Triple Fortification of Rice: Feasibility, Consumer Acceptance and In ...

Triple Fortification of Rice: Feasibility, Consumer Acceptanceand In-Vitro (Caco 2-Cell) Absorption Studies

Author:Thiruselvam, Nishaanthini

Publication Date:2015

DOI:https://doi.org/10.26190/unsworks/2727

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TRIPLE FORTIFICATION OF RICE: FEASIBILITY,

CONSUMER ACCEPTANCE AND IN-VITRO (CACO 2-CELL)

ABSORPTION STUDIES

By

Nishaanthini Thiruselvam

Supervisor: Associate Professor Jayashree Arcot

Co-supervisor: Dr. Janet Paterson

FOR FULFILLMENT OF THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN FOOD SCIENCE AND TECHNOLOGY

School of Chemical Engineering

The University of New South Wales

Australia

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1.

PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES

The--si$.10iuertation Sheet

Fi!::!l nmnc NISHA/\NTH NI

Ab'ore•;iation for ~gre'e ;;s grven in th~;; Un ._,~·siiy ::;:.ler.dar Oot:l:.u of Philcsphy

Scho~l: School )f Chem~ :.ng re~r ~.~

Titla: TRIPLE FORTIFICA- ION OF ~ICE =.:ASISIUTY. CONSUMER ACCEPTANCE A'ID IN· viTRO (C:ACO /·CEL- ) ASSORPn0.\1 STUDIES

=acohy FacJI!y of :.ngrr,eering

AbJ~;If::let 350 '"'Ord" m3xlmum: PLEASE TYPE,

R.ioe fcrti"l(::n:on '"'i th !l1tllt't:ole ,.,..;,;·o•' ,llt..ents (folic acd. trQil and P ca'otene) t y utilizing: two diffefent ,:)fOt::eSSes i>a•OOili'!g~ <:u' ilr' c em P•oce~s anc High Pt!!!l!isure 01ocessrnQ ~liPF) a nc·<'t:t pu;ce~.s l"ra~ t:een exanine~ h this l i!:!Sis. The fort ffica: on crocess was ad:fressed tfuough tou-t "' 1;:~1 ph:'lSe~r optlmls;.!t.:>r, n• fl."''hOiltrg r. ·~'~t:~ ~t, ~n(! use or I IPP to:r tort!fi~i'lhon •.-.tth m~ll1ifil!'! mler(lt'tul rl(!nlt; !)h)l~le!) .CJl(t'l'"l~l prepertJOt. ot tl"oC fondled flee, consume• nece;:::l"!:e !::Udf :'lrd I::!O•acc:essit:ll~· :me transpt>rl studieS.

Pa;b:,itn·g ))rocess wus optlnu:!-cd f~,· io·t.fy1119 n.;;c with lhrt:e rn..:·c·• uhu:n l:i us1ng ~he fo lo\.\'lllQ COII:ittrc·l: scaksr09 !he brown rice at ro<-c for 2 hours w~h the fo1t l'i<:;,sr1 se vt-;ou ;r ,\ 150 ·q:~ : DC· 62.5 rng: fe· 2:. ~i fol :>w::d by ste<~rnins; at 10o•c 'or 1 hour i:ml the:'! <1ir d;;•ing lttc noo at rocm te·npera:uu:: until ~·1 -: IIIIJ ':!'.•.I e w ·•t.:-ut d~>Jt,;r;>::U to 10 12%.Fol ic vcid shcrNt:C h glu~~t vpt<.~ke d Uflr¥.,1 p;,n boilin9. Nt~r cooking folic acid was

reu.·tne>::J tile lll!)nes: ~S<a'X:I 1::>11.-:·,#i ~'; l((:<"t •;SO':<): ar"(( tM 1' c .:;~rotene (35~1 Fer HPP ""~ ee$t ~fl(lr,ion tor rortllbtton was s~&Cted: treating milled •Nhite rioe 'Ai!h the ftjltifi:;:;nt : ·:>lu:it:l' a! 203 MPa fer 1 l"ou1 (noMherrnali an.j tl"en sir-dtj'ing :he pr0<:1essec rbe 10·12V, final mosture conrem). Fo11e a:::o !!.Mw<'l:1 , ,;:tt'<!M .J:ll:'lke nf.P.r t-IPP (41~~ f~ttnwec o:,· fi·C.'H<>iene :'ln<l rnr t2C% ea cl'! . ne1ent10"1 in cocked ti PP rloe follt>Wed tile :sa'Tic t.-cn::l as p:ar:>oil c: :: l it::.:- Fcl~e ;;c.::l (!;6%! <- Iron :~%:·~ '$ ·Garc:cnc ( ldYI: Rice fortifiCd Ot the :'N"C :cd"niques was assesMtd 'or pt,ysicod'iemicat pro.:)ertle3 to ~·ne€rslf. l r. (N ln!;e& n $tar<t'l cun"9 Iona ca;ion The proceM ot a<1e1ng nutrient$ Cl o 'lOt r~.a .. -e t~n impact 0t1 S.lard'l prcpertles in ttre foniHed t 'ij- I'Mo:::1 ~j r1ce v1as ~omp.etely gel~t•nisaCI v.'"'lle HPP rice showed OMIJI geta\in zaMn ·,.n.ct atre:te<J hydrolysis of search b'f " -am:.. lose .,.,. (.!' 1r tvr 1niluMced the releo5e o• ..,,t~mll\t;. ;rom tne r~U m;'! ~'il( eunrg eldr;aet•:>n ~HI ·~ '" \'ltrO cllget.:SO'"I. F'ortiflod p arboiled rioo ;•,•as diluted with whi:e r c.e- and ssse~d fo• eo b ur •,at al;on instrum&nt:dly in ccmpari~cr lo •,.tHte rice. which show'ed no sigr i(IC·ent diffaren<E be::.\·een t'lem. Tl'l~ dilutec foJtifieo rice '1•·s<s p~enteo t•j oons<Jmers f~r '.'isual M ::l laste aooeptante. Fortife-::1 diluted rioe uncooke~) w.;s ne» a!'> acr.ept.~hl~ AS .lr<.c'IO·.P.c ,·:hi:P. ru:.e (r:nmmf.rr.i:.l) le r.;>Mume·$ d~1e to n:>n·unifonniiy i r, ;;ppearan:;.e lnitiall;+ ;he pu'dta5e tntent was lo•.v (28%:+ fer lhe di!ul .?::l t::rtd.c:l rico H:h\-a .. ·er a"tar infcrm1ng ccns'~Mers about fnrtifce.:on arv.! :ha add bona I ru;riticral value ;lur-ch a se if'ltent for fortified rice ncreased ro !:1% impl:,'ul-;; trill .:.ll<":as.t 'lr.:lf \"'If lhe ccrcMvrrer:~. were w !ling ro buJ u·e rice 16% at C'.OI1SLm~rs prefP.tre!1 cooked forufied ric:.e m1xed wilh while nee p3rt CIJta·l:: w1.:n Tent•cr.erJ aoou; fo"t1f1cU on Tne bY.J·a·:cessib il• c·f fcnified rmcronLttremi us1ng bolh tecnmques w~ro higher for for.Jicd p:roc l:::c nee : FA· ~-6% BC· 12%) ' clbwed by HPP ( FA· 4:3%; BC· 2%) comp;;u;d 10 the fo."tifi: ant solution \FA· 80%; BC· 0.1 %) contain:ng the :::.:r e ccr:~ntr:mon of 11H.:iems excep; fc· iron (l::u bo ed ri~· 3 1% HFP nee - 42% and fcrt ifJCallt· 38%;.. Fon1tca:;on •udn mulliple mr::ronulrie;ns us ng p<.;:bol ing :;rocEi'5S 'Showed t:e!lel uanosp:n< tFA· 33:% BC· 2::1% and F~:titin 26 nglml) af:er n ;•ilrc :19;!stion and w<:uld be eas.e1 f<tf irnp~e··r ~t·e :<~tic ·

Declaration relating to (!!s.po,;ellon \)I projcel theslsfdlssorUtlon

I her<'!by gran: to the Uni·.·!irnlT'r (,f 1\~· ... · ~";:ct.t"' wn1e11; or 11. agen1:o: tM; rg l'lt t6 :!t~h1 ~·~ :lnd to ma.<e :'!V."3tl3bl!=! rrry thP.!tl !\ r. ~ d l r.~F,!n:ni;::r -."' ·hhOie or In port in the Ur..•~e r:;.~:y ihrMA':~. '"I ~u f:-r"r'!: of mea•n "':'hv ~~ M·e nfi~H krowt. ~ut"1ect t~ 1"\P. prt">\M:i-oM of th~ CepyrigM Act 196(! 1 ret,"' ln ;~ u prcpef'ti rights sucir .:s !:'Oicrn rtO'IlS. I nl:;o rela1n l lie ngl"t lo us::: •n Mu re W·:>lk.:; (sud" as ~rbc es Cl bookt») ill 01 part of lh1s !he:~::; er d,:;scrtabon.

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n.e Ul'lio.'(:r~lt>' reeognit:.e$ lh<tl ll'e,.., m;·y- t c ~"'«lpt10t'l;; l eir<A..ms.l~~s tC<Jl•ir r.g rewicllet:!!. M eo:>yl!'lfl o· eor.~NIOI'o$ er ''~· R~u~lt. tot f~t;IJ~on tcr <:~ pc r.o(t Of l-~ '" 7 ;·!'lft. '!= mMI be mae<t in .wilinG R.OQllQSts 'or t1 IO'"!.)l)r ~riOd t>f NMI'i~l.er M:o:y M eon~tder~:'l ln <':'lreepll<:t!~l cn=urrslarccs and require :he aOpro•,.;l ol th.~ Oo;;.n c ' G rodua:c Rosoarch.

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8. Acknowledgement

I would like to begin by thanking my primary Supervisor A/Prof. Jayashree Arcot for her

guidance during my PhD project. She has not only been my academic support but also my

emotional support whenever I needed her. She has always been very patient motivating and

enthusiastic and knowledgeable and also helped me relieve some financial burden by giving

me some teaching duties. I could not have asked for a better advisor and mentor for my PhD

study. I also would like to extend my gratitude to my co-supervisor Dr. Janet Paterson who

has added invaluable input for my work and also taken time to read my manuscripts and

provided feedback. I express my utmost gratitude to my supervisors who have helped shape

my PhD thesis and my professional life.

Besides my primary guides, I also worked in other Institutes and schools within UNSW who

have helped me with technical issues. My external supervisor Dr. Jagan Mohan from IICPT,

Thanjavur India, Dr. Judith Field, School of Biological Earth and Environmental Sciences,

UNSW, Dorothy Yu and Rabeya Akter Analytical Centre, UNSW, Dr. Andre Bongers from

BRIL- Lowy Cancer research centre, UNSW for assisting me in experimentation and

technical support

I also would like to appreciate the Technical staff from my school, Mr. Camillo Taraborrelli

who provided me lab consumables, Dr. Robert Chan for computer support, Dr. Victor Wong

for technical support for my chromatography issuesand Ms. Ik LingLau for administrative

support. They have been very patient that allowed a smooth run of experiments during my

PhD. I also would like to thank Dr. Karrie Kam who was my mentor during the initial stages

of my PhD who guided me and made me comfortable to initialize my work and to Dr. Maria

Veronica Chandra- Hioe who trained me on the cell culture work for my project. I also would

like to thanks Dr. Alice Lee and Dr. Patrick Spicer for helping me giving some teaching

opportunities to reduce my financial burden, Dr. Robert Driscoll, Dr. George Srzednicki, Dr.

Jian Zhao and Dr. Francisco Trujillo who have been on my review panel and guided my

project in the right direction. I also would like to thanks Siaw Wei who helped with my lab

work.

I am very grateful to have a wonderful bunch of friends and colleagues who made my PhD

journey colorful, provided insight and boosted me during my downtimes. My sincere thanks

to Xin Sun who has been a part of this journey since the beginning, Dr. Van Ho, Ghazaleh

Ghodsizad, Kitty Tang, Gib Uraipong, Na Wang and Dat Hyunh, Lydia and Yang Lu for

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sharing some light moments in the lab. Special thanks to Mr.Chellamariappan Manoharan for

his companionship and encouragement throughout my PhD study, Dr. Subathra Muthukumar

and Suganya Jeyaprakash for proof-reading my thesis writing.

This acknowledgement would be incomplete if I did not mention my father who has always

been my pillar of support for all these years. Words cannot express my gratitude towards him

for supporting me mentally and financially and made me achieve my goals. Without him and

my mothers‘ blessings I would never have been able to complete my PhD degree.

I sincerely thank everyone that I have mentioned once again for being a part of this long

journey and without you all this thesis would have been impossible. I would like to dedicate

this thesis to you all.

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9. ABSTRACT

Rice fortification with multiple micronutrients (folic acid, iron and β-carotene) by utilizing

two different processes: parboiling- an ancient process and High Pressure processing (HPP) –

a novel process has been examined in this thesis. The fortification process was addressed

through four vital phases: optimisation of parboiling process and use of HPP for fortification

with multiple micronutrients, physico-chemical properties of the fortified rice, consumer

acceptance study and bio-accessibility and transport studies.

Parboiling process was optimised for fortifying rice with three micronutrients using the

following condition: soaking the brown rice at 70°C for 2 hours with the fortificant

solution(FA- 150 mg; BC- 62.5 mg; Fe- 25 mg) followed by steaming at 100°C for 1 hour

and then air drying the rice at room temperature until the moisture content dropped to 10-

12%.Folic acid showed highest uptake during parboiling. After cooking folic acid was

retained the highest (98%) followed by iron (90%) and then β-carotene (35%).For HPP,

thebest condition for fortification was selected: treating milled white rice with the fortificant

solution at 200 MPa for 1 hour (non-thermal) and then air-drying the processed rice (10-12%

final moisture content). Folic acid showed highest uptake after HPP (41%) followed by β-

carotene and iron (20% each). Retention in cooked HPP rice followed the same trend as

parboiled rice- Folic acid (98%)> Iron (64%)>β-carotene (18%). Rice fortified by the

twotechniques was assessed for physico-chemical properties to understand changes in starch

during fortification. The process of adding nutrients did not have an impact on starch

properties in the fortified rice. Parboiled rice was completely gelatinised while HPP rice

showed partial gelatinization which affected hydrolysis of starch by α-amylase which in turn

influenced the release of vitamins from the rice matrix during extraction and in vitro

digestion.Fortified parboiled rice was diluted with white rice and assessed for colour

variation instrumentally in comparison to white rice, which showed no significant difference

betweenthem. The diluted fortified rice was presented to consumers for visual and taste

acceptance. Fortified diluted rice (uncooked) was not as acceptable as uncooked white rice

(coomercial) to consumers due to non-uniformity in appearance. Initially the purchase intent

was low (28%) for the diluted fortified rice. Howeverafter informing consumers about

fortification and the additional nutritional value, purchase intent for fortified rice increased to

51% implying that at least half of the consumers were willing to buy the rice. 76% of

consumers preferred cooked fortified rice mixed with white rice particularly when mentioned

about fortification. The bio-accessibility of fortified micronutrients using both techniques

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were higher for fortified parboiled rice ( FA- 96% BC- 12%) followed by HPP ( FA- 46%;

BC- 2%) compared to the fortificant solution (FA- 80%; BC- 0.1%) containing the same

concentration of nutrients except for iron (parboiled rice- 31%, HPP rice – 42% and

fortificant- 38%). Fortification with multiple micronutrients using parboiling process showed

better transport (FA- 33%; BC- 23% and Ferritin 26 ng/mL) after in vitro digestionand

would be easier for implementation.

