y n e s s ” c o n t r i b u t e s a b o u t 1 6 % t o t h e v a r i a t i o n i n h e

d o n i c v a l u e , w h e r e a s a c h a r a c t e r i s t i c l i k e

“ d o n e n e s s ” c o n t r i b u t e s o n l y a b o u t 2 % .

T h e m u l t i p l e R o f 0 . 9 7 a n d R 2 o f 0 . 9 4 a r e v e r y h i g h a n d i n d i c a t e t h a t 9 4 % o f t h e v a r i a b i l i t y o f t h e a v e r a g e h e d o n i c r a t i n g s

c o u l d b e a c c o u n t e d f o r b y t h e 1 3 s e n s o r y

c h a r a c t e r i s t i c s . T h e a d j u s t e d R v a l u e ( E z e k i a l , 1 9 4 1 ) t a k e s i n t o a c c o u n t t h e r a t i o o f t h e n u m b e r o f r i c e s a m p l e s t o

p r e d i c t o r v a r i a b l e s a n d t h u s g i v e s a n e s t i m a t e o f t h e d e g r e e o f p r e d i c t a b i l i t y i f s u c h a s t u d y w e r e c o n d u c t e d w i t h o t h e r

r i c e s a m p l e s a n d o t h e r h o u s e w i v e s .C o m p a r i n g t h e p e r c e n t c o n t r i b u t i o n

r e s u l t s o f t h e m u l t i p l e r e g r e s s i o n w i t h t h e

e a r l i e r i n f o r m a t i o n r e p o r t e d i n F i g u r e 1, r e v e a l s t h a t i n g e n e r a l t h e s a m e v a r i a b l e s a r e i n d i c a t e d a s i m p o r t a n t a n d u n i m p o r t a n t . H o w e v e r , t h e r e a r e d i f f e r e n c e s , e s p e c i a l l y i n t h e l e s s e r r o l e o f “ c h a l k i n e s s ” a n d t h e m o r e i m p o r t a n t r o l e o f “ r u b b e r -

i n e s s ” i n t h e m u l t i p l e r e g r e s s i o n t h a n i n t h e F i g u r e 1 r e s u l t s . T h e s e d i f f e r e n c e s c a n b e e x p l a i n e d i n p a r t b y t h e i n c r e a s e d n u m b e r o f s a m p l e s u t i l i z e d i n t h e r e g r e s s i o n ( 8 v s . 2 0 ) a n d b y t h e f a c t t h a t t h e m u l t i p l e r e g r e s s i o n t e c h n i q u e g i v e s t h e i n d e p e n d e n t c o n t r i b u t i o n o f e a c h v a r i a b l e . I f o n e w e r e t o u t i l i z e s u c h i n f o r m a t i o n i n d e v e l o p i n g n e w p r o d u c t s , i m p r o v i n g p r o d u c t s , o r f o r q u a l i t y c o n t r o l p u r p o s e s ,

Table 5—M ultip le regression analysis for predicting hedonic ratings using five sensory scales®

Variables DescriptionRegressioncoefficient

YHedonic Value

(Constant 3.139)X 2 Yellowness 0.157

x 4 Moistness HD.199X , Starchiness =0.057X , Doneness 0.520X„ Rice Flavor -0 .696

a M u ltip le R = 0 .8 9 6 ; A d jus ted R = 0 .87 0 ; R 2 = 0 .80 3 ; A d jus ted R 2= 0 .75 7 ; S .E . o f Est m a te = 0 .5 1 1.

t h o s e v a r i a b l e s w i t h t h e h i g h e s t p e r c e n t

a g e o f c o n t r i b u t i o n s h o u l d b e t h e o n e s

w h i c h o n e o p t i m i z e s , w h e r e a s t h e o n e s w i t h a l o w e r p e r c e n t a g e c o u l d b e g i v e n l e s s a t t e n t i o n w i t h t h e l e a s t l i k e l i h o o d o f

a f f e c t i n g c o n s u m e r p a l a t a b i l i t y .

A l t h o u g h t h e m u l t i p l e r e g r e s s i o n r e

s u l t s r e d u c e s t h e n u m b e r o f s e n s o r y v a r i

a b l e s f r o m 1 5 t o 1 3 , t h i s i s s t i l l a l a r g e n u m b e r o f s e n s o r y s c a l e s t o u t i l i z e i n

p r a c t i c e . A l s o , p r e d i c t i n g 2 0 c a s e s ( r i c e

s a m p l e s ) f r o m 1 3 v a r i a b l e s e v e n w i t h t h e a d j u s t m e n t g i v e n e a r l i e r c a n y i e l d a n i n f l a t e d m u l t i p l e R . I n o r d e r t o p r o v i d e a

s m a l l e r n u m b e r o f s c a l e s f o r p r e d i c t i o n , t h e r e g r e s s i o n p r o g r a m w a s s t o p p e d a f t e r f i v e v a r i a b l e s h a d b e e n i n c l u d e d . W h e r e o n e s t o p s s h o r t o f l a c k o f s t a t i s t i c a l s i g n i f i c a n c e i s a r b i t r a r y , b u t f i v e v a r i a b l e s

s e e m e d a r e a s o n a b l e n u m b e r f o r p r a c t i c a l t e s t i n g a n d t h e a d d i t i o n o f a s i x t h v a r i a b l e o n l y a d d e d 0 . 0 2 2 t o t h e R 2 . T h e

r e s u l t s o f t h i s a n a l y s i s a r e s h o w n i n T a b l e

5 . T h e h i g h m u l t i p l e R ( 0 . 9 0 ) w i t h o n l y

f i v e v a r i a b l e s i n d i c a t e s t h a t t h i s r e g r e s s i o n e q u a t i o n c o u l d b e u s e d w i t h r e a s o n a b l e c o n f i d e n c e i n p r e d i c t i n g r i c e h e d o n i c v a l u e a s m e a s u r e d i n t h i s s t u d y . T h e p e r

c e n t c o n t r i b u t i o n d a t a w a s n o t c a l c u l a t e d f o r t h i s s e t o f v a r i a b l e s s i n c e i t w o u l d n o t b e a s m e a n i n g f u l w i t h f e w e r c h a r a c t e r i s t i c s i n c l u d e d i n t h e a n a l y s i s .

S t u d i e s o f t h i s g e n e r a l t y p e a l s o g i v e d i r e c t i o n i n t h e d e v e l o p m e n t o f i n s t r u

m e n t a l m e a s u r e s w h i c h m i g h t b e u s e f u l i n t h e p r e d i c t i o n o f p a l a t a b i l i t y . D e v e l o p i n g

i n s t r u m e n t a l m e a s u r e s , e i t h e r p h y s i c a l o r c h e m i c a l , w h i c h a r e h i g h l y c o r r e l a t e d

w i t h s e n s o r y d i m e n s i o n s a n d t h a t a r e i m p o r t a n t t o p a l a t a b i l i t y w o u l d s e e m t o b e

a l o g i c a l w a y f o r p r o d u c i n g v a l i d m e a s

u r e s .I f o n e s p e c u l a t e s o n t h e g e n e r a l i z a b i l -

i t y o f t h e r e l a t i o n s h i p s f o u n d i n t h i s

s t u d y t o o t h e r r i c e s a m p l e s a n d o t h e r j u d g e s , i t w o u l d s e e m f a i r t o s a y i f p l a i n c o o k e d r i c e r e p r e s e n t e d t h e p r o d u c t s

s t u d i e d t h a t t h e r e s u l t s s h o u l d b e q u i t e s i m i l a r . O n t h e o t h e r h a n d , i f t h e r i c e p r o d u c t s w e r e u t i l i z e d i n r e c i p e s o r w e r e

f l a v o r e d i n s o m e w a y t h e r e l a t i o n s h i p s g e n e r a t e d i n t h e p r e s e n t s t u d y p r o b a b l y w o u l d n o t h o l d t r u e . A s t o t h e j u d g e s i n v o l v e d , c e r t a i n l y o n e c o u l d f e e l f a i r l y c o n f i d e n t t h a t t h e s e r e s u l t s w e r e g e n e r a l -

i z a b l e t o t h e m i d d l e c l a s s w h i t e h o u s e w i f e ( a v e r a g e a g e w a s 5 0 , i n c o m e $ 8 , 0 0 0 , r i c e

u s a g e o n c e a w e e k ) . I f o n e c o n s i d e r s m a l e s o r d i f f e r e n t e t h n i c g r o u p s , e s p e c i a l

l y o r i e n t a l s w h e r e r i c e c h a r a c t e r i s t i c s t h a t a r e d e e m e d a p p r o p r i a t e m a y d i f f e r , t h e

m u l t i p l e r e g r e s s i o n r e s u l t s w o u l d p r o b a

b l y c h a n g e . A l s o , c o n s u m e r s o f a d i f f e r e n t a g e s u c h a s c h i l d r e n m i g h t y i e l d a

d i f f e r e n t s e t o f r e g r e s s i o n w e i g h t s . H o w

e v e r , t h i s s t u d y c e r t a i n l y d e m o n s t r a t e s

t h e f e a s i b i l i t y o f s t u d y i n g c o n s u m e r s w i t h t h e p u r p o s e i n m i n d o f d e v e l o p i n g s e n

s o r y p r e d i c t i o n e q u a t i o n s w h i c h a c c o u n t

f o r a h i g h p e r c e n t a g e o f t h e v a r i a b i l i t y i n p a l a t a b i l i t y .

REFERENCESBatcher, O.M., Helmtoller, K.F. and Dawson,

E.H. 1956. Development and application of m ethods for evaluating cooking and eating quality of rice. Rice J. 59(13): 4.

Batcher, O.M., Deary, P.A. and Dawson, E.H. 1957. Cooking qualtiy of 26 varieties of milled white rice. Cereal Chem. 34(7): 277.

Dixon, W.J., Ed. 1970. “ Biomedical Com puter Programs, University of California Publications in Autom atic Com putation, No. 2 ,” p. 2 3 3. U niversity of California Press, Berkeley.

Ezekial, M. 1941. “ Methods of Correlation Analysis,” p. 218. John Wiley and Son, Inc., New York.

Kaitz, E.F. 1971. Hom emakers’ preferences and buying practices for selected po tato , rice and wheat products. Marketing Research Report No. 939, U.S. Dept, of Agriculture.

Pratt, P.M. 1960. Rice: Domestic consum ption in the United States. Bureau of Business Research, University of Texas, Austin.

Simpson, J.E., Adair, C.R., Kohler, G.O., Dawson, E.H., Deobald, H.J., Kester, E.B., Hogan, J.R ., Batcher, O.M. and Halleck, J.W. 1965. Quality evaluation studies of foreign and domestic rices. Tech. Bull. 1331, Agr. Research Service, U.S. Dept, of Agriculture.

Ms received 6 /28/73; revised 8 /2 1 /7 3; accepted 8/24/73.

This research was supported by a grant from the California Rice Advisory Board.