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10. Table of Contents

1. ACKNOWLEDGEMENT .............................................................................................. II

2. ABSTRACT .................................................................................................................. VII

3. TABLE OF CONTENTS .............................................................................................. IX

LIST OF TABLES ............................................................................................................... XV

LIST OF FIGURES ........................................................................................................ XVIII

LIST OF ABBREVIATIONS ........................................................................................ XXIII

LIST OF PUBLICATIONS ............................................................................................ XXIV

CHAPTER 1 ............................................................................................................................. 1

1. INTRODUCTION ............................................................................................................ 1

1.1 BACKGROUND ................................................................................................................... 1

1.1.1 Vitamin A deficiency ................................................................................................ 1

1.1.2 Iron Deficiency ........................................................................................................ 1

1.1.3 Folic Acid Deficiency .............................................................................................. 2

1.1.4 Rice- Medium of fortification .................................................................................. 2

1.2 AIMS ............................................................................................................................ 5

CHAPTER 2 ............................................................................................................................. 6

2. LITERATURE REVIEW ................................................................................................ 6

2.1 RICE AS A MEDIUM FOR FORTIFICATION ....................................................................... 6

2.1.1 Nutritional properties of rice ................................................................................... 8

2.1.2 Properties of modified rice starch ........................................................................... 8

2.2 PARBOILING TECHNIQUE .............................................................................................. 9

2.2.1 History ..................................................................................................................... 9

2.2.2 Overview of the Process .......................................................................................... 9

2.2.2.1 Soaking ............................................................................................................. 9

2.2.2.2 Steaming/Heating ........................................................................................... 10

2.2.2.3 Drying............................................................................................................. 10

2.3 HIGH PRESSURE PROCESSING (HPP) .......................................................................... 11

2.4 CHOICE OF MICRONUTRIENTS FOR FORTIFICATION ..................................................... 13

2.4.1 Importance of Vitamin A........................................................................................ 13

2.4.2 Vitamin A in the diet .............................................................................................. 14

2.4.3 Importance of Folic Acid ....................................................................................... 15

2.4.4 Importance of Iron ................................................................................................. 17

2.5 FACTORS TO BE CONSIDERED FOR FOOD FORTIFICATION ............................................ 18

2.6 COMMERCIALLY AVAILABLE FORTIFIED RICE ............................................................. 19

2.7 MANDATORY FOOD FORTIFICATION PROGRAMS (FOLIC ACID) .................................. 23

2.7.1 Mandatory Fortification in Australia & New Zealand .......................................... 23

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2.8 NOVELTY IN RICE FORTIFICATION ............................................................................. 24

2.8.1 Parboiling Process ................................................................................................ 24

2.8.2 High pressure processing ...................................................................................... 25

2.8.3 Cost Analysis for parboiling and HPP processes ................................................. 26

2.9 STUDY OF MORPHOLOGICAL AND PHYSICO-CHEMICAL PROPERTIES OF RICE ............. 31

2.9.1 Pasting Properties Using RVA .............................................................................. 31

2.9.2 Polarized light microscope .................................................................................... 32

2.9.3 X-Ray Diffraction .................................................................................................. 35

2.9.3.1 Diffraction of X-Rays..................................................................................... 35

2.9.3.2 Bragg‘s Law ................................................................................................... 35

2.9.3.3 X-ray Diffraction in rice ................................................................................. 36

2.9.4 Magnetic Resonance Imaging ............................................................................... 37

2.10 CONSUMER ACCEPTANCE STUDY ............................................................................... 38

2.11 BIOAVAILABILITY STUDIES USING CACO-2 CELLS (IN VITRO) .................................... 39

2.11.1 Introduction to Cell Culture............................................................................... 39

2.11.2 Previous studies using Caco-2 cells .................................................................. 40

2.11.3 Bioavailability of Iron in Parboiled Fortified Rice ........................................... 41

2.11.4 Bioavailability of β-carotene in carrots ............................................................. 43

2.11.5 Bioavailability of Folic Acid in Fortified Milk Products ................................... 43

2.11.6 Micronutrient Interaction in vitro ...................................................................... 44

2.12 CONCLUSION .............................................................................................................. 44

CHAPTER 3 ........................................................................................................................... 46

3. OPTIMIZATION OF FORTIFICATION OF RICE WITH FOLIC ACID, IRON

AND Β-CAROTENE BY THE PARBOILING METHOD ............................................... 46

3.1. INTRODUCTION ........................................................................................................... 46

3.2. MATERIALS AND METHODS ........................................................................................ 47

3.2.1. Brown Rice Samples .......................................................................................... 47

3.2.2. Fortificants used for Parboiling ........................................................................ 47

3.2.3. Parboiling Procedure ........................................................................................ 47

3.2.4. Parboiling procedure for Scale-up Studies........................................................ 49

3.2.5. Milling of Parboiled rice ................................................................................... 49

3.2.6. Moisture content in fortified rice and mass of solids that leached out after

soaking 50

3.2.7. Cooking of parboiled rice .................................................................................. 51

3.2.8. Sample extraction and analysis for the micronutrients ..................................... 51

3.2.8.1. Analysis of Folic acid ..................................................................................... 51

3.2.8.2. Analysis of β-carotene .................................................................................... 52

3.2.8.3. Analysis of Iron .............................................................................................. 53

3.2.9. Calculation of % concentration of micronutrients in the soak water before and

after the 2 hours soaking: % uptake and % retention of micronutrients in rice after

parboiling and cooking respectively................................................................................. 54

3.2.10. Statistical Analysis ............................................................................................. 55

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3.3. RESULTS AND DISCUSSION ......................................................................................... 56

3.3.1. Optimization of the soaking step of parboiling .................................................. 56

3.3.2. Moisture content of parboiled rice .................................................................... 57

3.3.3. Leaching of solids during the soaking step of the parboiling process ............... 59

3.3.4. Concentration of Nutrients during the soaking stage of the parboiling process

61

3.3.4.1. Folic acid ........................................................................................................ 63

3.3.4.2. Vitamin A (β-carotene) .................................................................................. 64

3.3.4.3. Iron ................................................................................................................. 64

3.3.5. Uptake of micronutrients in the fortified rice and effectiveness of Parboiling as

a method of fortification ................................................................................................... 65

3.3.6. Loss of nutrients on milling................................................................................ 69

3.3.7. Retention of micronutrients on cooking the fortified parboiled rice ................. 71

3.3.8. Pilot scale studies of experimental condition in comparison to conventional

parboiling condition ......................................................................................................... 74

3.3.9. Micronutrients interaction affecting uptake of each other during the parboiling

process 76

3.3.10. Nutrient concentration in dried parboiled rice after milling ............................. 76

3.3.10.1. Folic acid ........................................................................................................ 76

3.3.10.2. Vitamin A (β-carotene) .................................................................................. 77

3.3.10.3. Iron ................................................................................................................. 78

3.3.11. Efficacy of parboiling as a means of fortification ............................................. 80

3.4. CONCLUSIONS ............................................................................................................ 82

CHAPTER 4 ........................................................................................................................... 84

4. FEASIBILITY OF FORTIFYING RICE WITH FOLIC ACID, IRON AND Β–

CAROTENE BY HIGH PRESSURE PROCESSING (HPP) METHOD ......................... 84

4.1 INTRODUCTION ........................................................................................................... 84

4.2 MATERIALS AND METHODS ....................................................................................... 85

4.2.1 Preparation of rice and fortificants mixture.......................................................... 85

4.2.2 High Pressure Treatment of rice samples ............................................................. 86

4.2.3 Analysis of rice moisture content and micronutrient concentration post HPP

treatment ........................................................................................................................... 87

4.2.3.1 Analysis of moisture content in HPP processed rice...................................... 87

4.2.3.2 Analysis of concentration of micronutrients post HPP treatment .................. 88

4.2.3.3 Calculation of % uptake and % retention of micronutrients in fortified rice . 88

4.2.3.4 Data Analysis ................................................................................................. 88

4.3 RESULTS AND DISCUSSIONS ....................................................................................... 88

4.3.1 Moisture analysis of HPP rice............................................................................... 88

4.3.2 Concentration of micronutrients in fortified rice using HPP ................................ 90

4.3.3 Statistical Analysis of micronutrient concentration .............................................. 91

4.3.4 Loss of micronutrients due to longer treatment times and cooking ...................... 92

4.3.5 Efficacy of high pressure processing as a means of rice fortification................... 95

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4.4 CONCLUSIONS ............................................................................................................ 96

CHAPTER 5 ........................................................................................................................... 98

5. PHYSICO-CHEMICAL PROPERTIES OF RICE FORTIFIED BY THE

PARBOILING AND HPP TECHNIQUES ......................................................................... 98

5.1. INTRODUCTION ........................................................................................................... 98

5.2. MATERIALS AND METHODS ....................................................................................... 99

5.2.1. Optimization of method for studying gelatinization of starch in rice ................ 99

5.2.2. Instrumental colorimetric evaluation of fortified rice ..................................... 101

5.2.3. Rapid Viscoanalyser (RVA) of fortified rice flour samples ............................. 102

5.2.4. X-ray diffraction (XRD) of rice flour fortified by parboiling and HPP ........... 103

5.2.5. Polarized light microscopy and Differential Interference Contrast (DIC)

Microscopic images of starch granules in the fortified rice ........................................... 104

5.2.6. Real-time Magnetic Resonance Imaging (MRI) of water migration in rice

during parboiling ............................................................................................................ 105

5.3. RESULTS AND DISCUSSIONS: .................................................................................... 106

5.3.1. Degree of Gelatinization of fortified rice samples ........................................... 106

5.3.2. Colorimetric analysis of fortified processed rice............................................. 108

5.3.2.1. Total colour difference for parboiled rice and HPP rice– against uncooked

commercial white rice (ControlRaw) and parboiled rice (ControlParboil)....................... 111

5.3.3. RVA pasting curves .......................................................................................... 113

5.3.4. X-ray Diffraction (XRD) of fortified rice flour samples showing crystalline

patterns 119

5.3.5. Microscopic images of starch granules processed by parboiling and HPP

treatment ......................................................................................................................... 123

5.3.6. Real-time Magnetic Resonance Imaging of rice during parboiling process ... 131

5.4. CONCLUSIONS .......................................................................................................... 135

CHAPTER 6 ......................................................................................................................... 137

6. CONSUMER ACCEPTANCE STUDY OF FORTIFIED PARBOILED RICE .... 137

6.1 INTRODUCTION ......................................................................................................... 137

6.2 MATERIALS AND METHODS ..................................................................................... 138

6.2.1 Preparation of fortified parboiled rice ................................................................ 138

6.2.2 Micronutrient analysis of the fortified rice.......................................................... 138

6.2.2 Sensory evaluation of parboiled fortified rice: An Overview .............................. 138

6.2.3 Study 1: Visual consumer acceptance of uncooked fortified parboiled rice ....... 140

6.2.3.1 Samples preparation ..................................................................................... 140

6.2.3.2 Survey Methodology .................................................................................... 140

6.2.4 Study 2: Sensory evaluation (tasting) of cooked fortified rice after mixing with

unfortified white rice ...................................................................................................... 141

6.2.4.1 Sample Preparation ...................................................................................... 141

6.2.4.2 Cooking method ........................................................................................... 141

6.2.4.3 Survey Methodology .................................................................................... 142

xiii

6.2.5 Data Analysis ....................................................................................................... 142

6.2.5.1 Study 1: Visual Consumer acceptance of uncooked fortified parboiled rice

142

6.2.5.2 Study 2: Consumer acceptance of cooked fortified rice after mixing .......... 143

6.3 RESULTS AND DISCUSSION ....................................................................................... 143

6.3.1 Analysis of micronutrients in the fortified rice before and after dilution ............ 143

6.3.2 Study 1: Visual Consumer Acceptance of uncooked fortified parboiled rice ...... 146

6.3.2.1 Demographics and rice eating pattern of consumers ................................... 146

6.3.2.2 Degree of visual acceptance of fortified diluted uncooked rice ................... 148

6.3.2.3 Evaluation of purchase intent of uncooked diluted fortified rice with and

without nutritional information ................................................................................... 150

6.3.2.4 Evaluation of purchase intent of uncooked fortified rice- consumers who

were familiar with parboiled rice ................................................................................ 152

Study 2: Consumer acceptance of cooked fortified rice after mixing............................. 154

6.3.2.5 Consumer demographics and rice eating preferences .................................. 154

6.3.2.6 Degree of liking of cooked rice attributes .................................................... 154

6.3.2.7 Consumers‘ perception of attributes JAR (Just about right) ........................ 155

6.3.2.8 Evaluation of purchase intent of cooked rice ............................................... 158

6.4 CONCLUSIONS .......................................................................................................... 160

CHAPTER 7 ......................................................................................................................... 162

7. SHORT TERM RELATIVE BIO-ACCESSIBILITY AND ABSORPTION OF

FOLIC ACID, IRON AND Β – CAROTENE IN FORTIFIED PARBOILED AND

HIGH PRESSURE PROCESSED RICE: CACO-2 CELL STUDY ............................... 162

7.1 INTRODUCTION ......................................................................................................... 162

7.2 MATERIALS AND METHODS ..................................................................................... 163

7.2.1 Reagents for Cell Culture Resuscitation and Maintenance ................................. 163

7.2.2 Reagents for Cell Viability Assay ........................................................................ 163

7.2.3 Reagents for in vitro digestion ............................................................................. 164

7.2.4 Transport Study ................................................................................................... 164

7.2.5 Cell Culture & Maintainence Protocol ............................................................... 165

7.2.5.1 Procedure for resuscitation of frozen Caco-2 cells ...................................... 165

7.2.5.2 Sub-culturing of Caco-2 cells ....................................................................... 165

7.2.5.3 Cell Density .................................................................................................. 166

7.2.5.4 Freezing down Cells ..................................................................................... 167

7.2.6 Sample Preparation ............................................................................................. 167

7.2.6.1 In-Vitro Digestion Protocol .......................................................................... 167

7.2.6.2 Cell Viability Assessment ............................................................................ 168

7.2.7 Transport & Permeability Study.......................................................................... 169

7.2.7.1 Sample Preparation for Quantitative Analysis ............................................. 171

7.2.7.2 Folic acid analysis ........................................................................................ 171

7.2.7.3 β – carotene analysis .................................................................................... 172

7.2.7.4 Iron analysis ................................................................................................. 172

xiv

7.2.7.5 Calculations .................................................................................................. 173

7.3 RESULTS AND DISCUSSION ....................................................................................... 173

7.3.1 Cytotoxicity and cell viability .............................................................................. 173

Note: Mean absorbance values ±standard deviation of triplicate analysis ................... 174

7.3.2 In vitro-bio-accessibility of micronutrients from fortified rice ........................... 174

7.3.3 Transport Study ................................................................................................... 177