% £ J R ESEA R C H N O T E S

G. B E ETN ER , T. TSAO, A. F R E Y and J. H A R PER Dept, o f Agricultural Engineering, Colorado State University, F o rt Collins, CO 80521

A Research NoteDEGRADATION OF THIAMINE AND RIBOFLAVIN

DURING EXTRUSION PROCESSING

INTRODUCTION

C E R E A L P R O D U C T S a r e i m p o r t a n t s o u r c e s o f t h e B v i t a m i n s : t h i a m i n e ( B j ) a n d r i b o f l a v i n ( B 2 ) ( M i t c h e l l e t a l . , 1 9 6 8 ) . T h e p r o c e s s i n g c e r e a l s r e c e i v e c a n r e d u c e t h e q u a n t i t i e s o f B v i t a m i n s p r e s e n t . T h i a m i n e i s l i s t e d a s u n s t a b l e w h e n h e a t e d , w h i l e r i b o f l a v i n i s l i s t e d a s h e a t s t a b l e b u t u n s t a b l e w h e n e x p o s e d t o l i g h t .

E x t r u s i o n c o o k i n g o f c e r e a l s i s a n i n c r e a s i n g l y i m p o r t a n t f o o d o p e r a t i o n ( H a r p e r , 1 9 7 1 ) . H i g h t e m p e r a t u r e - s h o r t t i m e ( H T S T ) p r o c e s s i n g i s c h a r a c t e r i s t i c o f e x t r u s i o n . W i t h i t , i n v e s t i g a t i o n s c a n b e m a d e t o d e t e r m i n e t h e s t a b i l i t y o f t h e B

v i t a m i n s d u r i n g p r o c e s s i n g .M i l l i n g a n d h e a t p r o c e s s i n g r e d u c e t h e

B v i t a m i n c o n t e n t o f c e r e a l m a t e r i a l s

( S c h r o e d e r , 1 9 7 1 ) , w h i l e r a d i a t i o n c a n b e

a n a c c e l e r a t i n g f a c t o r ( D i e h l , 1 9 6 9 ) . T e i x e i r a e t a l . ( 1 9 6 9 ) u s e d a f i r s t o r d e r

r a t e e q u a t i o n w i t h t e m p e r a t u r e d e p e n d e n c e t o p r e d i c t t h e s t a b i l i t y o f t h i a m i n e

d u r i n g f o o d p r o c e s s i n g . T h i s r a t e e q u a t i o n w a s c o m b i n e d w i t h t h e u n s t e a d y s t a t e h e a t c o n d u c t i o n e q u a t i o n t o s t u d y t h i a m i n e d e g r a d a t i o n i n t h e c a n n i n g p r o c

e s s .M a t h e m a t i c a l m o d e l s o f t h e e x t r u s i o n

p r o c e s s ( H a r m a n n a n d H a r p e r , 1 9 7 2 ; H a r p e r e t a l . , 1 9 7 1 ; S c h e n k e l , 1 9 6 6 ; T a d m o r

a n d K l e i n , 1 9 7 0 ) d e s c r i b e t h e e x t r u d e r i n t e r m s o f f e e d , t r a n s i t i o n a n d m e t e r i n g z o n e s . P r e s s u r e s a n d t e m p e r a t u r e s a r e

h i g h e s t i n t h e m e t e r i n g z o n e ; h e n c e , t h i s z o n e i s t h e p r o b a b l e l o c a t i o n o f t h e g r e a t e s t d e g r a d a t i o n o f t h e B v i t a m i n s . T h e e x t r u d e r c a n b e d e s c r i b e d a s a t o r t u o u s

H T S T p a t h w h e r e l o n g i t u d i n a l t r a n s p o r t o f f o o d m a t e r i a l s o c c u r s w i t h s o m e m i x i n g . I n t h e t r a n s i t i o n s e c t i o n , c o o k i n g

b e g i n s a n d c o n t i n u e s t h r o u g h t h e m e t e r i n g s e c t i o n .

EXPERIMENTALA B R A B E N D E R plasticorder with variable speed D-C drive unit and tachom eter was used to extru d e corn grits. A 3 /4-in . diam (2 0 :1 length-to-diam eter ratio) barrel which had eight 1 /3 2-in. by 1 / 8 -in. longitudinal grooves was used. The screw used for the experim en ts had a 3 :1 Hite depth ratio.

T he m oisture co n ten t o f the corn grits was raised to the desired level by m ixing with distilled w ater and the vitam in desired for fo rtification in a P atterson-K elley liquid-solids twin shell blender m odel L B -P -8 . T he grits were then refrigerated in sealed contain ers for 18 hr before extru sion . M oisture co n ten t o f the samples

% MOISTURE (wet basis)

SCREW ROTATION

( r.p.m.)

BARRELTEMPERATURE

(°F)

Fig. 1—Independent variables and experimental conditions.

Table 1 —Experimental data and results

Temp ° F

Percent water before

extrusion

Experimental data

PercentScrew B t retained rpm Y ,

Percent B2 retained

y 2

300 13 125 60.9 100.0380 13 75 47.8 125.6300 16 75 90.1 87.0380 16 125 19.0 53.6

Results

f i. f i ,(Thiamine model) (R iboflavin model)

ßo 54.4 91.5

ßi 21.0 -1 .9

ßi 0.1 -21 .3

ßs 14.5 -1 4 .8

V olum e 3 9 ( 1 9 7 4 ) - J O U R N A L O F F O O D S C I E N C E - 2 0 1


before and after extru sion were determ ined using a vacuum drying oven.

V itam in assays were made using m icrob io logical m ethod s (A ssoc. V it. C hem ., 1 9 6 6 ; D ifco , 1 9 6 6 ) . T rip licate determ inations o f vitam in co n ten t o f each sample were made in all cases exce p t one, where only one determ ination showed m icrobio logical grow th. The pooled estim ates o f variance for the B , analysis data and B 2 analysis data were used to com pare individual d eterm inations w ith the m ean o f the three determ inations o f each sample. One d eterm ination o f B j and two d eterm inations o f B 2 were elim inated since the d ifference betw een these individual values and the m eans after removal was about three tim es or greater the pooled estim ate o f variance after rem oval. T he percent vitam in retained was calculated from the m eans. Based on the pooled estim ates o f variance, B t reten tion percentages have 1 1 % standard error w ith 10% standard error for B 2 .

An em pirical m athem atical m odel was developed to describe the vitam in reten tion during the extru sion o f corn grits fortified w ith B , and B 2 at 1 00 mg per kg. This m odeled the e x truder under various operating cond itions o f screw speed, barrel tem perature and m oisture co n ten t o f the m aterial. T he m odel was developed from a h alf fraction two level factorial experim ental design (B o x and Draper, 1 9 6 9 ). Figure 1 illustrates the three independent variables and experim ental conditions.

Table 1 lists the experim en tal data and final results. The em pirical m odel fit to the data is:

y = 0o + 0 iX , +/32 x 2 + (¡3 x 3

w here:

T (° F ) - 3 4 0 X‘ 4 0

% H2 0 - 1 4 . 5

= — l ! -----screw rpm - 1 0 0

T he tabulated data show the values o f the independent variables, tem perature, percent m oisture and screw rpm , used in the design. T he corresponding values o f the two dependent variables, B 2 retained and B 2 retained , are also show n. T he estim ated values o f £ are calculated from :

£ = [ X TX ] - ' [ x Ty ]

for each independent variable and displayed in T able 1. (3, are the constan ts for the thiam ine m odel and /32 the con stan ts for the riboflavin m odel.

RESULTS & DISCUSSIONT H E A V E R A G E r e t e n t i o n w a s l o w e r f o r

t h i a m i n e ( 5 4 % ) t h a n f o r r i b o f l a v i n ( 9 2 % ) . I n c r e a s e d d e g r a d a t i o n o f t h i a m i n e f r o m

h i g h e r t e m p e r a t u r e s ( 2 1 % l o w e r f o r 4 0 ° F i n c r e a s e ) a n d f a s t e r s c r e w r p m ( 1 5 % f o r 2 5 r p m i n c r e a s e ) i s v e r y s i g n i f i c a n t . R i b o

f l a v i n s h o w s v e r y s i g n i f i c a n t i n c r e a s e d d e g r a d a t i o n f r o m m o i s t u r e ( 2 1 % l o w e r

f o r 1 . 5 % m o i s t u r e i n c r e a s e ) a n d s c r e w r p m ( 1 5 % l o w e r f o r 2 5 r p m i n c r e a s e ) . T h e e f f e c t o f m o i s t u r e o n t h i a m i n e a n d

h e a t o n r i b o f l a v i n a p p e a r n o t s i g n i f i c a n t .T h e l i t e r a t u r e r e p o r t s t h a t t h i a m i n e is

h e a t l i a b l e w h i l e r i b o f l a v i n i s n o t . T h e s e r e s u l t s c o r r o b o r a t e t h e s e r e l a t i o n s . I n c r e a s e d m e c h a n i c a l w o r k a s e v i d e n c e d b y i n c r e a s e d s c r e w r p m d e c r e a s e s v i t a m i n r e t e n t i o n o f b o t h t h i a m i n e a n d r i b o f l a v i n . I t i s u n c l e a r w h e t h e r t h i s r e s u l t s f r o m m e c h a n i c a l s h e a r o r l o c a l i z e d m e c h a n i c a l h e a t g e n e r a t i o n , a l t h o u g h h e a t s h o u l d n o t a f f e c t r i b o f l a v i n . M o r e w o r k i s n e e d e d t o e x p l a i n w h y r i b o f l a v i n i s s e n s i t i v e t o m o i s t u r e a n d t h i a m i n e s e e m s n o t t o b e .

REFERENCESAssoc. Vit. Chem. 1966. “ M ethods of Vitamin

Assay.” Association of V itam in Chemists, Inc., Inter-science Publishers, New York,N.Y.

Box, G.E.P. and Draper, N.R. 1969. “ Evolutionary Operation, a Statistical M ethod for Process Im provem ent.” John Wiley & Sons, Inc., New York, N.Y.

Diehl, J.F . 1969. Combined effects of irradiation, storage and cooking on vitamins E and B, levels of foods. Irradiation Des Aliments (SACLAY) 10: 2.

Difco. 1966. “ Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures,” 9th ed. Difco Laboratories Inc., D etroit, Mich.

Harmann, D.V. and Harper, J.M. 1972. Effect of extruder geometry on torque and flow. Presented at 1972 Summer Meeting of American Society of Agricultural Engineers, Hot Springs, Arkansas, June, 1972. Subm itted for publication in ASAE Transactions.

Harper, J.M. 1971. Research needs in extrusion cooking and forming. Presented at 1971 Winter Meeting of American Society of Agricultural Engineers, Chicago, December, 1971.

Harper, J.M., Rhodes, T.P. and Wanninger, L.A. Jr. 1971. Viscosity m odel for cooked cereal doughs. Chem. Engr. Progress Symposium Series 67(108): 40.

Mitchell, H.S., Rynberger, H.J., Anderson, L. and Dipple, M.V. 1968. “ Cooper’s Nutrition in Health and Disease,” p. I l l , J.B. Lippin- co tt, Philadelphia.

Schenkel, G. 1966. “ Plastics Extrusion Technology and T heory.” American Elsevier Publishing Com pany, New York, N.Y.