7.3.3.1 Folic acid ...................................................................................................... 177

7.3.3.2 β-carotene ..................................................................................................... 178

7.3.3.3 Iron ............................................................................................................... 179

7.3.4 Improvement in the nutritional status after consumption of fortified rice .......... 181

7.4 CONCLUSIONS: ......................................................................................................... 183

CHAPTER 8 ......................................................................................................................... 184

8. CONCLUSION AND FUTURE WORK .................................................................... 184

8.1 EFFICIENCY OF PARBOILING AS A TECHNOLOGY TO FORTIFY RICE: ........................... 184

8.1.1 Feasibility of rice fortification with multiple micronutrients using HPP technology

186

8.1.2 Physicochemical properties of the rice starch due to the two different processing

methods ........................................................................................................................... 187

8.1.3 Acceptability of the fortified parboiled rice by consumers .................................. 187

8.1.4 Bio-accessibility of the micronutrients from the fortified rice ............................ 188

8.1.4.1 Limitations of the in vitro bio-accessibility and transport of micronutrients

using caco-2 cells ........................................................................................................ 189

8.2 RECOMMENDATIONS FOR FUTURE WORK .................................................................. 189

CHAPTER 9 ......................................................................................................................... 191

9. REFERENCES ............................................................................................................. 191

10. SUPPLEMENTARY SECTION ............................................................................. 216

xv

List of Tables

Table 1.1 Macro and micronutrients contribution of rice based on % RDI in the developing

countries (Kenny, 2001)............................................................................................................. 4

Table 2.1 Proximate composition of paddy (rough rice) and its milling fractions at

14%moisture (Eggum. Juliano & Maniñgat, 1982; Pedersen & Eggum, 1983; Juliano, 1985b)

.................................................................................................................................................... 8

Table 2.2 Comparison of Programs and Quality of rice- and Micronutrient-Premixes

(USAID, 2008) ......................................................................................................................... 21

Table 2.3 The Levels of folic acid fortification in countries with mandatory fortification

programs .................................................................................................................................. 23

Table 2.4 Energy consumption in parboiling processes (Roy et al., 2005) ............................. 28

Table 2.5 Installation costs of the parboiling process (US$) (Roy et al., 2005) ...................... 28

Table 2.6 Theoretical Fortification Formulation for Comparison Purposes (USAID, 2008) .. 29

Table 2.7 Comparison of the technologies for premixes production by extrusion, coating and

dusting3 (USAID, 2008)........................................................................................................... 30

Table 2.8 Table Capital and production cost of thermal and pressure processes (Sampedro,

2014) ........................................................................................................................................ 31

Table 3.1 Concentration of micronutrient addition into rice for Parboiling (per 300g of rice)

.................................................................................................................................................. 47

Table 3.2 Micronutrient mass dissolved in 600 mL of soaking solution for the parboiling

process (mg/300g of rice) ........................................................................................................ 50

Table 3.3 Conditions of ICP-OES instrument for Iron analysis .............................................. 54

Table 3.4 % of initial concentrations of micronutrient in soak water before and after

parboiling process and % residual concentration of micronutrients after 120 min of soaking 61

Table 3.5 Percentage uptake of micronutrients after the parboiling process in uncooked rice

showing concentration of micronutrients at different milling times at conditions A, B, C and

D. .............................................................................................................................................. 67

Table 3.6 % retention of micronutrients after cooking fortified parboiled rice (milled at 120s)

(concentrations of the fortificant added in mg/ 300g of rice) .................................................. 73

Table 4.1 HPP treatment conditions used for the rice fortification experiment ...................... 87

Table 4.2 Moisture content of HPP treated rice on wet basis .................................................. 89

xvi

Table 4.3 Concentration of folic acid (in d.w.b) in high pressure processed rice (uncooked

and cooked) (200MPa) at varying time intervals µg/g of rice ................................................. 91

Table 4.4 β-carotene concentration (in d.w.b.) in high pressure processed (200 MPa) fortified

rice at varying time intervals in µg/g ....................................................................................... 91

Table 4.5 Concentration of Na-EDTA (in d.w.b.) iron in high pressure processed rice (200

MPa) at varying time intervals in µg/g of rice ......................................................................... 91

Table 4.6 % uptake (in uncooked rice) and % retention (in cooked rice) of micronutrients in

the fortified rice before and after cooking at various soaking times ........................................ 94

Table 4.7 Concentration of micronutrients consumed from the fortified rice based on RDI (&

NIH-ODS, 2014 & NHMRC (Australia), 2006) ...................................................................... 96

Table 5.1 The RVA program for analysing rice sample ........................................................ 103

Table 5.2 Scan parameters for XRD measurement ................................................................ 104

Table 5.3 Activation energies for gelatinization in Rough rice (paddy) and brown rice (Bakshi

& Singh, 1980) ....................................................................................................................... 108

Table 5.4 Color parameters L* (Lightness), a* (Redness) and b* (Yellowness) of fortified

rice milled at different milling times at various concentrations ............................................ 110

Table 5.5 Colour difference between fortified rice and commercial white rice (Controlraw) and

parboiled rice (Controlparboil) .................................................................................................. 111

Table 5.6 Colour parameters L* (Lightness), a* (Redness) and b* (Yellowness) of HPP

fortified rice at different pressure and time treatment ........................................................... 111

Table 5.7 RVA data for fortified rice sample processed by parboiling and HPP techniques 115

Table 5.8 % Crystallinity (extracted from XRD) of rice sample treated by high pressure

processing and parboiling ...................................................................................................... 122

Table 6.1 Dietary Reference Intake for the selected micronutrients (Vitamin A, folic acid and

Iron) (Dietary reference intake: Elements and Minerals, (NIH-ODS, 2014 & NHMRC, 2006))

................................................................................................................................................ 144

Table 6.2 Socio-demographic characteristics of the participants of consumer acceptance

Study 1 and Study 2 ............................................................................................................... 147

Table 6.3 Rice consumption pattern of participants of the consumer acceptance study 1 and

study 2 .................................................................................................................................... 148

Table 6.4 Mean perceptions of appearance for uncooked rice sample- diluted fortified rice

and p-values (p≤0.05) of t-test on comparing the diluted fortified rice sample with Control –

commercial white rice (Tested by consumers in the Consumer Acceptance Study (colour

intensity, degree of liking of colour, uniformity of colour, overall appearance)) ................. 153

xvii

Table 6.5 Mean degree of liking score for sensory attributes (appearance, colour, odour,

texture, taste, aftertaste and overall liking) of rice samples tested by consumers in Phase 2 of

the Consumer Acceptance Study (n=54) ............................................................................... 155

Table 7.1 Absorbance values for the digesta samples and controls in the cell proliferation

assay after 2 hours incubation ................................................................................................ 174

Table 7.2 Concentration of folic acid, iron and β-carotene (µmol) in fortificant solutions

(control), fortified parboiled rice and HPP rice ..................................................................... 175

Table 7.3 Concentration of folic acid, β-carotene and iron present in the apical chamber of the

transwell in µg/monolayer ..................................................................................................... 177

Table 8.1 Parboiling process cost analysis (Roy, Shimizu, Shiina and Kimura, 2005)......... 185

xviii

List of Figures

Figure 1.1 Global rice production and consumption (1960- 2012) (USDA, 2013) .................. 3

Figure 2.1 Longitudinal cross-section of rice kernel (Juliano, 1993) ........................................ 6

Figure 2.2 Schematic diagram of discontinuous equipment for High Pressure Processing of

packaged food products (Knorr, 1993). ................................................................................... 12

Figure 2.3 Structure of folic acid showing the pteridine ring and the para-aminobenzoic acid

(PABA) (Eitenmiller, 2008)..................................................................................................... 16

Figure 2.4 Flow chart of local parboiling process in Bangladesh and West Bengal, India.

(Roy et al., 2005) ..................................................................................................................... 27

Figure 2.5 Model of amylose molecule with 1000 glucose units (a) and Model of amylopectin

molecule with 1000 glucose units in 30 branches (b) (Nikuni, 1957) ..................................... 33

Figure 2.6 Microscopic images of corn starch granules under normal light (A) and polarized

light (B); where I is untreated and II is pressure treated at 500 MPa for 20 min (Hibi et al.,

1993) ........................................................................................................................................ 34

Figure 2.7 Microscopic images of potato starch granules under normal light (A) and under

polarised light (B) where I is untreated and II is pressure-treated at 500 MPa for 20 min (Hibi

et al., 1993) .............................................................................................................................. 35

Figure 2.8 Diffraction of X-rays by crystal (Skoog and Douglas, 1980) ................................ 36

Figure 2.9 Iron retention rate in rice (as % of the un-rinsed) after rinsing (simulating rice

washing) in the Fe-fortified parboiled rice grains milled for 60 and 120 s, respectively, in the

3 rice cultivars tested (Prom-u-thai et al. 2008) ...................................................................... 41

Figure 3.1 Schematic representation of optimized parboiling condition ................................. 48

Figure 3.2 Moisture content of rice with different fortificants during soaking at 30-min time

interval. BC: β-carotene; Fe: iron; FA: folic acid (The error bars represent standard errors of

corresponding means from three replicates) ............................................................................ 58

Figure 3.3 Moisture content of rice after 2 hours soaking and 1 hour of steaming at 100 °C

(The error bars represent standard errors of corresponding means from three replicates) ...... 59

Figure 3.4 Total solids in rice soaking solution before and after parboiling for the different

soaking solutions (The bars represent standard errors of corresponding means from three

replicates) ................................................................................................................................. 60

Figure 3.5 Mean folic acid %loss in concentration in the soak solution after soaking for 120

minutes with different treatments (The bars represent standard errors of corresponding means

from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) .............................................. 63

xix

Figure 3.6 Mean β-carotene % loss in concentration in the soak solution after soaking for

120 minutes with different treatments (The bars represent standard errors of corresponding

mean values from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) ......................... 64

Figure 3.7 Mean iron % loss in concentration in the soak solution after soaking for 120

minutes with different treatments (The bars represent standard errors of corresponding means

from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) .............................................. 65

Figure 3.8 Concentration of micronutrients (d.w.b) in uncooked rice fortified by parboiling at

varying concentrations and milling times (0s, 60s and 120s) (Error bars indicate the standard

deviations between duplicates (A, B, C and D refers to the mass of the micronutrients added))

.................................................................................................................................................. 66

Figure 3.9 Concentration of micronutrients in the fortified rice bran (Condition A) during

milling (Error bars indicate the standard deviations between duplicates) ............................... 70

Figure 3.10 Schematic representation of mass balance (on a d.w.b.) in terms of nutrients

concentration and loss at key stages of parboiling in rice soaked with all the micronutrient at

condition A (FA- 150mg; BC- 62.5 mg; Fe – 25mg/600mL soaking solution used on 300g

rice) .......................................................................................................................................... 70

Figure 3.11 Retention of micronutrients in cooked rice fortified by parboiling at varying

concentrations (Error bars indicate standard deviation between replicate samples; A, B, C and

D refers to the concentration of the micronutrients added to the soaking water) .................... 71

Figure 3.12 Comparison of micronutrient concentration (in d.w.b) in uncooked rice using

experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4 hours) on

pilot scale (Error bars indicate standard deviation of duplicate samples) ............................... 75

Figure 3.13 Comparison of micronutrient retention in (in d.w.b.) cooked rice using

experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4 hours) on

pilot scale (Error bars indicate standard deviation of duplicate samples) ............................... 75

Figure 3.14 Mean folic acid concentration (in d.w.b) in dried parboiled rice with different

fortifications before and after milling at 60 s and 120 s (The bars represent standard errors of

corresponding means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) ........... 77

Figure 3.15 Mean vitamin A (β-carotene) concentration in (d.w.b) dried parboiled rice (11%)

with different treatments before and after milling at 60 s and 120 s (The bars represent

standard errors of corresponding means from replicates. BC: β-carotene; Fe: iron; FA: folic

acid).......................................................................................................................................... 78

xx

Figure 3.16 Mean iron concentration in (d.w.b.) dried parboiled rice with different

fortifications before and after milling at 60 s and 120 s (The bars represent standard errors of

corresponding means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) ........... 79

Figure 4.1 Flow chart of sample preparation for Hydrostatic High Pressure Processing

treatment .................................................................................................................................. 86

Figure 4.2 High pressure equipment used for the experiment ................................................. 87

Figure 4.3 % Moisture content of rice on a wet basis at different treatment times and

pressures used .......................................................................................................................... 90

Figure 4.7 Activity of α-amylase by testing glucose release from processed (fortified) and

unprocessed rice samples (Error bars represent standard error in replicates) .......................... 92

Figure 5.1 Optimization of KOH molarity to be added to study the degree of rice

gelatinization .......................................................................................................................... 100

Figure 5.2 % Gelatinization comparison between parboiled rice and HPP processed rice.

Untreated brown rice and white rice were used as controls (Error bars represent standard

errors in replicate samples) .................................................................................................... 106

Figure 5.3 RVA graph for rice treated at 200 MPa at different time intervals using high

pressure process ..................................................................................................................... 116


pressure process ..................................................................................................................... 117


pressure processing ................................................................................................................ 117

Figure 5.6 RVA graph for untreated rice and parboiled rice ................................................. 118

Figure 5.7 XRD pattern for parboiled and commercial white, brown and parboiled rice ..... 120

Figure 5.8 XRD pattern for HPP treated rice at 200 MPa for 1,2 and 3h .............................. 120

Figure 5.9 XRD pattern for HPP treated rice at 400 MPa for 1,2 and 3h .............................. 121

Figure 5.10 XRD pattern for HPP treated rice at 600 MPa for 1, 2 and 3h .......................... 121

Figure 5.11 a & b: Starch images for rice treated at 200 MPa for 1 hour (a. under differential

interference contrast; b. under cross-polarized light) ............................................................ 124

Figure 5.12 a & b: Starch images for rice treated at 200 MPa for 2 hours (a. under differential


Figure 5.13 a & b: Starch images for rice treated at 200 MPa for 3 hours (a. under differential




xxi

Figure 5.15 a & b: Starch images for rice treated at 400 MPa for 2 hours (a. under cross-

polarized light under; b. differential interference contrast) ................................................... 125

Figure 5.16 a & b: Starch images for rice treated at 400 MPa for 3 hours (a. under

differential interference contrast; b. under cross-polarized light) .......................................... 125



Figure 5.18 a & b: Damaged starch images for rice treated at 600 MPa for 2 hours (a. under

differential interference contrast; b. under cross-polarized light) .......................................... 126

Figure 5.19 a & b: Healthy starch grain images for rice treated at 600 MPa for 2 hours (a.

under differential interference contrast; b. under cross-polarized light) ................................ 126

Figure 5.20 a & b: Partially integrated starch Starch images for rice treated at 600 MPa for 3

hours (a. under cross-polarized light under; b. differential interference contrast) ................. 127

Figure 5.21 Figure 5.17 a & b: Completely damaged starch Starch images for rice treated at

600 MPa for 3 hours (a. under cross-polarized light under; b. differential interference

contrast).................................................................................................................................. 127

Figure 5.22 a & b: Starch images for parboiled rice milled at 0s (a. under differential


Figure 5.23 a & b: Starch images for parboiled rice milled at 60s (a. under differential


Figure 5.24 a & b: Starch images from in-house parboiled rice milled at 120s a. shows

birefringence in some starch granules under DIC while in b. it can be seen that there has been

a damaged starch granule ....................................................................................................... 128

Figure 5.25 a & b: Starch images from comemrical parboiled rice showing birefriengence (a.

under DIC and b. cross polarized light) ................................................................................. 128

Figure 5.26 a & b: Starch images from untreated rice showing birefriengence under a. DIC

and b. cross polarized light .................................................................................................... 129

Figure 5.27 Coronal section images of rice after parboiling process at different time intervals

at the optimized condition ...................................................................................................... 131

Figure 5.28 Sagittal section of soaked rice at different time intervals during the optimized

parboiling conditions ............................................................................................................. 132

Figure 5.29 a&b: Migration of water with and without micronutrients where A- is with Fe

and B – is without any micronutrients ................................................................................... 132

Figure 6.1 Schematic representation of fortified parboiled rice prepared for Consumer

Acceptance Study 1 and Study- 2 .......................................................................................... 139

xxii

Figure 6.2 Concentration of micronutrients (in d.w.b.) in fortified premix (cooked and

uncooked) and diluted fortified rice (cooked and uncooked) ................................................ 145

Figure 6.3 Preference of visual attributes investigated in Study 1 tested by participants (n=49)

presented on a hedonic scale .................................................................................................. 149

Figure 6.4 The distribution of purchase intent responses (%) in Consumer Acceptance Study -

STUDY 1 tested by participants (n=49). A) Before the notification of health claim; B) After

the notification of health claim .............................................................................................. 152

Figure 6.5 Bar graph showing consumer preference of cooked fortified rice compared with

unfortified rice ....................................................................................................................... 157

Figure 6.6 (a & b) Graph showing purchase intent of fortified rice before and after

mentioning about fortification ............................................................................................... 159

Figure 7.1 Schematic representation of Transwell permeable support with Caco-2 cells grown

on the membrane .................................................................................................................... 165

Figure 7.2 Diagrammatic representation of the chamber of a haemocytometer (Louis &

Siegel, 2011) .......................................................................................................................... 167

Figure 7.3 Confocal microscopy image of caco-2 cells showing tight junctions (Chandra-Hioe

et al., 2013) ............................................................................................................................ 175

xxiii

List of Abbreviations

Acronym Abbreviation

AM Amylose

ANN Annealing

ANOVA Analysis of Variance

AP Amylopectin

BC β-carotene

BR Brown Rice

d.w.b. Dry weight basis

DIC Differential Interference Contrast

DSC Differential Scanning Calorimeter

FA Folic acid

Fe Iron

HMT Heat and Moistutre treatment

HPP High Pressure Processing

IDA Iron Deficiency Anemia

IICPT Indian Institute of Crop Processing

Technology

JAR Just About Right

MPa Mega Pascals

MRI Magnetic Resonance Imaging

NTD Neural Tube Defects

RDI Recommended Dietary Intake

RVA Rapid ViscoAnalyser

TEER Trans Epithelial Electrical Resistance

VAD Vitamin A Deficiency

w.b. Wet basis

XRD X-Ray Diffraction

xxiv

List of Publications

Journal Articles

Chapter 3

Thiruselvam N, Cheong SW, Mohan J, Paterson J, Arcot J (2014) Micronutrients

Fortification of Rice by Parboiling: Lab Scale and Pilot Scale Studies. J Nutr Food Sci

4: 281,accepted and in press doi: 10.4172/2155-9600.1000281

Thiruselvam N, Mohan J, Paterson J, Arcot J Micronutrient fortification of Rice using

Parboiling: Scale-up Studies‖ at the 3rd International Conference of Food Technology

held at IICPT, Thanjavur, India during January 2013 and the paper was published in

the conference journal.

Conference Presentations

Oral presentation on ―Bio-accessibility of micronutrients from fortified rice using

caco-2 cell culture model‖ held at Washington DC, USA during May 12-15th

, 2014.

Oral Presentation on ―Physicochemical properties and sensory evaluation of fortified

rice‖ at the AIFST summer school held at The University of Queensland during 5-7th

Feb, 2014.

Poster Presentation on ―Folate stability in fortified rice in the presence of an

antioxidantduring parboiling process‖at the International Food Data Conference 2013

held at Granada Spain

Oral presentation on ―Multiple Micronutrient Fortification of Rice: What are the

Challenges?‖ at the 46th

AIFST Annual Convention during 14-16th

July, 2013.

Oral presentation on ―Micronutrient fortification of rice using Parboiling Technique‖

at the AIFST summer school held at The University of NSW during 6-8th Feb, 2013.

Oral presentation on ―A Comprehensive Approach to fortification of Rice with

Micronutrient using Parboiling‖ at the 2nd International Vitamin Conference held at

Copenhagen, Denmark during May 2012.

1

CHAPTER 1

1. INTRODUCTION

1.1 Background

Food fortification has been defined as the addition of one or more essential nutrients to a

food, whether or not it is normally contained in the food, for the purpose of preventing or

correcting a demonstrated deficiency of one or more nutrients in the population or specific

population groups (FAO/WHO 1994). In today‘s rapidly changing lifestyles populations have

become dependent on processed foods to attain nutritional adequacy and hence fortifying

foods with the aid (or during) of processing would be justifiable(LaChance and Bauernfiend,

1991; Hoffpauer and Wright, 1994).Micronutrient Malnutrition (MNM) is not spread

worldwide and not just confined to developing countries but also prevalent in industrialized

countries. Globally, the three most common forms of MNM are iron, vitamin A and iodine.

In the light of public health, MNM not only affects a vast population but is also a potential

risk factor for many diseases, and is the cause of high rates of morbidity and mortality

(Micronutrient Initiative and UNICEF, 2011). This chapter will briefly introduce the project

by explaining the importance of the micronutrients chosen for fortification, the medium of

fortification – rice and the aims of the study.

1.1.1 Vitamin A deficiency

Micronutrient deficiencies are common in developing countries such as Asia, Africa and the

Pacific. Vitamin A deficiency causes diseases such as xerophthalmia, Bitot‘s spot,

conjunctival xerosis, corneal lesion, xerosis, ulcer, scars and keratomalacia all of which can

lead to poor vision or total blindness if left untreated (WHO/UNICEF, 1996). For example,

during the year 2000, in India 5,049,139 people were affected by vitamin A deficiency out of

a total population of 841,523,272 (Food and Agricultural Organization of the United States,

1988).

1.1.2 Iron Deficiency

About one third of the world population suffers from iron deficiency anaemia (IDA)for

example India continues to be one of the countries with a high prevalence. National Health

Family Survey (NHFS, in 1998-1999) revealed that about 70-80% of children, 70% of

pregnant women and 24% of adult men were suffering from IDA (National Family Health

Survey, 2006).

2

1.1.3 Folic Acid Deficiency

Folic acid is one of the important B-complex vitamins essential for biosynthesis of DNA, and

it cannot be synthesized by the body and therefore needs to be obtained from dietary sources.

Folic acid deficiency leads to cardiovascular diseases, major depression, schizophrenia,

Alzheimer‘s disease, and some carcinomas such as colorectal, uterine, cervical, lung and

oesophageal (Gregory, 1996). Various studies and randomized trials over the last three

decades have shown that adequate intake of folate during early pregnancy reduces the risk of

abnormalities in early embryonic brain development and specifically the risk of

malformations of the embryonic brain/spinal cord, collectively referred to as NTDs (Czeizel

& Dudas, 1992; Cuskelly et al., 1996; Scholl & Johnson 2000;Kauwell et al., 2002&Öner et

al., 2006). Fortification of wheat flour to overcome folic acid deficiency diseases has proven

to be effective in many developed countries. Fortification of cereal grain products with folic

acid (100 to 150 µg/ day) resulted in a decrease in NTDs in the United States and Canada

(Bailey et al., 2001 and Berry et al., 2010). Pachón.et al. (2013) state that with the

widespread consumption of wheat flour-based products and advances in technology in the

milling industry, folic acid fortification should be adapted as a public health initiative in

Europe (Pachón et al., 2013). In Australia, two per thousand live births are affected by NTDs

attributed to folic acid deficiency. As a result, fortification of bread making flour with folic

acid ismandatory in Australia since September 2006 (Wesley & Dutta, 2009).

1.1.4 Rice- Medium of fortification

Figure 1.1 shows the growth in global rice production and consumption during the year 1960-

2012. Except for the decline in production during the years 2000 to 2003, there has been a

steady increase in rice production. There is also a consistent increase in rice consumption.

3

Figure 1.1 Global rice production and consumption (1960- 2012) (USDA, 2013)

Table 1.1 shows that rice provides a significant contribution of macro and micronutrients in

the diets of people from developing countries where rice is a staple food. Although rice seems

to contribute moderate suppliesof protein, lipids and other vitamin such as thiamin, niacin

and riboflavin and minerals such as iron, zinc and calcium, it is unable to provide for normal

requirements of humans. Yi et al., (2011) in a review showed that due to NTDs the economic

burden in terms of direct (e.g. drugs, hospitalizations and managing co-morbidities) and

indirect (e.g. loss of work time and costs due to premature loss of life) and caretakers‘ time

was significant. In another study conducted by Stein and Qaim in 2007, it was reported that in

India, due to deficiencies caused by the micronutrients vitamin A and iron, there was a loss of

2.5% to the Indian GDP. Therefore micronutrient deficiencies not only affect the

populations‘health but also an entire nation‘s economy. Hence, an economically beneficial

option of rice fortification with folic acid, iron and vitamin A could be a potential method to

address this public health issue.

4

Table 1.1 Macro and micronutrients contribution of rice based on % RDI in the developing countries (Kenny, 2001)

Consumption

(g/day)

Energy Protein Fat Calcium Iron Thiamine Riboflavin Niacin Zinc

(% of Recommended Daily Intake)

Bangladesh 441 76 66 18 3 8 18 14 25 30

China 251 30 20 17 2 4 10 8 17 17

India 208 31 24 4 1 4 8 6 4 15

Indonesia 414 51 43 8 3 7 17 13 8 29

Myanmar 578 74 68 20 4 10 23 17 20 40

Philippines 267 41 30 5 2 5 10 8 5 17

Sri Lanka 255 38 37 3 2 5 10 8 3 17

Thailand 285 43 33 5 2 5 12 9 5 21

Vietnam 465 67 58 14 3 8 19 14 14 34

5

Parboiling has been used as a successful fortification method in recent times where Prom-u-

thai (2008 & 2010) has utilized this method to fortify rice with iron and zinc individually.

Tyythan et al., (2007) and Kam et al., (2012) fortified rice with iodine and folic acid

respectively using the parboiling technique. Based on the results from the above studies it is

evident that through the parboiling process penetration of minerals and vitamin is significant

from the fortificant solution and the nutrients are able to penetrate through the outer layers

into the endosperm of the rice grain.

A comprehensive fortification program to combat all three deficiencies (folic acid, iron and

β-carotene) will be an ideal approach for those populations affected by all three deficiencies

as often encountered in developing countries. This study attempted to fortify rice with folic

acid, iron and β-carotene using two fortification techniques: Parboiling- an ancient technique

andhigh pressure processing (HPP)-a novel technique despite the difference in processing

properties for the first time.

1.2 Aims

1. To study the feasibility of fortifying rice with 3 micronutrients using parboiling;

performing pilot scale studies for the parboiling process; analysing the retention of

micronutrients after milling and cooking the rice; understanding the efficiency of

micronutrients uptake in the grain using the parboiling technique- Chapter 3.

2. To study the feasibility of fortifying rice with 3 micronutrients using HPP technique; loss

during cooking- Chapter 4.

3. To study the physico-chemical properties of fortified rice by using the Rapid

Viscoanalyser (RVA), microscopy of rice starch, changes in rice crystalline properties of rice

using X-ray diffraction (XRD) and migration of micronutrients using magnetic resonance

imaging -(MRI) Chapter 5.

4. To evaluate consumer acceptance of fortified rice using sensory analysis- Chapter 6.

5. Investigate the short-term relative bio-accessibility of the fortified micronutrients in the

parboiled and HPP rice using in vitro cell culture technique (Caco-2 cells)- Chapter 7.

6

CHAPTER 2

2. LITERATURE REVIEW

2.1 Rice as a medium for fortification

Rice (Oryza sativa L.) comes from the family Poaceae and is harvested as paddy rice, or

―spikelets‖ in botanical terms, where each spikelet consists of caryopsis, four bracts, and a

rachilla (Champagne et al., 2004). The major parts of rice grain are hull, pericarp, testa (seed

coat), embryo (germ) and endosperm (Champagne et al., 2004). The outer layer, called hull

consists of lemma and palea, where they differ in sizes, shapes, and numbers of vascular

bundles (Champagne et al., 2004). Brown rice consists of pericarp, testa and nucellus,

embryo (germ) and endosperm (Juliano, 1993). The pericarp runs along the embryo, dorsal

and the two lateral sides of the grain; testa lies beneath the pericarp; the embryo is located on

the ventral side of the grain; the endosperm consists of the aleurone and starchy endosperm,

where the latter constitutes the majority of the rice grain (Champagne et al., 2004).

Figure 2.1 Longitudinal cross-section of rice kernel (Juliano, 1993)

Rice is the staple food in many Asian countries like India, Myanmar, Bangladesh, Nepal,

Indonesia, Philippines, Sri Lanka and Vietnam where micronutrient deficiency diseases due

to vitamin A, iodine and iron is also common (Dexter , 1998). Rice accounts for nearly 22 %

7

of the world‘s energy intake (Bierlen et al. 1997). According to UNICEF (2009), it is noted

that most of the rice-consuming countries suffer from a higher incidence of low birth weight,

infant mortality and mortality under five. Vitamin A deficiency and nutritional anemia from

iron deficiency are also widespread in rice-consuming countries; vitamin A deficiency is

common particularly in South and East Asian countries such as Bangladesh, India, Indonesia,

Myanmar, Nepal, the Philippines, Sri Lanka and Vietnam (Juliano, 1993). Considering the

role of rice in the diets of these populations it is sensible to fortify rice to adapt a food-based

approach to prevent and control micronutrient deficiencies. Other criteria would be that the

nutrients should be stable under normal conditions of storage. It should also be well absorbed

and mixed with the appropriate vehicle and aesthetically acceptable (Rao, 1981). Therefore,

rice would be a good choice to fortify with Vitamin A, Folic acid and Iron as a

comprehensive fortification strategy as rice is consumed by large populations in India.

In the table below it can be seen that carbohydrates form the highest composition of nutrients

in rice. Milled rice has more carbohydrates compared to brown rice (Barber, 1972). Starch is

the major component of carbohydrate in rice- therefore it is an important determinant of

flavour and quality in rice. The two main polymers in rice starch granules are amylose (AM)

and amylopectin (AP). Other minor constituents associated with these are proteins namely

glutelin, prolamin, globulin and albumin in the rice endosperm (Singh et al., 2000). The

morphology of starch granules depends on the biochemistry of the chloroplast or amyloplast

in conjunction with the physiology of the plant (Badenhuizen, 1969). Rice starch granules are

small polygonal and angular-shaped (Singh et al., 2003) ranging between 3-10 µm (Ellis at

al., 1998) and exhibit unimodal distribution (Dang and Copeland 2004).