Schroeder, H.A. 1971. Losses of vitamins and trace minerals resulting from processing and preservation of foods. Am. J. Clin. Nutr. 24: 562.

Tadmor, A. and Klein, I. 1970. “ Engineering Principles of Plasticating Extrusion.” Van Nostrand Reinhold Company, New York,N.Y.

Teixeira, A.A., Dixon, J.R ., Zahradnik, J.W. and Zinsmeister, G.E. 1969. Com puter ortim ization of nutrient re tention in the therm al processing of conduction heated foods. Food Technol. 23(6): 845.

Ms received 6 /28 /73 ; revised 8 /31 /73 ; accepted9/6/73.______________________________________

The authors thank Pongsak Lim jaroenrat forassistance in extruding the samples, and MikeBoetger and Dale Felzien for assistance in thevitamin assay.

J. J. KUBALA, M. C. GACULA JR. and M. J. MORANArmour Food Research Laboratory, 801 West 22nd St., Oak Brook, IL 60521

A Research NoteDATA ANALYSIS: REGRESSION ANALYSIS WITH REPETITIONS OF THE INDEPENDENT VARIABLE

INTRODUCTIONO F T E N w e e n c o u n t e r e x p e r i m e n t a l s i t u a

t i o n s w h e r e t h e o b s e r v a t i o n s a r e p a i r e d ( X , Y ) , b u t t h e r e a r e m o r e o b s e r v a t i o n s i n o n e m e m b e r o f t h e p a i r t h a n i n t h e o t h e r . F o r e x a m p l e , i n t h e s t u d y o f a p a r e n t a n d i t s o f f - s p r i n g a d o m i n a n t t r a i t i n a p a r e n t m a y a p p e a r i n s e v e r a l o f i t s o f f s p r i n g . O r i n t h e c a s e o f s e n s o r y e v a l u a t i o n s e v e r a l o b s e r v a t i o n s a r e r e c o r d e d f o r e a c h t i m e p e r i o d . E a c h c a s e r e q u i r e s s o m e m a n i p u l a t i o n o f t h e d a t a i n o r d e r t o e q u a l i z e t h e n u m b e r o f o b s e r v a t i o n s b e t w e e n t h e v a r i

a b l e s . T h e a p p l i c a t i o n o f r e g r e s s i o n a n a l y s i s i n t h e a b o v e s i t u a t i o n s p r o v i d e s v a r i o u s w a y s i n w h i c h t o m a n i p u l a t e t h e d a t a . T h i s p a p e r i l l u s t r a t e s t h r e e m e t h o d s

o f d a t a m a n i p u l a t i o n a n d p r e s e n t s u s e f u l c r i t i q u e s o f e a c h m e t h o d .

EXPERIMENTALF O R SIM P L IC IT Y , we assumed that the relationship betw een X and Y is approxim ately linear defined by

Y j = a + bX j + e¡, i = 1 , 2 , . . . p j = 1, 2 , ... q

w here Y j is the dependent random variable, X j is the independent fixed variable and e ¡ represents an error com p on en t assumed to be independently and norm ally distributed. T he param eters a and b represent the in tercep t and slope, respectively , o f the regression line and are estim ated from the data.

In usual regression problem s p = q. However w here p A q , three m ethod s o f analysis are available to equalize the tw o variables:

(1 ) R epeatin g values o f X j for each Y ¡;(2 ) Averaging the Y ¡ ’s over an X j and re

gressing Y on X j ;(3 ) Sum m ing Y j and regressing Y ¡ on X j.

In each o f the m ethods certain statistical values play im p o rtan t roles. One value, the degrees o f freedom (D F ) available for the analysis, determ ines the accu racy o f the estim ate o f the residual. T he higher the degrees o f freedom o f the residual the m ore precise is the estim ate o f the random errors.

Tw o im p ortant statistics are the index o f determ ination (R 2 ) and the standard error o f the estim ate (S E ). T h e index shows how m uch o f the variation o f the response variable is pred icted by the predictor variable. T h e standard error o f the estim ate applies to the variation in predicting Y ¡. T he best accu racy is obtained w ith a high R 2 and a low SE .

T h e final sta tistic and perhaps the m ost im p ortan t is the co effic ie n t o f variation (C V ).

In this paper the C V is applied to the variation o f the Y ;’s w ithin each X j, and is denoted by (C V , X j) in the succeeding discussion. A high (C V , X j) indicates that the data m ight not be reprod ucib le in future experim en ts while a low (C V , X j) ind icates good rep rod ucib ility . A low (C V , XjT is the goal o f m ost researchers.

T aken altogeth er the ideal situation is a high num ber o f degrees o f freedom in the residual, a high R 2 , low S E and a low (C V , X j) . This paper exam ines the three aforem en tion ed regression m ethods in their application to off-flavor scores o f bologna. T he data are first exam ined in its original form and then are altered with certain restriction s to illustrate som e o f the points m entioned above.

RESULTS & DISCUSSIONT H E T H R E E R E G R E S S I O N m e t h o d s w e r e a p p l i e d t o t h e d a t a i n T a b l e 1 . T h e f i r s t m e t h o d , a s r e p o r t e d b y B r o w n a n d G a c u l a ( 1 9 6 4 ) , u s e s t h e i n d i v i d u a l o b s e r v a t i o n Y j , a n d r e g r e s s e s Y j t o X j p t i m e s . I n o t h e r w o r d s s i n c e p =/= q , t h e i n d e p e n d

e n t v a r i a b l e i s r e p e a t e d f o r e v e r y v a l u e o f Y j . B y t h i s m e t h o d t h e e s t i m a t e o f t h e

r e s i d u a l v a r i a n c e i s a t r u e e s t i m a t e o f t h e

v a r i a n c e o f t h e e x p e r i m e n t a l u n i t . S i n c e

t h e e x p e r i m e n t a l u n i t i s t h e i n d i v i d u a l p a n e l s c o r e , t h e r e s i d u a l v a r i a n c e e x p r e s s

e s t h e e x t e n t o f d i s a g r e e m e n t a m o n g p a n e l m e m b e r s w h i c h h o w e v e r , i s c o n f o u n d e d w i t h s a m p l e h e t e r o g e n e i t y . I f a

l a r g e d i s a g r e e m e n t e x i s t s a m o n g p a n e l

m e m b e r s , t h e R 2 o b t a i n e d i s g e n e r a l l y

l o w . M e t h o d o n e i s d e s c r i b e d b y t h e

m o d e l

Y j = a + b X j + e ,

w h e r e X j i s r e p e a t e d f o r e v e r v v a l u e o f

Y j .T h e s e c o n d m e t h o d i n v o l v e s t h e w i d e

l y u s e d r e g r e s s i o n o f t h e m e a n Y j o n t h e i n d e p e n d e n t v a r i a b l e X j . T h e m o d e l is

Y j = a + b X j + e i

w h e r e

P2Yj

a n d , p = n u m b e r o f o b s e r v a t i o n s ( p a n e l i s t s ) p e r e v a l u a t i o n . S i n c e m e t h o d 2 u s e s t h e m e a n o f t h e Y j ’s i t d o e s n o t a c c o u n t

f o r t h e v a r i a t i o n w i t h i n t h e Y j ’s a n d i s

o f t e n u s e d w h e n a h i g h v a r i a t i o n e x i s t s i n t h e d e p e n d e n t v a r i a b l e .

T h e t h i r d m e t h o d i s t h e r e g r e s s i o n o f t h e s u m o f Y j o n X j r e p r e s e n t e d b y t h e m o d e l

Y j = a + b X j + e j

w h e r e

PY j = 2 Y j

i

I t i s u s e d w h e r e t h e u s e o f s u m s a s a u n i t o f m e a s u r e m e n t a r e a p p r o p r i a t e s u c h a s t a k i n g l o t w e i g h t o f a g r o u p o f c a t t l e a s o p p o s e d t o i n d i v i d u a l w e i g h t s .

T o e v a l u a t e t h e e f f e c t s o f t h e s i z e o f t h e c o e f f i c i e n t o f v a r i a t i o n o n t h e s e e s t i m a t i o n p r o c e d u r e s , w e a l t e r e d t h e d a t a o n t h e 2 8 t h a n d 5 6 t h d a y s o f s e n s o r y

e v a l u a t i o n b y w i d e n i n g t h e r a n g e o f t h e

o b s e r v a t i o n s ( T a b l e 1 ) . A r e s t r i c t i o n w a s

i m p o s e d t h a t t h e s u m a n d t h u s t h e m e a n s o f t h e s c o r e s r e m a i n e d t h e s a m e . T h i s a l t e r a t i o n c r e a t e d a r i s e i n t h e c o e f f i c i e n t o f v a r i a t i o n f r o m 5 9 . 6 % t o 7 5 . 4 % o n t h e 5 6 t h d a y , a n i n c r e a s e o f b e t t e r t h a n 4 0 % ,

a l t h o u g h t h e a v e r a g e a n d t h e s u m f o r e a c h p e r i o d o f e v a l u a t i o n b e t w e e n t h e

t w o s e t s o f d a t a r e m a i n e d c o n s t a n t .T a b l e 2 s h o w s t h e l i n e a r r e g r e s s i o n

e q u a t i o n f o r t h e t h r e e m e t h o d s a p p l i e d t o t h e t w o s e t s o f d a t a . M e t h o d s 2 a n d 3

g i v e t h e s a m e r e s u l t i n b o t h d a t a s e t s a s e x p e c t e d . T h e r a t e o f c h a n g e i n d i c a t e d b y t h e r e g r e s s i o n c o e f f i c i e n t ( b ) i s v e r y c l o s e

f o r m e t h o d s 1 a n d 2 . T h e i n t e r c e p t ( a ) a n d t h e r e g r e s s i o n c o e f f i c i e n t f o r m e t h o d 3 w e r e l a r g e r t h a n t h o s e o f m e t h o d s 1 a n d 2 b y a f a c t o r o f p , t h e n u m b e r o f o b s e r v a t i o n s p e r e v a l u a t i o n . T h u s ,

_ m e t h o d 3 i n t e r c e p t

. _ m e t h o d 3 s l o p eP

= 0 > 2 5 1 0 2 = 0 . 0 4 2 5 1 7

w h i c h e q u a l s t h e p a r a m e t e r e s t i m a t e s o f m e t h o d s 1 a n d 2 . I t i s i n t h e u n i t u s e d f o r e x p r e s s i n g t h e r a t e o f c h a n g e t h a t d i f f e r s , a n d t h e r e f o r e s h o u l d b e p r o p e r l y i n t e r

p r e t e d .

V olum e 3 9 ( 1 9 7 4 1 - J O U R N A L O F F O O D S C I E N C E - 2 0 9


Table 1—Taste panel data on developm ent o f o f f fla vo r used toillu s tra te the concept

Age(days)

M e thod o f es tim a tion

1 2 3 1 2 3

U naltered data A lte re d data14 1 114 1 114 1 114 1 114 1 114 1 1.00 6 1 1.00 6

c o e ffic ie n t o f va ria tion % 0.0 0.028 2 228 2 1a28 1 128 1 128 3 2a28 2 1.83 11 4 a 1.83 11

c o e ffic ie n t o f va ria tion % 41.1 63 .856 2 256 3 2a56 4 3a56 1 156 2 256 5 2.83 17 7a 2.83 17

c o e ffic ie n t o f va ria tion % 59.6 75.4a Arbitrarily altered with a restriction that the sum of scores remains

the same.

Table 2—E ffe c t o f type o f expressing the data on the eq ua tion , the co rre la tio n c o e ffic ie n t and on the standard e rro r o f estim ate

M e thod o f es tim ation L inear regression equation R 2 f SE

1. Ind iv idua l

U naltered data

Y = 0 .5 000 + 0 .045252X 0.417 0.646 0.9312. Average Y = 0 .5000 + 0 .0424 49X 0.981 0.991 0 .1763. Sum Y = 3,0 + 0 .2 55102X 0.981 0.991 1.069

1. Ind iv idua lA lte red data

Y = 0 .5000 + 0 .0 4 2 5 1 7X 0.249 0.499 1.3662. Average Y = 0 .5000 + 0 .0 424 49X 0.981 0.991 0 .1 763. Sum Y = 3 .0 + 0 .255102X 0.981 0.991 1.069

AGE

Fig. 1—Regression o f o ff - f la v o r score on age by m ethods 1 (o l and 2 ('"-■)■ N um bers on the c irc le p lo t are num bers o f observations a t th a t p o in t.

M e t h o d 1 r e s u l t s in a l o w e r R 2 a n d a h ig h e r S E t h a n m e t h o d s 2 a n d 3 . T h is r e s u l t is d u e t o t h e h ig h c o e f f i c i e n t o f v a r i a t i o n w i t h in t h e in d iv id u a l age g r o u p s . S i n c e t h e a l t e r i n g o f t h e d a t a o n l y a f f e c t ed m e t h o d 1 , a c l o s e r i n s p e c t i o n o f t h e r e s u l t s is n e c e s s a r y , b u e t o t h e a l te r in g , t h e c o r r e l a t i o n c o e f f i c i e r r d r o p p e d f r o m 0 . 6 4 6 f o r t h e u n a l t e r e d d a ta t o 0 . 4 9 9 f o r t h e a l t e r e d d a ta ( T a b l e 2 ) . S i n c e b o t h o f t h e s e v a lu e s a re s i g n i f i c a n t , a l t h o u g h at d i f f e r e n t lev e ls , t h e d r o p is n o t t o o i m p o r t a n t . H o w e v e r , t h e R 2 v a lu e d r o p s f r o m 0 . 4 1 7 t o 0 . 2 4 9 . T h i s d r o p m e a n s t h a t t h e r e g r e s s io n e q u a t i o n f o r t h e alt e r e d d a ta e x p l a i n s 1 7 % le ss o f t h e v a r ia b i l i t y in t h e i n d e p e n d e n t v a r ia b le . F u r t h e r m o r e t h e S E in c r e a s e s f r o m 0 . 9 3 1 t o 1 . 3 6 6 w h i c h i n d ic a t e s a le ss a c c u r a t e e s t i m a t e o f t h e d e p e n d e n t v a r ia b le . T h e c o m b i n a t i o n o f t h e d r o p in R 2 a n d t h e r ise in S E m e a n s t h a t t h e r e g r e s s io n e q u a t i o n f o r t h e a l t e r e d d a ta is n o t as g o o d a p r e d i c t o r as t h a t f o r t h e u n a l t e r e d .

T h e sa l i e n t p o i n t in t h i s i l l u s t r a t io n is t h a t t h e w id e ly u s e d m e t h o d 2 w h i c h p r o d u c e s t h e s a m e a n s w e r s f o r b o t h se ts o f d a t a , d o e s n o t a c c o u n t f o r t h e v a r ia b i l i t y o f t h e in d iv id u a l o b s e r v a t i o n s as t h e d a t a u s e d in t h e r e g r e s s io n a r e b a s e d o n a v e ra g e s . I f t h e s e a v e ra g e s c o m e f r o m o b s e r v a t i o n s o f h ig h v a r i a b i l i t y , as in o u r a l t e r e d d a t a , t h e r e g r e s s io n m o d e l , a lt h o u g h f o u n d t o b e a d e q u a t e b y t h e size

o f t h e R 2 , w ill n o t b e as r e p r o d u c i b l e as t h a t f r o m d a ta o f l o w e r v a r i a t i o n . I n t h e c o m p a r i s o n o f r e s u l t s o f in d e p e n d e n t e x p e r i m e n t s b a s e d o n r e g r e s s io n l in e s , o n e c a n b e d e c e iv e d u n le s s t h e c o e f f i c i e n t o f v a r i a t i o n is p r o v id e d . O n e m a y o b t a i n i d e n t i c a l l in e s b e t w e e n e x p e r i m e n t s b e c a u s e o f t h e a v e ra g in g p r o c e s s o r b y t h e s u m m i n g p r o c e s s b u t o b v i o u s l y w i l l fa v o r t h o s e e x p e r i m e n t s w i t h a lo w e r c o e f f i c i e n t o f v a r i a t i o n . T h e r e f o r e , i t is s u g g e s t ed t h a t w h e n u s in g m e t h o d 2 , t h e r e s e a r c h e r s h o u ld p r o v id e t h e c o e f f i c i e n t o f v a r i a t i o n f o r e a c h p e r io d .

T h e u s e o f m e t h o d 1 g ives t h e t r u e v a r i a b i l i t y o f t h e d a ta . T h i s v a r ia b i l i t y is a t t r i b u t e d t o s a m p le h e t e r o g e n e i t y and p a n e l d i s c r e p a n c y . M e t h o d 1 g ives m o r e w e ig h t t o in d iv id u a l j u d g e m e n t t h a n m e t h o d 2 . O n t h e c o n t r a r y , m e t h o d 2 g ives m o r e w e ig h t t o t h e c o n s e n s u s o f t h e p a n e l o f j u d g e s as t h e a n a ly s i s is b a s e d o n a v erag es .

F i g u r e 1 s h o w s t h e r e g r e s s io n l in e a n d t h e d a ta p o i n t s f o r m e t h o d s 1 a n d 2 . M e t h o d 3 w ill n o t b e d is c u s s e d as i t is r a r e ly u s e d in s e n s o r y d a ta a n a ly s is . T h e l in e s a re d r a w n as o n e b e c a u s e o f t h e i r c l o s e n e s s . T h e s p r e a d o f t h e in d iv id u a l s c o r e s w i t h t i m e r e s u l t s in l o w R 2 f o r m e t h o d 1. T h e e s t i m a t e o f t h e r e g r e s s io n p a r a m e t e r s a r e p r a c t i c a l l y i d e n t i c a l b u t d i f f e r c o n s i d e r a b l y in t h e e s t i m a t e o f R 2 .

T h e g u id e l in e s f o r t h e u s e o f m e t h o d s

1 a n d 2 in s e n s o r y d a ta a n a ly s i s c a n b e g e n e r a l iz e d as f o l l o w s :

( 1 ) M e t h o d 1 is u s e d i f t h e n u m b e r o f e v a l u a t i o n p e r io d s ( N ) is s m a l l , t h e c o e f f i c i e n t o f v a r i a t i o n in e a c h p e r io d ( C V , X j ) is r e a s o n a b l y s m a l l , a n d t h e s a m p l e s a re r e la t iv e ly h o m o g e n e o u s . A l o w c o e f f i c i e n t o f v a r i a t i o n i m p l i e s g o o d a g r e e m e n t a m o n g p a n e l m e m b e r s a n d g o o d h o m o g e n e o u s s a m p le s .

( 2 ) M e t h o d 2 is r e c o m m e n d e d i f N is la rg e a n d a h ig h ( C V , X j ) is p r e s e n t in t h e d a ta as a r e s u l t o f s i g n i f i c a n t v a r i a t i o n d u e t o p a n e l i s t s , s a m p l e s , a n d i n t e r a c t i o n s b e t w e e n p a n e l i s t s , s a m p l e s a n d t i m e p e r io d s . T h e c o e f f i c i e n t o f v a r i a t i o n ( C V , X j ) o f t h e d a ta ( Y ) s h o u ld b e p r o v id e d w h e n u s in g t h i s m e t h o d t o g iv e t h e r e a d e r s i n f o r m a t i o n a b o u t t h e t r u e v a r i a b i l i t y o f t h e d a t a n o t e n t e r e d in t h e r e g r e s s i o n a n a ly s is .

( 3 ) I n o t h e r c a s e s , i f ( C V , X j ) is s m a l l a n d N is la rg e , m e t h o d 1 o r 2 m a y b e a p p r o p r i a t e . I f ( C V , X j ) is la rg e a n d N is s m a l l , i n c r e a s e N a n d u s e m e t h o d 2 .

REFERENCESB ro w n , C .J . a n d G a c u la , M .C . J r . 1 9 6 4 . E s t i

m a te s o f h e r i t a b i l i t y o f b e e f c a t t l e p e r f o r m a n c e t r a i t s b y r e g r e s s io n o f o f f s p r i n g o n s ir e . J . A n im a l S c i. 2 3 : 3 2 1 .

M s re c e iv e d 6 / 2 8 / 7 3 ; r e v is e d 8 / 2 7 / 7 3 ; a c c e p te d 8 / 2 8 / 7 3 .

STEPHEN E. CRAVEN and H. S. L IL LA R DUSDA Richard B. Russell Agricu ltura l Research Center, ARS, P.O. Box 5677, Athens, GA 30604.

A Research NoteEFFECT OF MICROWAVE HEATING OF PRECOOKED CHICKEN

ON Clostridium perfringens

INTRODUCTION

A N A N N U A L R E P O R T f r o m t h e C e n t e r f o r D ise a s e C o n t r o l ( C D C , 1 9 7 0 ) im p l i c a t e d Clostridium perfringens in 2 9 . 7 % o f f o o d p o is o n in g c a s e s c o m p a r e d w i t h 2 0 . 4 % f o r Salmonella a n d 1 9 . 8 % f o r Staphylococcus. R e d m e a t s a n d p o u l t r y and t h e i r c o m b i n a t i o n s w i t h o t h e r f o o d p r o d u c t s a re c o n s i d e r e d t h e m o s t l ik e ly v e h ic le s o f C. perfringens f o o d p o is o n in g . D u n c a n ( 1 9 7 0 ) r e p o r t e d t h a t p o u l t r y a n d p o u l t r y p r o d u c t s w e r e i n v o lv e d in 3 2 % o f C. perfringens f o o d p o is o n in g o u t b r e a k s in t h e 1 0 - y r p e r io d c o v e r e d b y h is s t u d y .