8

Table 2.1 Proximate composition of paddy (rough rice) and its milling fractions at

14%moisture (Eggum. Juliano & Maniñgat, 1982; Pedersen & Eggum, 1983;Juliano,

1985b)

Rice

fraction

Crude

protein

(g N x 5.

95)

Crude

fat (g)

Crude

fibre

(g)

Crude

ash (g)

Available

carbohydrates

(g)

Neutral

detergent

fibre (g)

Energy

content

Density

(g/ml)

Bulk

density

(g/ml) (kJ) (kcal)

Rough

rice

5.8-7.7 1.5-2.3 7.2-

10.4

2.9-5.2 64-73 16.4-19.2 1580 378 1.17-

1.23

0.56-

0.64

Brown

rice

7.1-8.3 1.6-2.8 0.6-1.0 1.0-1.5 73-87 2.9-3.9 1520-

1 610

363-

385

1.31 0.68

Milled

rice

6.3-7.1 0.3-0.5 0.2-0.5 0.3-0.8 77-89 0.7-2.3 1460-

1 560

349-

373

1.44-

1.46

0.78-

0.85

Rice bran 11.3-

14.9

15.0-

19.7

7.0-

11.4

6.6-9.9 34-62 24-29 670-1

990

399-

476

1.16-

1.29

0.20-

0.40

Rice hull 2.0-2.8 0.3-0.8 34.5-

45.9

13.2-

21.0

22-34 66-74 1110-

1 390

265-

332

0.67-

0.74

0.10-0.1

2.1.1 Nutritional propertiesof rice

Rice is rich in energy and a good source of protein; contains reasonable concentrations of

thiamine, riboflavin, niacin and vitamin E in the bran layer (Juliano, 1993). Normally, the

level of the vitamin is 2-10 times higher in brown rice than in milled rice (Champagne et al.,

2004). In contrast, the rice grain does not contain any vitamin C, D or A (Juliano, 1993). To

produce milled rice, bran layers, polish (sub-aleurone), germ (embryo) and a small part of the

endosperm are removed. After milling, rice results in loss of nutrients, while starch content

(90% dry weight) is higher in milled rice than in brown rice (Champagne et al., 2004).

2.1.2 Properties of modified rice starch

Starch gelatinization is an important aspect in food modification and it is applicable in

cooking, baking and extruding starch-based foods (Zobel, Young and Rocca, 1988).

Gelatinization is a complex phenomenon that obeys first order kinetics (Riva , Piazza &

Schiraldi , 1991) and alters the physical, chemical and nutritional properties of starch. It also

affects heat diffusivity, viscosity, rheological behaviour, swelling and deformation of original

shape of starchy products and susceptibility to enzymatic digestion (Tsai Li & Lii 1997).

Gelatinization is a dynamic process which disrupts the crystalline and molecular order within

the starch granule (Yifang , Yongzhong , Qifa , Mei & Harold , 2001).

9

The swelling property of starch can be attributed to AP (Tester & Morrison, 1990) where AM

tries to restrict this (Park et al., 2007; Patindol et al., 2007). The variation in the unit chain

length of the AP influences swelling and pasting properties. Presence of lipids and other

morphological structures also affect the solubility and swelling power of starch granules

(Gaillard & Bowler 1987; Singh et al., 2003). The changes that occur to rice starch molecules

upon gelatinization can be studied based on pasting properties. It involves further swelling of

granules, leaching of molecular components from the granules and eventual disruption with

the application of shear force.

2.2 Parboiling Technique

2.2.1 History

Parboiled rice is predicted to have originated in ancient India but it is unknown how and why

it was exactly started (Bhattacharya, 2006). However, parboiled rice reduces the breakage of

rice during milling (Bhattacharya, 1969) and is favoured by South Asians due to its‘ hardened

texture after parboiling (Bhattacharya, 2006). In early 20th

century, parboiled rice gained a lot

of importance as it was seen to preventberiberi (Bhattacharya, 2006). At present, parboiled

rice is popular among the low-income people of Bangladesh, India, Nepal, Pakistan, Sri

Lanka, and parts of West Africa and contributes to one-fifth of the world‘s rice consumed

(Bhattacharya, 1985). Parboiling is desirable not only because of improved nutritional value

but because of improved cooking and processing properties which are desirable from an

industrial point of view (Bhattacharya, 2006).

2.2.2 Overview of theProcess

2.2.2.1 Soaking

The first step in parboiling is soaking, where the paddy is hydrated by soaking in water to

allow gelatinization on subsequent heating (Juliano, 1993). At low temperatures, the paddy

does not absorb water quickly and reaches equilibrium of ~30% moisture content (wet basis)

(Bhattacharya, 2006). Unlike at low temperatures, water absorption of the paddy at high

temperatures increase exponentially after an initial lag period due to starch gelatinization

(Bhattacharya, 2006). Consequently, the ‗swollen‘ starch leaks out of the grain and the grain

deforms as a result of exceeding ~30-32% moisture (Bhattacharya, 2006). Therefore, two

important factors that affect soaking are temperature and time. To achieve practical soaking,

rice is soaked at ~70°C for a certain period of time depending on the gelatinization

temperature (GT) of the variety of rice (Bhattacharya, 2006). According to Biswa and Juliano

10

(1988), it is found that low-GT varieties require soaking temperature of less than 70°C to

avoid over-hydration.

2.2.2.2 Steaming/Heating

Steaming aims to gelatinize the starch of the soaked paddy (Juliano, 1993). As described by

Ayamdoo et al. (2013), covering the rice during steaming creates pressure that aids in inward

movement of water molecules into each rice kernel. According to Wimberly (1983),

gelatinization of starch causes certain changes such as creating translucency and makes the

grain hard, as well as resistant to breakage during milling. Therefore, it results in easier

removal of the husk during milling but more difficult bran removal. Other physiochemical

changes include starch retrogradation, pigment transformations and enzyme deactivation,

where these changes improve flavour and cooking characteristics of the rice (Ayamdoo et al.,

2013).

2.2.2.3 Drying

Drying of steamed paddy takes place at an airy place. During the first stage, moisture

reduction occurs quickly, dropping from 36 to 18% followed by tempering before proceeding

with drying to 14% (Wimberly, 1983). Throughout the process, solute concentrates in the rice

kernel and the gelatinous amylose starch are compressed, where the latter results in easier

husk milling (Ayamdoo et al., 2013).

After parboiling, gelatinized starch and protein bodies occupy the empty spaces in the rice

endosperm thereby giving better consistency and hardness to rice kernel. Therefore, the

kernel becomes translucent and offers great resistance to breakage during milling. Parboiled

rice takes longer time to cook than normal rice and also the color is slightly yellowish to

brown. During parboiling, dextrinisation and destruction of lipase enzyme can occur.

Diffusion and heat-sealing of B-vitamin and other water soluble nutrients to the endosperm

also occur thereby improving the nutritive value of rice. However, heat treatment of rice can

cause destruction of some anti-oxidants resulting in rancidity of parboiled rice during long-

term storage (Nawab & Pandya, 1974).

In a study conducted by Chukwu & Oseh in (2009),the effect on nutritional values of rice

(Oryza sativa) at different parboiling temperatures was analysed. Standard laboratory

conditions, instruments and methods of AOAC (Association of Official Analytical Chemists)

nutritional guidelines were used to obtain the proximate composition of non-parboiled rice

11

and rice that was steam-parboiled at 80, 100 and 120ºC. The results showed that parboiling

leads to variation in the nutritional contents of rice as demonstrated for protein content which

shows a decrease from 6.61% to 5.29% after the parboiling. The results obtained for vitamins

A and C also showed a decrease in values after parboiling at different temperatures of 80, 100

and 120ºC. It was concluded that parboiling rice adversely affects some of the nutritional

content of the product. The moisture content of the rice decreased with gradual increase in

temperature (Chukwu & Oseh, 2009). Parboiled rice is mainly consumed in parts of South

Asia especially, India, Bangladesh and some parts of Pakistan. About 60% of total rice

production in India is through parboiling (Pillayar, 1990).

Traditional drying of parboiled rice involves sun drying. Paddy is laid out on a cemented

floor in a 10-30 mm thick layer. It is continuously stirred with a spiked plank or by feet. After

certain duration the rice is heaped and covered by mats and this process is called tempering.

After tempering for 2-3 hours, the paddy is spread out again and dried for 2 days in sunshine

until the desirable moisture content is reached. The parboiling process incorporates 30 % of

moisture in the rice and this has to be reduced to 13% by the drying process which usually

takes 6 hours in Indian summer and 8-10 hours during the winters in India. Mechanical

dryers, bag dryers and batch dryers, continuous flow dryers, rotating drum dryers, and

modern paddy dryers are the commonly used dryers (Juliano, 1993).

2.3 High Pressure Processing(HPP)

High pressure processing (HPP) is a novel technology which involves treating a product

under a pressure of 100 MPa and above. The transmission of pressure is uniform and

instantaneous and this results in the product being homogenous as well. Conventionally it is

used for the production of ceramics, steels, superalloys and synthetic metals. This technology

gained popularity in the early eighties in biological and food systems. Hite (1899 )tested the

high pressure system for improving milk stability. But it was 70 years later that the first

pressurized jam product was marketed in Japan in 1993 (Konrr 1993; Mertens 1993).

The unit of pressure for high pressure processing is Megapascal (MPa). 1 MPa is equal to

9.869 atm which is equal to 10 bar. Pressure can alter the conformation of secondary, tertiary,

quaternary and supramolecular structures of nucleic acids, polysaccharides and proteins

(Cheftel and Cuiliolo, 1997).

12

Figure 2.2 Schematic diagram of discontinuous equipment for High Pressure Processing

of packaged food products (Knorr, 1993).

The principle of high pressure processing is isostatic transmission of pressure throughout the

food. This prevents deformation of the food products under high pressure and at the same

time the pressure is transmitted instantaneously. This allows homogenous processing

independent of the product size and shape. Another added advantage of this method is that it

takes energy to reach the desired pressure but once the pressure is reached, there is no energy

loss. These properties of high pressure give added advantages in contrast to heat processing.

Pressurization can also be done at low temperatures such as room temperature, refrigeration

and freezing temperature and by doing so the nutrient content and sensory properties can be

preserved. High pressures influence weak interaction bonding in foods and can keep the

covalent bond of primary macromolecules intact (Knorr 1993; Mertens 1993; Heremans

1995).

The most common application of high pressure processing is enhancing food safety by the

killing of microorganisms. High pressure processing can eliminate bacteria and bacterial

spores. When the pressure is between 400 to 600 MPa, vegetative cells can be killed with

temperatures ranging from 5 to 90°C for 10-30 min (Barbosa-Canovas et al., 1998).

Therefore products with good microbiological quality and greater safety and longer shelf life

can be produced. Novel products can be generated by pressurizing food ingredients and

modifying their functionality. This has an effect on the physical properties of the food and

13

induces textural changes different from usual ones. High pressure technology is relatively

new in food science and can be explored more in the nutritive, microbiological, physico-

chemical and sensory aspects.

2.4 Choice of micronutrients for fortification

2.4.1 Importance of Vitamin A

Vitamin A includes pre-formed structures (retinol) and oxidized forms of retinols (retinoic

acid), where they are needed to maintain a healthy vision (Thurnham, 2007). Vitamin A

deficiency(VAD) causes xerophthalmia (dry eyes), which eventually leads to night blindness,

increased morbidity and mortality in children (Sommer and West, 1996). India is one of the

developing countries in which VAD is very common and needs most attention (Food and

Agricultural Organisation of the United Nations, 1988). Serum retinol analysis was done for

children between 3-5 years in the year 2000 and it was found that southern Tamil Nadu

accounted for about 42% of VAD when compared with other states in India. In order to

reduce the prevalence of VAD in India, supplementation and fortification programs arenow

being carried out for young children and lactating women. However, no food fortification

programs have been implemented other than some health drinks (UNICEF, Multiple Indicator

Cluster Survey, 2000).

WHO reported that between 1987 and 1995, vitamin A deficiency increased in prevalence

from 39 countries to 60 countries based on the ocular manifestations of xerophathlmia or

deficient serum (plasma) retinol concentrations (<0.35 µmol/l) (WHO, 2009). In addition, it

indicated that night blindness and biochemical vitamin A deficiency (serum retinol

concentration <0.70 µmol/l) have affected a staggering amount of preschool children, as well

as pregnant women. Globally, night blindness is estimated to affect 5.2 million preschool

children and 9.8 million pregnant women; low serum retinol concentration (<0.70 µmol/l)

affected another 190 million preschool children as well as 19.1 million pregnant women

(WHO, 2009b). To sum up, the affected populations correspond to 33.3% of the preschool

population and 15.3% of pregnant women in populations at risk of vitamin A deficiency, i.e

in low-income countries such as Africa and South-East Asia (WHO, 2009b).

14

Vitamin A deficiency usually results from malnutrition, but can also be due to abnormalities

in intestinal absorption of retinol or carotenoids. Some of the more serious manifestations of

vitamin A deficiency include (Bates, 1995):

Blindness due to inability to synthesize adequate quantities of rhodopsin. Moderate

deficiency leads to deficits in vision under conditions of low light ("night blindness"),

while severe deficiency can result in severe dryness and opacity of the cornea

(xeropthalmia).

Increased risk of mortality from infectious diseaseswhich has been best studied in

malnourished children, but is also seen in animals. In such cases, supplementation

with vitamin A has been shown to substantially reduce mortality from diseases such

as measles and gastrointestinal infections.

Abnormal function of many epithelial cells, manifest by such diverse conditions as

dry, scaly skin, inadequate secretion from mucosal surfaces, infertility, decreased

synthesis of thyroid hormonesand elevated cerebrospinal fluid pressure due to

inadequate absorption in the meninges.

Abnormal bone growth in animals which can result in malformations and, when the

skull is affected, disorders of the central nervous system and optic nerve

2.4.2 Vitamin A in the diet

Vitamin A is a fat-soluble vitamin and an isoprenoid compound mostly obtained from animal

sources. Retinoids are a class of compounds that show vitamin A activity. They differ

structurally from all trans-retinol. Dietary vitamin A is found in the form of provitaminA in

plants. Long chain retinyl esters are mainly found in animals. Both forms of vitamin A are fat

soluble, therefore, normal fat digestion and absorption is essential. Provitamin A can be

found in various forms in nature such as α. Β, γ carotene and cryptoxanthin. β-carotene is

predominantly found in nature. About 50% of dietary carotenoids are absorbed. Low fat

intake reduces absorption of vitamin A. β –carotene is absorbed by enterocytes mainly by

passive diffusion. Vitamin E and other anti-oxidants protect β –carotene from oxidation of

double bonds. In a normal diet, about 1/6th

of the ingested β –carotene is converted to retinol.

Preformed vitamin A is present as retinyl esters in the diet. Retinyl esters are hydrolysed by

pancreatic lipase or cholesterol esterase (Rodriguez- Amaya, 1997; Booth et al., 1992 &

Butití Palm, 1975).

15

Vitamin A deficiency (VAD) is the leading cause of preventable blindness in children. It also

increases the risk of disease and death from severe infections. In pregnant women VAD

causes night blindness and may increase the risk of maternal mortality. Vitamin A deficiency

is a public health problem in more than half of all countries, especially in Africa and South-

East Asia, hitting young children the hardest and pregnant women in low-income countries

(World Health Organization, 1998).

2.4.3 Importance of Folic Acid

Folic acid (water soluble vitamin B9) is a pharmaceutical form of folate that is used for food

fortification, which is important for cell division and reduction of neural tube defects in

babies (Truswell, 2007). It cannot be synthesized by the body and therefore it needs to be

obtained from dietary sources. The major sources of folic acid are fruits, green leafy

vegetables, cow‘s liver, egg yolk, and legumes. Folic acid deficiency leads to cardiovascular

diseases, and some carcinomas such as colorectal, uterine, cervical, lung and

oesophagus.Inadequate intake of folic acid early in pregnancy, results in neural tube defects

(Oner et al., 2006). Recent studies indicate that the daily intake of folate in women in rural

areas of India and that of lower socioeconomic strata in Delhi is less than one third of the

400-micrograms of folate per day required to prevent birth defects. The data showed that the

incidence of NTD was among the highest at 6.57-8.21 per 1000 live births. Fortification of

wheat flour to overcome folic acid deficiency diseases has been implementedbut is yet to

reach the rural population. Rice would be another choice forfortification with folic acid

(Wesley & Dutta, 2009).

Folate helps produce and maintain new cells (Kamen, 1997).This is especially important

during periods of rapid cell division and growth such as infancy and pregnancy. Folate is

needed to make DNA and RNA, the building blocks of cells. It also helps prevent changes to

DNA that may lead to cancer (Fenech Aitken , Rinaldi, 1998). Both adults and children need

folate to make normal red blood cells and prevent anemia. Folate is also essential for the

metabolism of homocysteine, and helps maintain normal levels of this amino acid (Zittoun,

1993). In its naturally occurring form folate is unstable in food storage and preparation.

(Eitenmiller &Landen, 1999). On the other hand folic acid is stable and can be used a

supplement and for food fortification (Eitenmiller & Landen, 1999; O‘Broin, Temperley,

Brown , Scott, 1975; Temple and Montgomery, 1984).

http://ods.od.nih.gov/factsheets/showterm.aspx?tID=267







16

Figure 2.3 Structure of folic acid showing the pteridine ring and the para-aminobenzoic

acid (PABA) (Eitenmiller, 2008)

In the year 1965 the relationship between folic acid and neural tube defects was hypothesized

(Hibbard, Hibbardand Jeffcoate, 1965). Neural tube defects (NTDs) are an embryonic

underdevelopment of the neural tube which fails to close resulting in damage of the exposed

underlying neural tissue. There is significant morbidity and mortality depending on the

severity and the location of the lesion. Several Randomized Control trials (RCT) were

performed to explain the relationship between folic acid deficiency and NTDs. In the research

conducted by British Medical Research Council it was found that in pregnant women with a

past history of NTD the incidence of NTD recurrence was reduced by 70% by taking 400 μg

of folic acid daily (Medical Research Council (MRC) Vitamin Study Research Group, 1991).

In 1991 the Centre for Disease Control and Prevention recommended that women with a

history of past NTD-affected pregnancy should consume 4000 μg of folic acid per day

starting at the time of planning for pregnancy. However encouraging women to start

consuming supplements has limitations in terms of a public health programs since in the USA

about 50% of pregnancies are unplanned. The neural tube closes 28 days after conception

which is very early (Finer & Henshaw, 2006) and therefore women who are planning to get

pregnant should start an early dose of folic acid supplementation i.e. during the planning

stage for pregnancy. Education campaigns that encourage women to increase their use of

supplements have not been very effective in reaching the highly affected population (Ray,

Singh, & Burrows, 2004) Therefore it has been suggested that well- implemented mandatory

fortification programs might help reduce the discrepancy (Dowd & Aiello, 2008). Mandatory

fortification programs have been implemented in many countries to maximize their effect and

reduce the cost associated with prevention programs such as education campaign and other

interventions that require behavioural changes (Flour Fortification Initiative, 2010).

17

One of the ways to evaluate the effect of folate fortification programs is to measure the blood

folate levels in the target population. Folate deficiency can be defined as a serum folate

concentration of <7nmol/L (~3 ng/mL) or a red blood cell folate concentration <315 nmol/L

(~140 ng/mL) (Institute of Medicine, 1998). Homocysteine concentration can be used as a

biomarker for folate status in conjunction with blood folate concentrations (Selhub, Jacques,

Dallal, 2008). In an Irish cohort study it was found that the greatest reduction in NTDs was

observed only when blood folate concentration was much higher than the level set for folate

deficiency (Daly et al., 1995).

In Australia, it is mandatory to fortify all bread-making flour with folic acid, where 3 slices of

bread (100g) contains an average of 120 µg of folic acid (FSANZ, 2007). According to Allen

et al. (2006), folate deficiency is common in populations that consume a high intake of

refined cereals that are low in folate as well as a low intake of leafy greens and fruits that are

high in folate.

2.4.4 Importance of Iron

Apart from vitamin A deficiency, approximately 1.6 billion people are anemic worldwide

(WHO/ CDC, 2008). It is reported that iron-deficiency anemia is prevalent in Asia, where

60% of women and over 30% of men are anemic (MacPhail, 2007). Iron is important for

oxidative metabolism and cell functions (NHMRC, 2006). In children, iron- deficiency

anemia can cause impaired psychomotor development; pregnant women are likely to have

premature birth, low birth weight babies and increased perinatal mortality (MacPhail, 2007).

Unlike in Asia, the prevalence of iron-deficiency anemia among women and men in Europe

and North America is less than 5% and 2%, respectively (MacPhail, 2007). Compared to poor

populations, wealthier people have greater access to a variety of fruits and vegetables and

micronutrient-rich foods such as meat and eggs.

United Nations (2002, Vienna)estimated by that about 34% of the world‘s population suffers

from iron deficiency and 80% of this population belonged to developing countries. The

paradoxical fact about iron is that although it is one of the most abundant elements in the

planet its deficiency represents the most common nutritional problems. The reason for this is

that most of the ingested iron has low solubility thereby resulting in low bioavailability

(United Nations, 2002).

Iron is essential for the human body as it integrates many bio-molecules with various

biochemical and physiological functions. Iron deficiency affects most pregnant women. Other

18

consequences of iron deficiency are absorption of toxic metals, low resistance to infections,

and irregular maintenance of body temperature and modification of physical activity, fitness

and work capability (FAO/WHO, 1988).

Iron is present in foods in two forms namely, heme iron which is derived from flesh foods

like fish, meat etc. and non-heme iron which is the inorganic form present in plants like

cereals, pulses, legumes, grains, nuts and vegetables. Heme iron has a better absorption rate

than non-heme iron. Iron absorption can be enhanced by intake of fats and oils, vitamin A,

muscle tissue and ascorbic acid in diet and by reducing foods containing high phytic acid and

tannic acid, other polyphenols, calcium and animal proteins content (Hurrel and Egli, 2010)

(FAO/WHO, 1988). Daily requirement for iron varies based on age. For adults in the age

group of 19-51 the iron requirement is 8 mg/day for men and 18 mg/ day for women

(National Institute for Health, 2007).

2.5 Factors to be considered for food fortification

Fortification of foods with micronutrients involves the basic principle of choosing

theappropriate medium for fortification. The medium should be consumed by the target

population on a daily basis and in adequate intake. Therefore, rice was chosen for

micronutrient fortification as it is the staple food in several developing countries.

Fortification of rice with these micronutrients would improve the nutritional status of the

population suffering from micronutrient deficiency.

In order to fortify foods with nutrients with an expanding range of fortificant compounds and

technologies the need to choose the best medium for fortification becomes a practical

problem in establishing fortification programs. The following factors must be considered to

fortify foods with nutrients (FAO/WHO, 1988):

Consumed by a large majority of target population in adequate intakes

Manufactured in large scale at few centers to supervise efficiently

Nutrient must be stable under normal conditions of storage

Relatively low cost

Have a constant consumption pattern with a low risk of excess consumption

Centrally processed with minimal stratification of the fortificant

Minimal interaction between the fortificant and carrier food

Contained in most meals with availability unrelated to socio-economic status

Linked to energy intake

19

The colour of the food may not change much due to the addition of fortificants. In the case of

iron, commonly used compounds are ferrous sulphate and ferrous fumarate. In recent

research it has been suggested that sodium-iron EDTA is a better fortificant than the other

two as it has iron from the EDTA-complex that remains bioavailable even in the presence of

iron absorption inhibitors and also it does not show adverse effects on humans. The Joint

FAO/WHO Expert Committee on Food Additives (JECFA) has given sodium iron-EDTA

tentative approval for use as a fortificant. In the case of vitamin A fortification, if the

moisture content of the food is greater than about 7-8% it is known to adversely affect the

vitamin A stability. Therefore, the choice of medium for fortification should meet these

criteria (Food and Drug Administration, 1996).

2.6 Commercially available fortified rice

Fortified rice products are commercially available namely, Nutririce and Ultra Rice. The

Ultra Rice micronutrient delivery system employs a unique mechanism for incorporating

vitamin and minerals within an extruded ―rice grain‖ made from rice flour to minimize

nutrient losses. These fortified extruded grains (called ―Ultra Rice grains‖) resemble natural

milled rice in size, shape, and density. After drying, Ultra Rice grains are blended with local

rice, typically in a ratio of 1:100. When cooked, the fortified rice has the same taste, colour,

and texture as unfortified rice. Ultra Rice is fortified with iron, thiamin, zinc, and folic acid

(PATH, 2007).

NutriRice process uses broken rice kernels from the rice mill for production of rice flour and

mixing with vitamin and minerals. To resolve the problems of segregation and washing off

vitamin and minerals, the mixture is formed by extrusion into reconstituted rice kernels. The

fortified reconstituted kernels are then added to the natural whole rice kernels. NutriRice is

fortified with vitamin B1, Vitamin B2, Vitamin B6, Iron, Zinc and Folate. The above two

mentioned fortified products use extrusion as a means of fortification (Global Alliance for

Improved Nutrition GAIN, 2009).

Table 6 gives a comprehensive summary of the existing rice fortification techniques

including the above mentioned. It was concluded that the hot extrusion technique was the

best for premixes excluding the fact that it confers the rice with a strong colour. Rice

premixes produced by cold extrusion technique can be utilized by people who are less

concerned about the rice whiteness and therefore, this rice was successful in Costa Rica. The

disadvantage of the coating technique is that the fortificants give colour to the grain making it

20

less acceptable. Moreover, in The Philippines, the adhesion of fortificant layer on to the rice

in the locally developed method was very weak and therefore leading to heavy loss during

washing and cooking (USAID, 2008).

The dusting technology which is claimed to be the easiest method resulted in a strong

vitaminodour because of the high concentration of micronutrients in the premix. However

after diluting it with the retail rice in the ratio of 1:1600, the odour was not noticeable

(USAID, 2008).

21

Table 2.2 Comparison of Programs and Quality of rice- and Micronutrient-Premixes produced by two different existing rice

fortification technologies (USAID, 2008)

Extrusion and Dusting Technology Coating Technology

Company

(Country)

Type of

Program

Description of Premix Company

(Country)

Type of Program Description of Premix

DSM/ Buhler

& COFCO

(China)

Aimed at

high-end

market

consumers.

White and stable.

A product with β-

carotene is yellowish

and unstable after 3

months at 30°C.

Wright

(USA for the

Philippines)

Aimed at social-

programs under

government

administration.

Product with a strong golden dark

color and off-taste, which is very

easily distinguished from unfortified

kernels. Losses have been reported

after rinse-wash preparation for

cooking.

Superlative

Snacks

(Philippines)

Aimed at

high-end

market

consumers.

Premix has an off

white colour that is

distinguishable upon

close inspection.

CLG-Health

Food

Products

(Philippines)

Responding to

national regulation,

but indeed with a

very small

coverage.

The premix has a grayish color with

dust and strong off-taste. It is

distinguishable from non-fortified

rice. Stability has not been measured,

but losses during the rinse-wash

preparation for cooking might be

large.

22

Vigui- with

DSM support

(Costa Rica)

National

program

mandate by

government

Premix is a rice-shaped kernel of

yellowish colour with no

apparent odour that becomes soft

in contact with water. It is

possible to differentiate from the

non-fortified rice based on

colour. Claims that losses during

washing and cooking are

minimal.

Group NTQ

and Kuruba

with

Fortitech

support

(Costa Rica)

National program

mandate by

government

Slightly yellowish in colour with

some vitamin-like odour. It is

possible to differentiate the rice-

premix from the non-fortified rice

based on colour. Stability of the

micronutrient layer over the grain

surface is attained through a 5-layer

coating process. Losses during

washing have been claimed to be less

than 5%.

Wright &

RPC

(USA)

Required if

claim of

enrichment

is done.

Compulsory

in some

states.

White powder with strong

vitaminodour. It has the

common characteristics of

micronutrient premixes for

cereal fortification. It can

segregate from the rice grain.

Rinse-washing for cooking will

remove the micronutrient premix

Wright &

RPC

(USA)

Required if claim

of enrichment is

done. Compulsory

in some states.

It is claimed that there is no product

odour or colour change if the coating

process is done correctly. Claims

shelf life is 2 years, but storage time

is typically 3 months.

23

2.7 Mandatory Food Fortification Programs (Folic Acid)

Mandatory fortification of wheat flour with folic acid is currently in place in 53 countries

although in many cases regulations are not in place (Centre for Disease Control and

prevention, 2010). Maximum fortification level and the Legal Minimum Level of folic acid to

be used to fortify flour have been established (Allen, de Benoist, Dary, Hurrell, 2006),

Table 2.3 The Levels of folic acid fortification in countries with mandatory fortification

programs

Country Fortification Level Year of Implementation

United States (Food and

Drug Administration, 1996) 140 μg/100g 1998

Canada (Canada Gazette

Food and Drug Regulations,

2008)

150μg/100g 1998

Costa Rica (Chen & Rivera,

2004) 180 μg/100g 1998

Chile (Hertrampf & Cortes,

2004) 220 μg/100g 2000

South Africa (Sayed, Bourne,

Pattionson, Nixon,

Henderson, 2008)

150 μg/100g 2003

2.7.1 Mandatory Fortification in Australia & New Zealand

FSANZ (2007) recommendedthat Australia and New Zealand implement mandatory

programs to fortify bread with folic acid. Australia implemented the mandatory fortification

in September 2009. Cereals and cereal products require that all wheat flour for making bread,

with the exception of flour represented as organic, must be fortified with folic acid from 13

September 2009. The level of fortification required is between 2 and 3 milligrams of folic

24

acid per kilogram of wheat flour for making bread. Folic acid is the only permitted form of

folate that can be used to meet these requirements.

2.8 Novelty in Rice Fortification

2.8.1 Parboiling Process

From the results obtained from the study by Prom-u-thai (2008), it was stated that iron

fortified parboiled rice provided a preliminary basis for further investigation of this

innovative technique. This was further continued by their research group (2009) which

inferred that parboiled rice had a great potential for zinc fortification. Iodine, another mineral

was also successfully fortified in rice by the parboiling method (Tulyathan, 2007). This led to

the possibility of fortifying rice using this technique with vitamin.