M e a d a n d I m p e y ( 1 9 7 0 ) , G i b b s ( 1 9 7 1 ) an d L il la rd ( 1 9 7 1 ) i s o la t e d C. perfringens f r o m v a r io u s s o u r c e s in p o u l t r y p r o c e s s in g p la n ts . U sin g e n r i c h m e n t t e c h n i q u e s , L i l la rd ( 1 9 7 1 ) i s o la t e d t h e o r g a n is m in f u r t h e r - p r o c e s s e d p o u l t r y p r o d u c t s an d i n g r e d ie n t s . T h e p r e s e n c e o f C. perfringens in p r e c o o k e d c h i c k e n , t h o u g h n o t f r e q u e n t , r e p r e s e n t s a p o t e n t i a l s o u r c e o f f o o d p o is o n in g . M ic r o w a v e o v e n s a n d p r e c o o k e d c h i c k e n are u s e d i n c re a s in g ly in c h a in f o o d - s e r v ic e e s t a b l i s h

m e n t s a n d p u b l i c i n s t i t u t io n s . B e c a u s e r e h e a t in g c h i c k e n in m ic r o w a v e o v e n s i n v o lv e s o n l y a f r a c t i o n o f t h e t i m e r e q u ire d in r e h e a t in g b y o t h e r m e t h o d s , th is s t u d y w as u n d e r t a k e n t o d e t e r m i n e t h e e f f e c t o f m ic r o w a v e r e h e a t in g o n C. perfringens s p o r e s a n d v e g e t a t iv e ce l ls .

MATERIALS & METHODS

V E G E T A T I V E C E L L S o f C. perfringens C D C s t r a in 7 9 4 7 w e re p r e p a r e d b y th e t r a n s f e r o f a 2 4 -h r c u l tu r e i n t o t u b e s c o n ta in in g 1 0 m l o f th io g ly c o l l a t e b r o t h w i th d e x t r o s e (T B D , D if c o ) a n d in c u b a t in g a n a e r o b i c a l ly in a n a t m o s p h e r e c o n ta in in g 8 0 % n i t r o g e n , 1 0 % h y d r o g e n a n d 1 0 % c a r b o n d io x id e , v /v ( L i l la rd , 1 9 7 1 ) a t 3 5 ° C f o r 16 h r . T h e 1 6 -h r c u l tu r e w a s w a s h e d th r e e t im e s w i th a n a q u e o u s s o lu t io n c o n ta in in g 0 .1 % p e p t o n e a n d 0 .1 % s o d iu m t h i o g ly c o l la te p r io r t o i n o c u la t i o n . S p o r e s w e re p r e p a r e d b y tw o c o n s e c u t iv e t r a n s f e r s o f a1 6 -h r T B D c u l t u r e o f v e g e t a t iv e c e l ls i n t o 10 m l o f T B D u s in g a 1% in o c u lu m a n d 4 h r i n c u b a t i o n p e r io d s a t 3 5 ° C . F r o m th e l a s t 4 - h r c u l t u r e , 0 .1 m l p o r t i o n s w e re i n o c u l a t e d in to t u b e s c o n ta in in g 1 0 m l o f t h e s p o r u l a t i o n m e d iu m o f K im e t a l. ( 1 9 6 7 ) a n d i n c u b a te d f o r 16 h r a t 3 5 ° C . T h e c u l tu r e w a s w a s h e d th r e e t im e s w i th

d e io n iz e d w a te r . 2 0 -m l p o r t i o n s w e re t r e a t e d w i th a s o n i f i e r ( B r a n s o n W 1 8 5 ) a t 5 0 w f o r 2 0 m in to d i s r u p t v e g e t a t iv e c e l ls . T h e s p o r e s w e re w a s h e d th r e e t im e s , s u s p e n d e d in d e io n iz e d w a te r a n d s to r e d a t 4 ° C .

E i t h e r v e g e t a t iv e c e l ls o r s p o re s w e re i n o c u l a t e d i n t o c o m m e r c ia l ly p r e p a r e d , b r e a d e d a n d b a t t e r e d , p r e c o o k e d , c h ic k e n th ig h s (h e ld a t 6 ° C ) . E a c h th ig h w a s in o c u la t e d w i th a s y r in g e in n u m e r o u s p la c e s im m e d ia t e ly b e lo w th e b r e a d in g w i th 0 .8 m l o f i n o c u lu m ( 0 .4 m l p e r s id e ) . F o r e a c h t r e a t m e n t , s ix i n o c u l a t e d th ig h s ( m e a n t o t a l w t 4 3 3 . 2 g ) w e re p la c e d in a s te r i le p y r e x d is h a n d h e a t e d in a 2 4 5 0 M H z V a r ia n m ic ro w a v e o v e n (M o d e l # T C S - 2 .5 ) w i th a r o t a r y t u r n t a b l e . E x p o s u r e t im e s o f 4 5 - 9 0 sec a t a p o w e r s e t t i n g o f 2 k w w e re u s e d t o a t t a in m a x im u m i n t e r n a l t e m p e r a tu r e s r a n g in g f r o m 4 9 - 8 4 ° C . M a x im u m t e m p e r a tu r e s w e re m e a s u r e d w i th m e r c u r y th e r m o m e te r s a f t e r t r e a t m e n t . U n h e a te d i n o c u la t e d c o n t r o l s w e re p r e p a r e d f o r e a c h m ic ro w a v e t r e a t m e n t . A f te r c o o l in g to r o o m t e m p e r a t u r e , e a c h s a m p le w a s d e b o n e d a s e p t i c a l l y a n d p la c e d in a s e p a r a te s te r i l e b l e n d o r j a r . D i lu t io n s w e r e m a d e u s in g th e p e p to n e - s o d i u m t h io g ly c o l l a t e s o lu t io n a n d b le n d e d f o r 1 m in a t h ig h s p e e d . F o r s p o re c o u n t s a 4 m l p o r t i o n o f a 1 /5 d i l u t i o n o f e a c h b l e n d e d s a m p le w a s h e a t s h o c k e d in a w a te r b a th a t 7 5 ° C f o r 2 0 m in .

Fig. 1—Percent surviva l o f C. perfringens 7947 vegetative cells fro m microwave-heated inocu la ted chicken. Ino cu la : 1.5—3 .0 X 10s cells/g ( • • ) . No detectable survivors f *■). Inocu la : 3 .0 —6 .6 X 703 cells/g( o --------o j. N o detectable survivors I '■).

Fig. 2 -P e rc e n t surviva l o f C. perfringens 7947 spores in m ic ro wave-treated chicken heated to various in te rn a l temperatures. (A ) P rio r to heat shock: inocu la 1.2—1.4 X 10“ spores/g; (B) A fte r heat shock ¡75° C fo r 2 0 m in ) : inocu la 6 .4 X 10" — 1.0 X 10s spores/g.



S u l f i t e - p o ly m y x in - s u l f a d ia z in e (S P S ) a g a r , ( A n g e lo t t i e t a l . , 1 9 6 2 ) w a s u s e d f o r p o u r p la te s f r o m s e r ia l d i lu t i o n s . P la te s w e re i n c u b a te d a n a e r o b i c a l ly a t 3 5 ° C f o r 4 3 h r . C o u n t s w e re o b t a i n e d b y d i r e c t p la t in g o n S P S a g a r . P e r c e n ta g e s o f v ia b le c e l ls a n d s p o re s s u rv iv in g t r e a t m e n t w e re b a s e d o n c o m p a r i s o n s t o c o u n t s o f u n t r e a t e d c o n t r o l s . P e r c e n ta g e s w e re p l o t t e d o n a l o g a r i t h m i c s c a le a s a f u n c t io n o f i n t e r n a l t e m p e r a tu r e . C o o k e d m e a t m e d iu m (D i f c o ) w a s u s e d f o r e n r i c h m e n t (D o w e ll a n d H a w k in s , 1 9 6 8 ) t o d e t e c t lo w le v e ls o f c e lls a n d c e l ls t h a t m a y h a v e b e e n d a m a g e d b y h e a t t r e a t m e n t .


M I C R O W A V E H E A T I N G o f p r e c o o k e d c h ic k e n r e s u l t e d in c o n s i d e r a b l e v a r ia t io n s in i n t e r n a l t e m p e r a t u r e s o f p ie c e s w i th in a g iven t r e a t m e n t , as w e l l as b e t w e e n r e p l i c a t i o n s o f t r e a t m e n t s , e v e n t h o u g h p ie c e s w e r e o f s im ila r w e ig h t . D i f f e r e n c e s in t h e c o n f o r m a t i o n a n d c o m p o s i t i o n o f p ie c e s a n d t h e p r e s e n c e o f “ c o ld s p o t s ” in t h e o v e n w e r e p r o b a b l y r e s p o n s i b l e f o r t h e s e v a r ia t io n s , ev e n t h o u g h a r o t a r y t u r n t a b l e in t h e o v e n s h o u ld h a v e m in im iz e d t h e p r o b l e m o f u n e v e n e x p o s u r e t o m ic r o w a v e s . L a c k o f u n i f o r m d is t r ib u t i o n w i th in m ic r o w a v e ov e n s h a s b e e n r e p o r t e d b y o t h e r in v e s t i g a t o r s ( B a l d w i n e t a l . , 1 9 7 1 ; V a n Z a n t e , 1 9 6 6 ; L a c e y a n d W in n e r , 1 9 6 5 ) .

M ic r o w a v e h e a t i n g o f i n o c u l a t e d , c o m m e r c i a l ly p r e c o o k e d c h i c k e n a d v e rse ly a f f e c t e d v e g e t a t iv e ce l ls o f C. perfringens 7 9 4 7 w h e n t h e m a x i m u m i n t e r n a l t e m p e r a t u r e ra n g e d f r o m 4 9 - 8 4 ° C ( F i g . 1). In s a m p le s i n o c u l a t e d w i th 1 0 5 c e l l s / g o r 103 c e l l s /g , su rv iv a l e x c e e d e d 1 0 % w h e n in t e r n a l t e m p e r a t u r e s r e m a i n e d b e lo w 5 5 ° C . W h e n in t e r n a l t e m p e r a t u r e s e x c e e d e d 5 5 ° C , su rv ival d e c r e a s e d r a p id ly at b o t h levels o f i n o c u l a t i o n . S t a t i s t i c a l a n a ly s is o f t h e d a ta s h o w e d n o s ig n i f i c a n t d i f f e r e n c e b e t w e e n t h e s lo p e s o f t h e t w o

l in e s . V i a b l e ce l l s w e r e n o t d e t e c t e d b y d ir e c t p la t in g o r b y e n r i c h m e n t w h e n i n t e r n a l t e m p e r a t u r e s e x c e e d e d 7 0 ° C in c h ic k e n i n o c u l a t e d w i t h 1 0 s c e l l s / g o r w h e n i n t e r n a l t e m p e r a t u r e s e x c e e d e d 6 4 ° C in c h i c k e n i n o c u l a t e d w i th 1 0 3 ce l l s /g . B r y a n ( 1 9 6 9 ) r e p o r t e d t h a t c o o k e d m e a t s h o u ld n o t b e w a r m e d u p , b u t s h o u ld b e h e a t e d t o an i n t e r n a l t e m p e r a t u r e o f a t l e a s t 7 4 ° C t o d e s t r o y C. perfringens v e g e t a t iv e ce l ls . O u r d a ta i n d i c a t e t h a t m ic r o w a v e h e a t i n g o f p r e c o o k e d c h i c k e n t o 7 0 ° C s h o u ld be s u f f i c i e n t t o k i l l o r r e d u c e t o u n d e t e c t a b l e lev e ls v e g e ta t iv e ce l ls o f s t r a in 7 9 4 7 .