Extensive research on rice fortification has been conducted in UNSW (de Ambrosis et al.,

2004, Mulia 2010 and Gunawan 2010 and Kam et al.,2012). Parboiling model for

fortification of rice with folic acid using different times and temperatures have been

extensively studied by Kam et al., 2013 and also the physical and chemical characteristics,

consumer acceptance of the fortified rice. Fortification of β-carotene and iron in rice was

studied by Gunawan and Mulia, respectively (2010). From the above studies it has been

found that the optimum condition for the maximum concentration of each nutrient

individually was found to be 70 °C soaking temperature for 2 hours followed by steaming at

100°C for an hour and shade drying until the moisture content dropped to 14% c.a.

Kam, Arcot and Adesina (2012) have reported on folic acid fortification using parboiling at

70°C for 1, 2 and 3 h using four different concentrations of folic acid. The dried parboiled

rice was milled at 3 different durations i.e. 0s, 60s and 120s. A multifactorial model was

developed to describe the residual folate concentration and it was found that soaking and

milling were significant factors for folic acid fortification. The optimum soaking time was

found to be 1.97 hours and folate uptake rate followed 1st order kinetics. The rate of natural

rice hydrolysis and folate uptake by rice was both time-dependent (Kam, Arcot & Adesina,

2011). From the above literature resources, feasibility of fortifying rice with three

micronutrients was therefore explored through the present study.

25

2.8.2 High pressure processing

High pressure processing (HPP) technology is a novel food processing technique. This novel

technique could be a potential method in delivering fortified rice. Being a novel techniqueit

has not been applied to food fortification. HPP is used in food processing. It involves

application of hydrostatic pressure which can cause gelatinization of egg yolk and white by

the application of 1000-7000 atm pressure at 25-30°C (Yaldagard, Mortazavi, Tabatabaie ,

2008). The application of HPP is independent of sample size and geometry. HPP is

commonly used in food industries for(Knorr, 1993):

1. Inactivation of microorganisms and enzymes

2. Modification of biopolymers

3. Quality retention, such as colour and flavour

4. Changes in product functionality

HPP causes minimal changes to fresh foods as the food is not subjected to thermal

degradation. HPP processed foods have a fresher taste, better appearance, texture and

nutrition. It can be used at high as well as low pressures and it is beneficial for heat sensitive

products(Knorr, 1993).

In a typical HPP process, the product is packaged in a flexible container such as a pouch or

plastic bottle and is loaded onto a high pressure chamber filled with a pressure-transmitting

hydraulic fluid. The hydraulic fluid in the chamber is usually water which is pressurized with

a pump, and this pressure is transmitted through the package into the food itself. Pressure is

applied for a specific time, usually 3 to 5 minutes. The processed product is then removed

and stored/distributed in the conventional manner. Because the pressure is transmitted

uniformly, food retains its shape, even at extreme pressures. And because no heat is needed,

the sensory characteristics of the food are retained without compromising microbial safety

(Ramaswamy, 2006).

Being a novel technique, the usefulness is yet to be explored in detail. However, few

researchers in Japan have tried to treat rice with HPP technique and analysed the property of

rice. Huang, Jao, Hsu, in 2009 analysed the combined effect of pressure (200-500 MPa) and

temperature (20, 40 and 50˚C) on the water uptake and gelatinization of japonica rice variety.

When the pressure was greater than 200 MPa, at all temperatures the moisture content and

volume of grains increased. The highest degree of gelatinization was observed to be 73% at

500 MPa at 50˚C for 120 min. Gelatinization did not occur at pressures below 300 MPa and

26

temperature of 20 or 40˚C. Therefore, it can be said that at higher temperature and pressure

gelatinization of rice occurs using HPP equipment (Huang, Jao, Hsu, 2009).

Yamakura et al., (2005)studied theeffect ofsoaking followed by high pressure treatment on

the cooked quality of rice. In this study, milled rice grains which were pre-soaked in water at

25-550C for 30 min was subjected to HPP treatment at 400 MPa for 10 min and then soaked

in water overnight. The physical and chemical properties of the treated rice were then

analysed. The viscosity of milled grains increased with the soaking process. Total sugar

increased and in internal structure of rice grains changed after HPP treatmentin soaked rice.

The structural change of rice seemed to promote water penetration and brought about higher

degree of swelling in rice granules. This might result in high degree of gelatinization and

higher digestibility of HPP-treated cooked rice. HPP treatment of rice resulted in denaturation

of water soluble proteins and increased free amino acids. Therefore, HPP treated rice proved

to be an advantageous method for cooked rice with better palatability (Yamakura et al.,

2005). Based on literature collected so far in terms of treating rice using HPP fortification of

rice would be novel to explore.

2.8.3 Cost Analysis for parboiling and HPP processes

In a study conducted by Roy, Shimizu, Shiina and Kimura (2005), to evaluate the local

parboiling processes, cost and energy analysis was done. In the eastern part of India (West

Bengal) and Bangladesh, the production of parboiled rice is the main income of many people

in the local area. The commonly used parboiling processes are vessel, small-boiler and

medium-boiler. The major source of energy for this process is biomass and this was

contributed by rice husk. The following paragraph will explain about the common local

parboiling processes and their respective cost and energy analysis.

The local parboiling process is presented in the flow chart in Figure 2.4. The vessel process is

a direct heat method of parboiling at a household level to produce parboiled rice for

consumption. This is the major source of income for these families on a daily basis. The

capacity of the vessel varies from 0.5 to 1.2 tonnes/batch (t/batch) and this varies with the

business capital (Roy et al., 2005).

27

On a commercial scale, a small-boiler process is used. For this process, two conical hoppers

made of aluminium sheets are used for pre-steaming and steaming so that the steam

generation would be continuous and simultaneously applied to the paddy. For the drying

process the home yard itself is used as a drying yard and if the place is too small another

place could also be used. The capacity for this batch processing varied from 2-4 t/batch which

depends on the business capital and on the drying yard area (Roy et al., 2005). .

In the case of a medium-boiler process the purpose is completely dedicated to

commercialization. Two conical hoppers are used as a set for pre-steaming and steaming. The

larger set is used for pre-steaming and the smaller one is used for the steaming process itself.

The capacity of this process varies from 5-10 t/batch (Roy et al., 2005).

Figure 2.4 Flow chart of local parboiling process in Bangladesh and West Bengal, India.

(Roy et al., 2005)

The energy consumption for each of the local parboiling processes described above has been

presented in the Table 2.4. It was observed that the medium-boiler required the lowest energy

and the small-boiler had the highest requirement. However, the energy consumption was

found to be lower than the boilers parboiling process which required 4200 MJ/t where hot

28

soaking was done to the paddy and this is considered to be a modern method (Tiwary & Ojha,

1981). On a lab-scale traditional parboiling process the energy requirement varied from 1400-

2442 MJ/t depending upon the treatment times (Roy et al., 2003a). As it can been seen from

Table (2.4), small-boiler process is a more energy intensive process compared to the vessel

and medium boiler. This was attributed to the usage of two-barrel boiler which caused loss of

heat energy through gaps between the barrels during parboiling and also to the boiler

installation and lower quantity of paddy parboiled and limited capacity of the drying yard.

Therefore, it can be suggested that to reduce the cost of energy a large scale boiler could be

used.

Table 2.4 Energy consumption in parboiling processes (Roy et al., 2005)

Process Biomass consumption, kg/t Total Energy consumption,

MJ/t

Pre-steaming Steaming

Vessel 71.9 113.7 2583

Small- boiler 87.2 109.6 2758

Medium- boiler 43.1 75.1 1659

Table 2.5 Installation costs of the parboiling process (US$) (Roy et al., 2005)

Process Vessel or boiler Hopper Soaking

tank

Tube-well Initial

investment

Cost/unit Used

Units

Life

year

Cost/unit Units

used

Life

year

Cost Life

year

Cost Life

year

Vessel 5.32 2 0.5 42.55 10 21.28 10 397.47

Small-

boiler

42.55 1 1.0 42.55 2 10 148.94 10 21.28 10 1281.87

Medium-

boiler

1170.21 1 1.0 63.83 4 10 638.29 10 212.77 10 5101.09

29

In comparison to the vessel process, boiler process needs a higher installation and investment

cost (Table 2.5). Table 2.6 shows the theoretical fortification formulation from which the

choice of micronutrients relevant to the present study has been presented. It is recommended

that the fortificant should be mixed in the ratio of 1:5000 with the unfortified white rice. If

the fortificants were to be added in the given concentrations, it is suggested that the formula

needs an addition of about 110 g of fortificant mix per metric ton of fortified rice and the

major weight comes from iron. The fortificant mix as per this table costs approximately US$

9 which means that each metric tonne of rice would contain an equivalent of US$ 1.8 of

fortificant mix regardless of the delivery method being a premix or micronutrients mix. From

the table, it is evident that iron is the most expensive fortificant ($1.04) followed by vitamin

A ($ 0.53) and folic acid ($ 0.22).

Table 2.6 Theoretical Fortification Formulation for Comparison Purposes (USAID,

2008)

Micronutrients

(Fortificants)

Micronutrient

content added

to rice

(mg/kg)

Fortificants

to be added

in the

fortified

rice 1(g/MT)

Cost of

fortificants

in the

fortified

rice 2(US$/MT)

Fortificants

in 1

kg

fortificant

mix

(g)

Fortificant

cost in

the

fortificant

mix

(US$/kg)

Vitamin A (Dry

form- 250,000

IU/g)

1.0 13.3 0.53 67 2.68

Folic Acid 1.0 1.1 0.22 6 1.10

Iron

(Micronized

Ferric

Pyrophosphate)

24 96 1.04 484 5.21

Total - 110.4 $1.79 557 $8.99

Note: 1Fortificants are the micronutrient source. These values were calculated dividing the

micronutrient level by the proportion of the micronutrient in the fortificant.

2Using the usual prices in the international market in 2007.

Existing fortification models were compared using a theoretical fortification formula to

understand cost effectiveness of these models. These models were obtained from

DSM/Buhler and COFCO in China (hot extrusion), Vigui in Costa Rica (cold extrusion),

Group NTQ in Costa Rica (coating), and Wright or RPC in the USA (dusting). The

production capacities and therefore the capital costs were different but the capital cost of

30

premix production was found to be in decreasing order of investment i.e. hot extrusion, cold

extrusion, coating, and dusting.

Table 2.7 Comparison of the technologies for premixes production by extrusion, coating

and dusting3 (USAID, 2008)

Technology Dose on rice

(kg/MT)

Annual

Production

(MT)

Capital Cost

(US$)

Recurrent

fortification

costs (US$-

thousands

per year)

Premix

cost

(US$/kg)

Hot Extrusion 4

10 1,500 3,880,000 1724 $ 1.15

Cold

Extrusion 5

10 730 770,000 762 $ 1.05

Coating 6 10 430 300,000 389 $ 0.90

Dusting 7 1 2,500 100,000 11509 $ 4.60

3 This formulation assumes that 10 kilograms of rice-premix for the coating or extrusion

technologies, or one kilogram of micronutrient premix for the dusting technology, are added

to one metric ton of retail unfortified rice. The dilution rates are 1:100 and 1:1000,

respectively. In both cases, the final dilution rate of the fortificants in the rice is 1:5000,

because ~200 grams of the combined fortificants are needed to achieve the proposed

micronutrient levels as described in Table 2.6. The rice-premix is a dilution 1:50 of the

fortificant mix (5000/100 = 50); and the micronutrient mix is a dilution of 1:5 of the same

fortificant mix (5000/1000 = 5). Price of the fortificant mix is assumed as US$15/kg. Price of

broken rice is assumed as US$0.30/kg, and whole rice as US$0.50/kg. The coated technology

uses half broken rice and half whole rice.

4 Using the model of DSM/Buhler and COFCO in China.

5 Using the model of Vigui in Costa Rica.

6 Using the model of the Group NTQ in Costa Rica

7 Using the model of Wright or RPC in USA assuming that process costs are equivalent to

US$1/kg micronutrient premix.

31

Table 2.8 Table Capital and production cost of thermal and pressure processes

(Sampedro, 2014)

Capital cost

Process parameters Unit of measure Thermal HPP

Heat exchanger $ 18,000 2,000

HPP equipment $ - 2,495,000

Process pumps $ 12,000 12,000

Total equipment

cost

$ 66,000 2,545,000

Total Capital cost $ 132,000 5,090,000

Production cost

Total electricity cost $ 3,000 70,000

Total energy cost $/year 8,000 70,000

Compared to thermal processing high pressure processesing is a novel technology. Therefore

it is likely that the capital cost would decrease in the future when they are widely used in food

industries. From the above table it can be seen that the major costs are driven by energy

consumption, and capital cost. The overall cost for HPP is higher than parboiling in terms of

capital and production. Since parboiling is more established than HPP it is likely that the

former method is more economically feasible than HPP.

2.9 Study of Morphological and Physico-chemical properties of Rice

2.9.1 Pasting Properties Using RVA

Rice is classified according to the grain dimensions, amylose content and amylograph

viscosities of rice, degree of gelatinization properties of extracted starches and texture of

cooked rice. Based on these properties rice can be classified into waxy and non-waxy types.

Reddy and co-workers in 1993 proposed that textural changes in cooked rice are attributed to

the fine structure of amylopectin. Starch retrogradation occurs at 40C storage (Lu et al.,

1997; Mohamed et al., 2006) which leads to harder texture and is not desirable (Kadan et al.,

2001; Yu et al., 2010). During starch retrogradation the starch polymer realigns in a more

orderly fashion and creates a more crystalline structure thereby making it less susceptible to

enzymatic breakdown (Bjӧrck, 1996; Holm et al., 1988; Ching et al., 2006). During the

32

gelatinization process the inter- and intra-molecular hydrogen bonds between starch granules

are disrupted and hence retrograded starch has lower GI value (Erlingnen et al., 1994;

Fredriksson et al., 2000; Chung et al., 2006).

Starch is characterized often by RVA for pasting properties, DSC for gelatinization, XRD for

changes in crystalline states. Starches that have undergone ANN and HMT exhibit higher

pasting temperature and stability but lower pasting viscosity (Knutson 1990; Gunaratane and

Hoover 2002; Singh et al., 2005; Vermeylen et al., 2006). In nature, A-type crystals are

present in cereals and legumes. B-type crystals occur natively in tubers. The variation in the

crystal type is attributed to the water content and the alignment of the water molecules are

packed in the crystals (Imberty et al., 1991).

2.9.2 Polarized light microscope

A starch is made up of two types of glucose chains: amylose and amylopectin and they

constitute the main energy storage bodies in plants (Torrence and Barton, 2006). In

archaeology starch is mainly identified by cross-polarized light microscopy which has 200-

400 times magnification. This technique allows real time examination of starch in plant

microfossils (Piperno, 2006). Starch is a very fragile organic material and is very soft. Due to

this property when it undergoes several changes during pre and post-harvest and also during

processing such as milling, grinding, cooking, parching and drying they are prone to damage

(McGee, 1984). Very few studies have been conducted to see the physical effects of cooking

on starch grain alteration that are visible under light microscope (Lamb and Loy, 2005;

Samuel, 2000; del Pilar Babot, 2003).