M i c r o w a v e h e a t i n g o f i n o c u l a t e d c h ic k e n s t i m u l a t e d g e r m i n a t io n a n d o u t g r o w t h o f s p o r e s w h e n t h e m a x i m u m i n t e r n a l t e m p e r a t u r e w as 4 9 —8 4 ° C ( F ig . 2 A ) . W h e n i n o c u l a t e d s a m p le s w e re m ic r o w a v e h e a t e d , b l e n d e d a n d h e a t s h o c k e d a t 7 5 ° C f o r 2 0 m in ( F i g . 2 B ) , survival w as lo w e r t h a n f r o m b l e n d e d p o r t io n s o f i n o c u l a t e d c o n t r o l s t h a t w ere o n ly h e a t s h o c k e d . M ic r o w a v e h e a t i n g a p p a r e n t l y i n i t i a t e d g e r m i n a t io n w i th s u b s e q u e n t k i l l in g o f t h e g e r m in a t in g sp o r e s u p o n e x p o s u r e t o h e a t - s h o c k t e m p e r a tu r e s ( 7 5 ° C / 2 0 m in ) . T h e h ig h e r t h e t e m p e r a t u r e a t t a i n e d d u r in g m ic r o w a v e e x p o s u r e t h e g r e a t e r w as t h e n u m b e r o f s p o r e s w h ic h g e r m i n a t e d a n d , t h u s , t h e g r e a t e r w as t h e n u m b e r k i l le d o n h e a t s h o c k in g .

G o l d b l i t h a n d W a n g ( 1 9 6 7 ) an d L e c h o w i c h e t a l . ( 1 9 6 9 ) s t a t e d t h a t th e d e s t r u c t iv e e f f e c t o f m ic r o w a v e s o n b a c te r ia l ce l ls a n d s p o r e s w as d u e t o t h e r m a l in f l u e n c e s . B y c o m p a r i n g b le n d e d s p o r e - i n o c u l a t e d s a m p le s h e a t e d b y m ic r o w a v e s w i t h s u c h s a m p l e s h e a t e d in an o i l b a t h , w e s h o w e d t h a t g e r m i n a t io n o f C. perfringens s p o r e s u p o n m ic r o w a v e h e a t i n g is d u e t o a t h e r m a l e f f e c t .

W e c o n c l u d e f r o m th is s t u d y t h a t h e a t in g p r e c o o k e d c h ic k e n b y m ic r o w a v e s t o in t e r n a l t e m p e r a t u r e s u p t o 8 4 ° C is n o t

a d e q u a t e t o e l i m i n a t e t h e p o s s i b i l i t y o f C. perfringens f o o d p o is o n in g .

REFERENCES

A n g e lo t t i , R . , H a ll , H .E ., P o t e r , M .J . a n d L e w is ,K .H . 1 9 6 2 . Q u a n t i t a t i o n o f C lo s t r i d iu m p e r f r in g e n s i n f o o d s . A p p l . M ic r o b io l . 1 0 : 1 9 3 .

B a ld w in , R .E . , F ie ld s , M .L ., P o o n , W .C . a n d K o r s c h g e n , B . 1 9 7 1 . D e s t r u c t i o n o f S a l- m o n e l la e b y m ic ro w a v e h e a t in g o f f i s h w i t h i m p l i c a t i o n s f o r f i s h p r o d u c t s . J . M ilk F o o d T e c h n o l . 3 4 : 4 6 7 .

B ry a n , F .L . 1 9 6 9 . W h a t t h e s a n i t a r i a n s h o u ld k n o w a b o u t C lo s t r id iu m p e r f r i n g e n s f o o d - b o r n e i l ln e s s . J . M ilk F o o d T e c h n o l . 3 2 : 3 8 1 .

C D C . 1 9 7 0 . F o o d b o r n e o u t b r e a k s . A n n u a l S u m m a r y 1 9 7 0 . U .S . D e p t , o f H e a l t h , E d u c a t io n a n d W e lfa re , C e n t e r f o r D is e a s e C o n t r o l , A t l a n t a , G A 3 0 3 3 3 .

D o w e l l , V .R . a n d H a w k in s , T .H . 1 9 6 8 . “ L a b o r a t o r y M e th o d s in A n a e r o b ic B a c t e r io lo g y .” P u b l ic H e a l t h S e rv ic e P u b l i c a t i o n N o . 1 8 0 3 ,U .S . G o v e r n m e n t P r in t in g O f f ic e , W a s h in g t o n , D .C .

D u n c a n , C .L . 1 9 7 0 . C lo s t r i d iu m p e r f r i n g e n s f o o d p o is o n in g . J . M ilk F o o d T e c h n o l . 3 3 : 3 5 .

G ib b s , P .A . 1 9 7 1 . T h e in c id e n c e o f C lo s t r id ia in p o u l t r y c a r c a s s e s a n d p o u l t r y p ro c e s s in g p la n t s . B r. P o u l t . S c i. 1 2 : 1 0 1 .

G o ld b l i th , S .A . a n d W a n g , D .I .C . 1 9 6 7 . E f f e c t o f m ic ro w a v e s o n E s c h e r ic h ia c o l i a n d B a c illu s s u b t i l i s . A p p l . M ic r o b io l . 1 5 : 1 3 7 1 .

K im , C .H ., C h e n e y , R . a n d W o o d b u r n , M . 1 9 6 7 . S p o r u l a t i o n o f C lo s t r i d iu m p e r f r in g e n s in a m o d i f i e d m e d iu m a n d s e le c te d f o o d s . A p p l . M ic r o b io l . 1 5 : 8 7 1 .

L a c e y , B .A . a n d W in n e r , H .I . 1 9 6 5 . E f f e c t s o f m ic ro w a v e c o o k e r y o n t h e b a c t e r i a l c o u n t s o f f o o d . J . A p p l . B a c te r io l . 2 8 : 3 3 1 .

L e c h o w ic h , R .V . , B e a u c h a t , L .R . , F o x , K .I . a n d W e b s te r , F .H . 1 9 6 9 . P r o c e d u r e f o r e v a l u a t in g t h e e f f e c t s o f 2 4 5 0 - m e g a h e r t z m ic ro - w a v e s u p o n S t r e p t o c o c c u s f a e c a l is a n d S a c c h a r o m y c e s c e r e v i s ia e . A p p l . M ic ro b io l . 1 7 : 1 0 6 .

L i l la r d , H .S . 1 9 7 1 . O c c u r r e n c e o f C lo s t r id iu m p e r f r in g e n s in b r o i l e r p r o c e s s in g a n d f u r t h e r p ro c e s s in g o p e r a t i o n s . J . F o o d S c i. 3 6 : 1 0 0 8 .

M e a d , G .C . a n d I m p e y , C .S . 1 9 7 0 . T h e d i s t r i b u t i o n o f C lo s t r id ia i n p o u l t r y p ro c e s s in g p la n t s . B r. P o u l t . S c i. 1 1 : 4 0 7 .

V a n Z a n te , H .J . 1 9 6 6 . D e t e r m i n a t i o n o f c o o k in g p o w e r d i s t r i b u t i o n in e l e c t r o n i c r a n g e s . J . H o m e E c o n . 5 8 : 7 9 2 .

M s r e c e iv e d 6 / 1 9 / 7 3 ; r e v is e d 9 / 1 4 / 7 3 ; a c c e p te d9 / 1 9 / 7 3 . _____________________________________

R e f e r e n c e t o a c o m p a n y o r t r a d e n a m e d o e s n o t im p ly e n d o r s e m e n t b y t h e U S D A .

T h e a u t h o r s t h a n k R .I . H e g g e f o r h is c a p a b le t e c h n ic a l a s s is ta n c e a n d R .T . T o le d o a n d V ic to r C h e w f o r t h e i r h e l p f u l s u g g e s t io n s .

A . F. S C H IN D L E R , A . N. A B A D IE , J. S. G EC AN , P. B. M IS L IV E C and P. M. B R IC K E Y

Div. o f M ic ro b io log y , F o o d and D rug A d m in is tra tio n , W ashington, D C 20204

A Research NoteMYCOTOXINS PRODUCED BY FUNGI ISOLATED FROM INSHELL PECANS

INTRODUCTION

D U E T O T H E V A R I E T Y o f h a r v e s t in g m e t h o d s a n d p o s t h a r v e s t h a n d l i n g t e c h n iq u e s , p e c a n s m a y b e s u b j e c t e d t o m a n y e n v i r o n m e n t a l c o n d i t i o n s c o n d u c i v e t o fu n g a l i n f e c t i o n a n d g r o w t h . P e c a n s g a t h e red as t h e y fa l l f r o m t h e t r e e s a re p r a c t i c a l l y m o l d - f r e e ; h o w e v e r , i f t h e y a re le f t o n d a m p g r o u n d f o r a w e e k o r m o r e a h ig h p e r c e n t a g e o f m o l d y k e r n e l s m a y d e v e lo p . I t h a s a ls o b e e n d e m o n s t r a t e d t h a t t h e r e is a d i r e c t r e l a t i o n s h i p b e t w e e n t h e t i m e o f e x p o s u r e o f p e c a n s t o w e t so i l a n d k e r n e l m o l d i n g , a n d t h a t n u t s fa l l in g t o t h e g r o u n d in p a s t u r e s a re m o r e l ik e ly t o m o l d t h a n t h o s e f a l l in g in n o n p a s t u r e o r c h a r d s ( W o o d r o f f , 1 9 6 7 ) .

T h e m o i s t u r e c o n t e n t o f p e c a n s d r o p s f r o m 3 0 % t o 8 % as t h e n u t s m a t u r e o n t h e t r e e s a n d n o r m a l l y d e h is c e . A k e r n e l m o i s t u r e lev e l o f 4 . 5 % o r less is n e c e s s a r y t o p r e v e n t m o l d i n g d u r in g s t o r a g e ( W o o d r o f f , 1 9 6 7 ) . In c o m m e r c i a l p r a c t i c e t h e d e c r e a s e in m o i s t u r e is o b t a i n e d b y a r t i f i c ia l d ry in g . M o i s t u r e c o n t r o l is p r o b a b ly t h e m o s t i m p o r t a n t f a c t o r in p r o p e r h a r v e s t in g , s t o r in g o r p r o c e s s in g o f p e c a n s .

L i l la rd e t a l . ( 1 9 7 0 ) r e p o r t e d t h a t 8 5 o f 1 2 0 A s p e r g i l l u s f l a v u s g r o u p i s o la t e s c u l t u r e d f r o m s u r f a c e s t e r i l i z e d p e c a n m e a t p ie c e s u s e d in c o m m e r c i a l b a k e r y p r o d u c t s a n d l o c a l s u p e r m a r k e t s p r o d u c e d a f l a t o x i n . D o u p n i k a n d B e l l ( 1 9 7 1 ) i s o la t e d a v a r i e t y o f m o l d s in c l u d i n g A . f l a v u s a n d A . o c h r a c e u s f r o m s u r f a c e s t e r i l ized m e a t s o f s t o r e d in s h e l l p e c a n s . C h ip le y a n d H e a t o n ( 1 9 7 1 ) , b y c o n t r a s t , f o u n d o n l y t w o fu n g i , A . c l a v a t u s a n d T r i c o t h e c i u m sp . , in a s e p t i c a l l y sh e l le d p e c a n s , a l t h o u g h c o m m e r c i a l l y sh e l le d p e c a n m e a t s c o n t a i n e d n u m e r o u s a n d varied m ic r o o r g a n i s m s .

F u n g a l i n f e c t i o n a n d d e t e r i o r a t i o n o f p e c a n s r e s u l t in e c o n o m i c lo s s t o t h e g r o w e r , s h e l le r o r c o n s u m e r . T h e i r t o x i c m e t a b o l i t e s a lso p r e s e n t a h a z a r d t o h u m a n s . S o m e s p e c ie s o f A s p e r g i l l u s and P e n i c i l l i u m , c o m m o n so il f u n g i , p r o d u c e t o x i n s s u c h as o c h r a t o x i n , a f l a t o x i n a n d s t e r i g m a t o c y s t in , as w e l l as p e n ic i l l i c a c id , p a t u l in a n d i s l a n d i t o x i n s (C ie g le r e t al .,1 9 7 1 ) . T h e r e f o r e , w h e n p e c a n s w e r e c o l l e c t e d d u r in g a 1 9 6 7 - 6 8 s u r v e y , t h e y w e r e e x a m i n e d t o d e t e r m i n e t h e p r e s e n c e a n d t y p e s o f m o ld s .

Table 1 —Fungi isolated fro m m o ld y inshell pecans (Carya illoensis)

OrganismIMo. o f isolates

A lte rn a ria spp. 19A sperg illus spp. 42

A . aw am ori 1A. cheval ie r i 1A . niger 6A. ochraceus 2A. parasiticus 1A . repens 21A . versicolor 10

Cladosporium spp. 4Fusarium spp. 10M e/anconium sp. 1M ucor sp . 1P en ic illium spp. 78

P. aurantio -v irens 1P. b rev i-com pactum 7P. c itr in u m 6P. cyaneo-fu lvum 1P. c y d o p iu m 9P. cyc lo p iu m var. ech inu la tum 2P. expansum 16P. frequentans 26P. is land icum 1P. m u lt ic o lo r 3P. oxa licum 1P. pa litans 2P. s teck ii 1P. tardum 1P. w o rtm a n n i 1

Pestalotla sp. 1R hizopus s to lo n ife r 4Trichoderm a v iride 3

Tota l 163

EXPERIMENTAL

E A C H o f 1 0 ,8 0 0 in s h e l l p e c a n s , f r o m s e v e n d i f f e r e n t m a n u f a c tu r e r s , w a s s e p a r a te ly c r a c k e d o p e n a n d th e p e c a n m e a t s w e re e x a m in e d m a c r o s c o p ic a l ly .

T h e p r e s e n c e o f m o ld in in s h e l l p e c a n s w a s d e t e r m in e d b y th e f o l lo w in g m a c r o s c o p ic m e t h o d o f t h e U .S . F o o d a n d D ru g A d m in i s t r a t i o n : “ A n u t k e r n e l is c la s s if ie d a s m o ld y i f i t c o n ta in s a n y c o n s p ic u o u s f r u i t i n g m o ld o r i f i t c o n ta in s a n y m o ld a f f e c t in g m o r e t h a n o n e - f o u r th o f i t s s u r f a c e o r a n a g g re g a te a r e a g r e a te r t h a n

o n e s q u a r e c e n t im e t e r . T h e p r e s e n c e o f m o ld m a y b e v e r i f i e d b y m a g n i f i c a t i o n b u t t h e a re a a f f e c t e d m u s t b e d e t e r m in e d w i t h o u t m a g n i f i c a t i o n ” ( F o o d & D ru g A d m in i s t r a t i o n , 1 9 6 8 ) . S e le c te d p e c a n s f r o m s a m p le s o f m o ld y n u t s w e re e x a m in e d u n d e r a d is s e c t in g m ic r o s c o p e a n d is o la te s o f t h e m o ld w e re t r a n s f e r r e d to n u t r i e n t a g a r t e s t t u b e s la n t s f o r s to r a g e a n d e v e n tu a l i d e n t i f i c a t i o n ( T a b le 1). I s o la te s w e re i d e n t i f i e d a c c o r d in g to G i lm a n ( 1 9 5 7 ) .

A f t e r i d e n t i f i c a t i o n m o s t o f th o s e i s o la te s w h ic h w e re p o t e n t i a l p r o d u c e r s o f s t e r ig m a to c y s t i n , a f l a t o x i n , o c h r a t o x i n o r p a tu l in w e re s tu d i e d f o r t h e i r a b i l i t y to s y n th e s iz e th e s e m y c o to x in s . F o r a ll m y c o t o x i n s e x c e p t p a tu l in , e a c h i s o la te w a s g r o w n a t t h e a m b ie n t l a b o r a t o r y t e m p e r a t u r e o f 2 2 ° ± 1 ° C in 5 0 0 -m l w id e - m o u t h E r l e n m e y e r f la s k s o n s te a m s te r i l iz e d r ic e ( 5 0 g + 5 0 m l w a te r ) a n d o n s te a m s te r i l iz e d s h r e d d e d w h e a t ( 2 5 g + 2 5 m l w a te r ) s u b s t r a te . A ll c u l tu r e s w e re a l lo w e d t o g r o w u n t i l a ll o r m o s t o f th e s u b s t r a t e w a s m o ld y . F la s k c o n t e n t s w e re e x t r a c t e d b y h e a t in g w i th 2 0 0 m l o f c h lo r o f o r m o n a s te a m b a t h u n t i l h o t c h lo r o fo r m v a p o r s w e r e d r iv e n t h r o u g h th e c o t t o n p lu g o f t h e f la s k . T w o m o r e e x t r a c t i o n s w e re m a d e w i t h 1 5 0 -m l p o r t i o n s o f u n h e a t e d c h lo r o f o r m . A ll e x t r a c t s w e re f i l t e r e d t h r o u g h S & S N o . 5 8 8 f i l t e r p a p e r s a n d e v a p o r a t e d to d ry n e s s o v e r s te a m . M y c o to x i n s w e re d e t e r m in e d q u a n t i t a t i v e ly ( T a b l e 2 ) b y t h in - l a y e r c h r o m a t o g r a p h ic a n a ly s i s o n p l a t e s c o a t e d w i th a b o u t 0 .2 5 m m th ic k n e s s o f S i l ic A R T L C -7 G (M a l- l i n c k r o d t C h e m ic a l W o rk s , S t . L o u is , M o .) . A u th e n t i c m y c o t o x i n s w e r e u s e d a s r e f e r e n c e s t a n d a r d s . P la te s w e re d e v e l o p e d as fo l lo w s : fo r s t e r ig m a to c y s t in , w i th b e n z e n e : a c e t i c a c id : m e th y l a lc o h o l ( 9 0 : 5 : 5 ) a n d f o r a f l a t o x i n w i th

c h l o r o f o r m : m e t h y l a lc o h o l ( 9 3 : 7 ) , b o t h in e q u i l i b r a t e d t a n k s ; f o r o c h r a t o x i n , w i th b e n - z e n e : a c e t i c a c id ( 9 : 1 ) in a n u n e q u i l i b r a t e d ta n k .

R e p r e s e n ta t i v e Penicillium expansum i s o la te s w e re a s s a y e d f o r p a tu l in p r o d u c t i o n b y in o c u la t in g a p p le s (v a r . G o ld e n D e l ic io u s ) a n d a l lo w in g r o t t e n a r e a s to d e v e lo p . T h e a p p le s w e re t h e n m a c e r a t e d in a b l e n d e r w i th 5 0 0 m l o f e t h y l a c e t a t e , t h e e x t r a c t w a s f i l t e r e d , a n d th e f i l t r a t e w a s d r ie d in a f la s h e v a p o r a t o r u n d e r v a c u u m . T h e e x t r a c t s w e re s p o t t e d o n S i l i c A R T L C - 7 G F p la te s a lo n g w i th a n a u t h e n t i c p a tu l in s t a n d a r d . P la te s w e re d e v e lo p e d in a n e q u i l i b r a t e d t a n k w i th a b e n z e n e : a c e t i c a c id : m e th y l a lc o h o l ( 9 0 : 5 : 5 ) s o lv e n t s y s te m .

A d d i t i o n a l t e s t s w e re u n d e r t a k e n to d e t e r m in e th e s u i t a b i l i t y o f p e c a n s as a s u b s t r a t e fo r a f l a to x in , o c h r a t o x i n a n d s t e r ig m a to c y s t in p r o d u c t i o n . I s o la te s k n o w n to p r o d u c e th e s e m y c o to x in s w e r e u s e d . 2 0 m l o f w a te r p lu s 5 0 g o f p e c a n p ie c e s w e re p la c e d in e a c h o f a n u m b e r o f 5 0 0 -m l E r l e n m e y e r f la s k s , a u to c la v e d , a n d i n o c u la t e d w i th th e a p p r o p r i a t e m o ld is o la te . E ig h t o f th e f la s k s w e re n o t i n o c u la t e d .



Table 2 —M yco to x in s produced by m e ld cu ltu res isolated fro m m o ld y pecans and grow n at 22° ± 1°C on rice and shredded w heat

Pecansample

Genus and species

(N o. o f isolates)

D iv. o f M ic ro b io log y

cu ltu re co llec tio n No.

TLCresults®

m l 2 flasks'3

1 A. versico lor M-1101 4 ,000

2 A . ochraceus M -1100 NegativeA . versico lor M -1105 NegativeP. ex pansum (2) - Negative0

3 A . sydo w i M -1108 Negative

4 A . versicolor M-1103 8,500A . versicolor IVI-1104 1,400A . versico lor M -1106 1,013

5 A . versicolor M -1107 4 ,500P. expansum (2) - Negative0

6 P. expansum - Negative07 A. parasiticus M-1099 21,120

A . versico lor M-1102 8,750P. expansum (3) - Negative0

a Sterigmatocystin for A. versicolor and sydowi isolates; aflatoxin B, G and M for A. parasiticus isolate; patulir for P. expansum isolates; ochratoxin for A. ochraceus isolate

b Total of 50g rice and 25g shredded wheat c Grown on apple

F la s k s w e re r a n d o m ly p la c e d in a B O D i n c u b a t o r a t 2 7 ° C f o r 15 d a y s . A ll t r e a t m e n t s w e re r e p l ic a te d f o u r t im e s . E x t r a c t io n s a n d m y c o - t o x i n a s s a y s o f t h e c o n t e n t s o f a ll f la s k s w e re p e r f o r m e d a s in p r e c e d in g e x p e r im e n t s in w h ic h r ic e a n d s h r e d d e d w h e a t s u b s t r a t e s w e re u s e d .

M y c o to x i n s w e re c h e m ic a l ly c o n f i r m e d a c c o rd in g to t h e m e th o d s o f N e s h e im e t a l.( 1 9 7 3 ) fo r o c h r a to x in s A a n d B , P r z y b y ls k i( 1 9 6 9 ) fo r a f l a to x in s B , a n d G , , S ta c k a n d R o d r ic k s ( 1 9 7 1 ) fo r s t e r ig m a to c y s t in , a n d S ta c k e t a l. ( 1 9 7 2 ) f o r a f l a to x in M , .


A T O T A L o f 1 6 3 m o l d i s o la t e s w e re r e c o v e r e d a n d i d e n t i f i e d . N in e g e n e r a and a t le a s t 2 4 s p e c ie s o f m o l d s o c c u r r e d , w i t h A l t e r n a r i a spp . ( 1 9 i s o la t e s ) , A s p e r g i l l u s sp p . ( 4 2 i s o la t e s in c lu d in g 9 s p e c ie s ) a n d P é n i c i l l i u m sp p . ( 7 8 i s o la t e s in c lu d in g 15 s p e c ie s ) p r e d o m i n a t i n g ( T a b l e 1). M o s t o f t h e s p e c ie s w e r e c o m m o n so il o r g a n is m s ( G i l m a n , 1 9 5 7 ) .

In a d d i t i o n , f iv e o f t h e s p e c ie s i d e n t i f ied are i m p o r t a n t s t o r a g e o r g a n is m s : A s p e r g i l l u s r e p e n s a n d A . c h e v a l i e r i ( C h r i s t e n s e n a n d K a u f m a n , 1 9 6 5 ) , P é n i c i l l i u m c y c l o p i u m , P. v i n d i c a t u m an d P. b r e v i - c o m p a c t u m (M is l iv e c a n d T u i t e ,1 9 7 0 ) . T h e r e p o r t e d t e m p e r a t u r e an d re la t iv e h u m i d i t y r e q u i r e m e n t s o f P. f r é q u e n t o n s a n d P. e x p a n s u m (M is l iv e c an d T u i t e , 1 9 7 0 ) i n d ic a t e t h a t t h e y a lso are s t o r a g e o r g a n is m s . T h e s e d a ta su g ge st i m

p r o p e r h a r v e s t in g a n d / o r h o ld in g p r a c t i c e s .

W h e n g r o w n o n s h r e d d e d w h e a t o r r i c e , s ix o f t h e se v e n A . v e r s i c o l o r i s o la t e s in v e s t ig a t e d p r o d u c e d s t e r i g m a t o c y s t i n , t h e o n e A . p a r a s i t i c u s i s o la t e p r o d u c e d a f l a t o x i n , and n o o c h r a t o x i n w a s p r o d u c e d b y A . o c h r a c e u s . N o p a t u l in w as p r o d u c e d b y P. e x p a n s u m g r o w n o n ap p les ( T a b l e 2 ) . A l t h o u g h all e ig h t P é n i c i l l i u m e x p a n s u m i s o la t e s t e s t e d w e r e n e g a t iv e f o r t h e p r o d u c t i o n o f p a t u l in , b e c a u s e o f t h e re la t iv e ly h ig h i n c i d e n c e o fP. e x p a n s u m t h is t o x i c m e t a b o l i t e a lso m a y p r e s e n t a p r o b l e m in p e c a n s .

W h e n g r o w n o n p e c a n s u b s t r a t e s all t h r e e A s p e r g i l l u s s p e c ie s p r o d u c e d t h e i r r e s p e c t iv e m y c o t o x i n s . A v e ra g e r e c o v e r y (pig p e r 5 0 g o f p e c a n s ) w a s : f o r a f la t o x i n s , 3 , 2 5 0 B 1; 1 , 5 0 0 B 2 , 3 3 3 G j , 2 0 0 G 2 a n d 4 2 M ( ; f o r o c h r a t o x i n s , 4 1 , 6 5 0 A, an d 2 5 , 0 0 0 B ; f o r s t e r i g m a t o c y s t i n , 4 3 7 . A l l c h e m i c a l c o n f i r m a t i o n s w e r e p o s i t iv e f o r t h e r e s p e c t iv e m y c o t o x i n s . T h e s e f i n d in g s e x p a n d a n d , in s o m e in s t a n c e s , c o n f i r m t h o s e o f L i l la rd e t al . ( 1 9 7 0 ) a n d D o u p - n ik a n d B e l l ( 1 9 7 1 ) . N o m y c o t o x i n s w e r e d e t e c t e d in t h e e ig h t f la s k s c o n t a in i n g u n - in o c u la t e d p e c a n s .

T h e p r e s e n c e o f m y c o t o x i n s in p e c a n s w o u ld c o n s t i t u t e a h e a l t h h a z a r d . O u r d a ta in d ic a t e t h a t s t e r i g m a t o c y s t i n and a f l a t o x i n , a n d p o s s ib ly p a t u l in a n d o c h r a -

t o x i n , a re m o s t a p t t o o c c u r ( T a b l e s 1 a n d 2 ) . A ls o , p e c a n s w e r e d e m o n s t r a t e d t o b e a s u i t a b le s u b s t r a t e f o r p r o d u c t i o n o f a f l a t o x i n s , o c h r a t o x i n s a n d s t e r i g m a t o c y s t in .

T h e F o o d & D r u g A d m i n i s t r a t i o n , d u r ing i t s s u r v e i l la n c e p r o g r a m s , h a s e n c o u n t e r e d a f l a t o x i n c o n t a m i n a t i o n o f p e c a n s s a m p le d in t h e o p e n m a r k e t . T h e P e c a n S h e l te r s A s s o c i a t i o n , a le r t e d t o t h is f i n d ing, h a s u n d e r t a k e n a n a c t i v e r e s e a r c h p r o g r a m t o l o c a t e a n d e l i m i n a t e t h e c a u s e . R e c e n t A O A C a d o p t i o n o f a n a l y t i ca l m e t h o d s ( A n o n y m o u s , 1 9 7 3 ) f o r o c h r a t o x i n a n d s t e r i g m a t o c y s t i n h a s p aved t h e w a y f o r f u t u r e s u r v e y s f o r t h e s e m y c o t o x i n s .

REFERENCES

A n o n y m o u s . 1 9 7 3 . C h a n g e s in o f f i c i a l m e t h o d s o f a n a ly s is . J . A s s o c . O f f ic . A n a l . C h e m . 5 6 : 4 8 6 .

C h ip le y , J .R . a n d H e a to n , E .K . 1 9 7 1 . M ic r o b ia l f lo r a o f p e c a n m e a t . A p p l . M ic r o b io l . 2 2 : 2 5 2 .

C h r i s te n s e n , C .M . a n d K a u f m a n , H .H . 1 9 6 5 . D e te r io r a t i o n o f s to r e d g ra in s b y f u n g i . A n n . R e v . P h y t o p a t h o l . 3 : 6 9 .

C ie g le r , A ., K a d is , S . a n d A jl, S .J . 1 9 7 1 . “ M ic r o b ia l T o x i n s , ” V o l 6 . A c a d e m ic P re s s , N e w Y o rk .

D o u p n ik , B . J r . a n d B e ll, D .K . 1 9 7 1 . T o x i c i ty t o c h ic k s o f A s p e rg i l lu s a n d P e n ic i l l iu m s p e c ie s i s o la te d f r o m m o ld y p e c a n s . A p p l . M ic r o b io l . 2 1 : 1 1 0 4 .

F o o d & D ru g A d m in i s t r a t i o n . 1 9 6 8 . U n p u b l is h e d M e th o d M 1 6 A , S e p t . , 1 9 6 8 , D iv . o f M ic r o b io lo g y , F o o d & D ru g A d m . , W a s h in g t o n , D .C .

G ilm a n , J .C . 1 9 5 7 . “ A M a n u a l o f S o i l F u n g i , ” 2 n d e d . T h e I o w a S t a t e U n iv e r s i ty P re s s , A m e s , Io w a .

L i l la rd , H .S ., H a n l in , R .T . a n d L i l la r d , D .A . 1 9 7 0 . A f la to x ig e n ic i s o la te s o f A s p e rg i l lu s f la v u s f r o m p e c a n s . A p p l . M ic r o b io l . 1 9 : 1 2 8 .

M is liv ec , P .B . a n d T u i t e , J . 1 9 7 0 . T e m p e r a tu r e a n d r e la t iv e h u m i d i t y r e q u i r e m e n t s o f s p e c ie s o f P e n ic i l l iu m i s o la te d f r o m y e l lo w d e n t c o r n k e r n e l s . M y c o lo g ia 6 2 : 7 5 .

N e s h e im , S . , H a r d in , N .F . , F r a n c i s , O .J . a n d L a n g h a m , W .S . 1 9 7 3 . A n a ly s is o f o c h r a t o x i n A a n d B a n d t h e i r e s te r s in b a r le y , u s in g p a r t i t i o n a n d t h i n l a y e r c h r o m a t o g r a p h y . 1 . D e v e lo p m e n t o f t h e m e t h o d . J . A ss o c . O f f ic . A n a l . C h e m . 5 6 ( 4 ) : 8 1 7 .

P r z y b y ls k i , W . 1 9 6 9 . U n p u b l i s h e d d a t a . H e a l th P r o t e c t i o n B ra n c h , D e p t , o f N a t io n a l H e a l th & W e lfa re , O t t a w a , O n ta r io , C a n a d a .

S ta c k , M . a n d R o d r ic k s , J .V . 1 9 7 1 . M e th o d s fo r a n a ly s is a n d c h e m ic a l c o n f i r m a t i o n o f s t e r ig m a to c y s t i n . J . A s s o c . O f f ic . A n a l . C h e m . 5 4 ( 1 ) : 8 6 .

S ta c k , M .E ., P o h la n d , A .E ., D a n tz m a n , J .G . a n d N e s h e im , S . 1 9 7 2 . D e r iv a t iv e m e t h o d f o r c h e m ic a l c o n f i r m a t i o n o f i d e n t i t y o f a f la t o x i n M , . J . A s s o c . O f f ic . A n a l . C h e m . 5 5 ( 2 ) : 3 1 3 .

W o o d r o f f , J . 1 9 6 7 . “ T re e N u ts : P r o d u c t i o n , P ro c e s s in g , P r o d u c t s , ” V o l 2 . A v i P u b l i s h in g C o ., I n c . , W e s tp o r t , C o n n .

M s r e c e iv e d 7 / 3 1 / 7 3 ; r e v is e d 1 0 / 1 6 / 7 3 ; a c c e p te d 1 0 / 2 4 / 7 3 .

P r e s e n te d a t t h e 3 3 rd A n n u a l M e e tin g o f t h e I n s t i t u t e o f F o o d T e c h n o lo g is t s i n M ia m i B e a c h , J u n e 1 0 —1 3 , 1 9 7 3 .

W e t h a n k J o h n D a n tz m a n , D iv . o f F o o d C h e m is t r y & T e c h n o lo g y , F o o d & D ru g A d m in i s t r a t i o n , f o r t h e t h in - l a y e r c h r o m a to g r a p h ic a n a ly s is o f p a tu l in .

Journal of food Science 1974 Volume.39 No.1

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