33

Figure 2.5 Model of amylose molecule with 1000 glucose units (a) and Model of

amylopectin molecule with 1000 glucose units in 30 branches (b) (Nikuni, 1957)

Rice starch grains are small, subangular, faceted and compound. The hilum is centric and the

Maltese crosses are radially symmetric but faint. The size of individual grains range from 3-

10 µm. Light microscope was used to see changes in starch microstructure on baking and

staling of wheat bread by Hug- Iten, Handschin, Conde-Petit and Escher(1999). When

observed under polarized light microscope, starch granules show a Maltese cross. When the

filter of the polarized light is adjusted to an additional λ/4, positive birefringence of starch

granules was observed which theoretically indicates a radial orientation of the principal axis

of the crystallites (Gallant, 1974; Gallant et al., 1992). Native starch is about 15-48%

crystalline and therefore it is not the primary sequence of starch polymers. Starch crystals are

made up of semi-crystalline and crystalline shells which are between 120 to 400 nm thick at

the lowest level of structure. According to Kassensbecj, (1975); Oostergetel & van Bruggen,

(1989); Jenkins et al., (1993) the crystalline shells in starch consists of amorphous and

crystalline lamellae that alternates with each other as a sequence. Robin et al (1974) and

French (1984) stated that the side chain clusters represent the crystalline regions whilst the

branched regions represented amorphous regions of amylopectin molecules. Amylopectin

b a

34

forms the side chain clusters (80-90%) and the inter-cluster connections form the remaining

10-20%

Effect of high pressure processing (600 MPa) on starch and the susceptibility to amylase was

studied by various researchers. High pressure was applied to induce gelatinization in cereals

such as wheat and pulse such as smooth pea (Muhr et al., 1982). The study found that upon

applying 400-500 MPa at 45-50°C on wheat and corn starch showed high susceptibility to

amylase treatment, however potato starch was resistant to high pressure treatment and

therefore only showed slight susceptibility (Hayashi and Hayashida, 1989). Changes in

structure of cassava starch after heating with water was observed using scanning and

transmission electron microscopy (Gracia, Colonna, Bouchet, Gallant, 2006). Effect of high

pressure processing on crystalline structure of corn and potato starch was studied by Hibi et

al., (1993). There is limited information on the changes of rice starch crystallinity after HPP.

Therefore in the current study this gap was tried to be addressed.

Figure 2.6 Microscopic images of corn starch granules under normal light (A) and

polarized light (B); where I is untreated and II is pressure treated at 500 MPa for 20

min (Hibi et al., 1993)

A B

35

Figure 2.7 Microscopic images of potato starch granules under normal light (A) and

under polarised light (B) where I is untreated and II is pressure-treated at 500 MPa for

20 min (Hibi et al., 1993)

2.9.3 X-Ray Diffraction

2.9.3.1 Diffraction of X-Rays

When X-Radiation passes through a sample matter, the electric vector of the radiation

interacts with electrons in the atoms of matter to produce scattering. When X-rays are

scattered by the order of crystals in the sample, constructive and destructive interference

occurs among the scattered rays because the distances between the scattering centres are of

the same order of magnitude as the wavelength of irradiation. As a result of this the

phenomenon of diffraction occurs (Skoog et al., 1980).

2.9.3.2 Bragg’s Law

When an incident beam of X-ray strikes on a surface of a crystal at an angle say θ a part of

the beam is scattered by the layer of atoms at the surface. The other part that is unscattered

penetrates to the second layer where another fraction is scattered and the rest is passed

through to the third layer and so-on. X-ray diffraction is based on the spacing between layers

of atoms which should be uniform and same as the wavelength of radiation and the scattering

centres must be spatially distributed in a highly regular way (Skoog et al., 1980).

A B

Typical

Maltese

corss pattern

36

Figure 2.8 Diffraction of X-rays by crystal (Skoog and Douglas, 1980)

Bragg in 1912 treated crystals with X-rays as shown in the figure above. A narrow beam of

radiation strikes the crystal surface at an angle θ; scattering occurs as a result of interaction of

radiation located at A and C. Therefore it can be said that nλ= BC+CD where n is an integer.

However, BC=CD= d sin θ where d is the interplanar distance of the crystal. Due to

constructive interference of the beam, at angle θ nλ= 2d sin θ which is the fundamentally

important Bragg equation. X-rays appear to be reflected from the crystal only if the angle of

incidence satisfies the following condition (Skoog et al., 1980).

sin θ = nλ/2d.

At all other angles, destructive interference occurs.

2.9.3.3 X-ray Diffraction in rice

Parboiling process imparts vital changes to the rice components. Starch gelatinization which

takes place during the heating step in parboiling affects the organoleptic properties of cooked

parboiled rice. Innate starch is generally in a semi-crystalline structure. With the aid of wide-

angle X-ray scattering (WAXS) it was deduced by Imberty & Pérez (1988) that rice is

arranged on a monoclinic crystal lattice (A-type). The structure of the A-type crystal is

affected depending on the heat and moisture treatment during the parboiling process.

(Biliaderis et al., 1993 & Priestly, 1976). According to Bhattacharya (1985), parboiled rice

should have limited presence of white ―bellies‖ or residual A-type crystals in the centre of the

rice kernels. During the drying stage of the parboiling process, retrogradation of the starch

occurs resulting in the formation of A-type or more hydrated B-type crystalline units.

The second change that occurs during the heating step of parboiling process is the

complexation between the lipid and the amylose molecules (Priestly, 1976). The firmer

37

texture of the parboiled rice compared to the normal white rice could be attributed to the level

of crystalline amylase-lipids complexes formed during this stage. This complex is observed

to be stable in the cooking process (Biliaderis et al., 1993; Ong and Blanshard, 1995 &

Priestly, 1977).

The interaction and formation of disulphide bonds between rice proteins has also been

reported during the parboiling process which can be stated as the third change that is also

partly responsible for the changes in cooking properties (Derycke et al., 2005). Apart from

the above mentioned, Pillayar & Mohandoss (1981) reported that the cooling and drying

process and the changes in the rice kernels associated with the process have significant

impact on the properties of the cooked parboiled rice.

Therefore, in this study the variation in the rice crystal changes due to the parboiling process

and HPP was analysed using XRD as the two processes have different effects on the rice

starch. Since the micronutrients adherence was confined to the starchy endosperm of the rice

and the outer layers of the rice are milled away, this study was performed to understand the

variation in the concentrationof nutrients and their interaction with the rice starch.

2.9.4 Magnetic Resonance Imaging

Parboiled rice absorbs high water and therefore leads to lower number of broken grains and

increases the milling yield. This is an essential parameter ofquality for rice grain postharvest

(Miah et al., 2002). When the moisture in the grain is low it results in poor quality after

processing such as cooking. Therefore moisture is a vital quality parameter in rice (Yanase

and Ohtsubo, 1986). Starch is the main component of the rice grain and therefore the degree

of starch gelatinisation brought about by hydration and heating is directly related to the

cooked rice texture (Maruyama and Sakamoto, 1992).

Moisture content of whole grains is usually measured gravimetrically which directly

correlates with the quality of milled and cooked rice. However along with moisture content,

moisture distribution is also essential as the rice grain by itself is inhomogeneous therefore

the distribution of water and diffusion into the grain is also inhomogeneous (Horigane et al.,

2006). Horigane et al. in 2001 measured moisture distribution in cooked spaghetti samples

using magnetic resonance imaging (MRI) and high resolution three-dimensional magnetic

resonance (3D-MR).

38

In Japan an essential preliminary step in cooking rice is to soak rice in excess water which is

a factor in determining cooking quality of rice (Koide et al., 2001 Okuno and Adachi, 1992).

According to Seki and Kainuma (1982), insufficient water penetration into rice grain during

the soaking step results in a hard textured cooked grain as the starch does not fully swell and

gelatinise. For biomaterials such as foods, 3D- MRI is generally performed using pulse

sequence of spin echo (SE) or gradient echo (GE) or single-point imaging (SPI). The most

common method used however is SE which can produce high quality image with good signal

to noise ratios and high spatial resolutions (Callaghan, 1991). SPI is a new imaging technique

that can be used for foods with low moisture content and semi-solid samples (Balcolm et al.,

1996). Jenner et al., (1988) was the first to conduct kinetic study of water movement into

wheat grains using MRI during the intermediate stage of development which had a moisture

content of 62-68%. The total duration of soaking was 1.6 h.

MRI has been used for several cereals such as barley endosperm during storage (Gruwel et

al., 2002) and steeping (Molina-cano et al., 2002) and wheat grain was studied by Song et al

(1998). Development of barley and rice grain was studied by Glidewell (2006) and Horigane

et al. (2001) respectively. Since rice has a moisture content of 13-35%, a fast imaging

technique with high sensitivity for water proton signals is vital. With the aid of high-spatial-

resolution 3D-MR images, water penetration can be observed from all directions. With low

moisture foods, MR images did not show moisture distribution successfully and thus it

required longer measurement time. Real time rice imaging during cooking was achieved by

turbo-spin technique by Mohoric et al(2004) which is a fast imaging technique. However this

was not possible again at low moisture content. Samples with low moisture content have very

short spin relaxation times (T2) of water protons and the echo times (Te) should also be

shortened during measurement (Song et al., 1998).

2.10 Consumer Acceptance Study

Consumer acceptance is the ultimate measure of end-quality of a newly developed product in

terms of preferences based on which the product‘s market is driven. This complex

terminology of consumer acceptance encloses several elements such as sensory descriptors,

packages, food appearance, food labels and health claims and consumer‘s experiences and

knowledge towards the product (Heinemann et al., 2006). It is always a challenge when

value-added foods and functional foods are marketed when it comes to consumer acceptance

as they may not be welcomed by customers (Siro et al., 2008). This scenario can be

explained with an example of Danish consumers who were sceptical about functional food as

39

the food was perceived to be unnatural and the consumers were anxious about the taste of the

product (Poulson, 2009). This scenario was an exemption in the United States where several

fortification programs have been successfully implemented and adopted since 1924

(Backstrand, 2002).

Parboiled rice that was a prototype was evaluated for consumer acceptance against three local

samples and one imported sample by Tomlins et al., 2007. The panel was asked to assess the

appearance of the rice. The end results suggested that the consumers were against the local

parboiled rice which was described as brown, black heads. This shows that appearance is an

important factor for consumer acceptance. The colour of cooked rice is a negative factor for

consumer acceptance (Tomlins et al., 2005). Overall, texture (Tomlins et al., 2005), grain

size and uniformity (Heinemann et al., 2006) are vital factors for consumer acceptance of

parboiled rice.

Apart from appearance, taste is also a major influencing factor for consumer acceptance

(Food Insight, 2010). Folic acid fortified parboiled rice was subjected to consumer evaluation

by Kam et al., (2012) and it was concluded that in terms of visual acceptance an informed

health claim of the fortified rice enhanced consumer‘s purchase intent of the fortified rice.

After cooking and tasting the rice, it was reported that more than 50% of the consumers were

willing to purchase the fortified rice that was mixed with unfortified rice and cooked. This

shows evidence for the acceptance of fortified parboiled rice by consumers. Rice in general

does not create a strong sensory stimulus in terms of tasting (Heinemann et al., 2006).

Although it is described as bland, taste still plays an important role in consumer acceptance

(Diako et al., 2010).

2.11 Bioavailability Studies using Caco-2 cells (in vitro)

2.11.1 Introduction to Cell Culture

The cost of performing animal and human trials for micronutrient bioavailability has shown

limited progress over the past few decades (vanCampen & Glahn, 1999). Therefore an

invitro model which utilises intestinal epithelial cells that can mimic gastric and intestinal

digestion would be able to fill the gap in this area (Glahn et al., 1996-2000). This model

which makes use of caco-2 cells has been approved for bio-accessibility studies at a

conference held by the Office of Dietary Supplements, the National Institute of Health and

the American Society of Nutritional Science (Wood and Tamura, 2001).

40

Cell culture has recently been used extensively as an in vitro method to assess human

bioavailability. In vivo studies have the drawback of dealing with complex systems in which

it is difficult to determine the relative importance of different factors. Therefore, in vitro cell

lines can overcome this problem by identifying an appropriate cell line that can suit the

purpose of our study. Caco-2 cells which are human adenocarcinoma cells have the ability to

differentiate into enterocyte of the microvilli with similar morphological and biochemical

characteristics. These cells can differentiate spontaneously and form polarised monolayers

with well-formed brush border and also produce the associated enzymes. Therefore, these cell

lines could be an appropriate model for the study of transport mechanism related to intestinal

barrier and can be used to study the absorption of nutrients (Pinto et al. 1982 & Ismail M.,

1999)

2.11.2 Previous studies using Caco-2 cells

Several studies have been conducted and reported using Caco-2 cells for analysing uptake,

bioavailability and bio-accessibility of nutrients in various foods. In a study conducted by

Fleshman et al in 2011 Caco-2 cells were used to determine β-carotene bio-

accessibility/bioavailability in stored honeydew and musk melon tissues which were grown

under same conditions. Musk melons and honeydew are good sources of β-carotene.

However, its bio-accessibility/bioavailability was unknown and therefore Caco-2 cells were

used to deduce this. It was found that in orange-fleshed honeydew melons the bio-

accessibility of β-carotene was 3.2±0.3%, bioavailability using Caco-2 cells was found to be

11%.

In another study conducted by Alminger et al.(2012) Caco-2 cells were used to determine the

bioavailability of lycopene and β-carotene in differentially processed soups. Since human

studies are labour intensive and due to the limitations in the number of samples,in vitro

models were sought as a replacement for human study. In this paper in vitro data from 2

humans were compared to results from in vitro bio-accessibility studies. The food sample

used was differentially processed soup which contained carrots, tomatoes and broccoli.

Caco-2 cell uptake model was used by Thakkar, Dixon, Dixon & Failla (2007) for

understanding β-carotene micellarization and β-carotene uptake in different cultivars of

cassava. Cassava is a staple food in Africa where the annual consumption exceeds 80 kg per

capita (Aerni, 2006).

41

Several lines of evidence indicate higher bioavailability of added folic acid than naturally

occurring folates in many foods. As stated previously, the study by Cuskelly et al. (1996)

indicates similar bioavailability of folic acid in fortified foods and dietary supplements. In

addition, labeled folate added to various cereal-grain foods also exhibited bioavailability

similar to that of folic acid in aqueous solution (Pfeiffer et al. 1997), and folate in fortified

breakfast cereal has been shown to be effective in raising folate status in humans (Malinow et

al. 1998).

2.11.3 Bioavailability of Iron in Parboiled Fortified Rice

Prom-u-Thai.,Huang ., Fukai. and Rekasem (2008) fortified rice with iron and found that it

increased the iron content in rice by 10 to 50 fold depending on grain properties among

different rice varieties. The retention rate of Fe in white rice ranged from > 50% to nearly

100% despite rinsing rice with water several times before cooking.

Figure 2.9 Iron retention rate in rice (as % of the un-rinsed) after rinsing (simulating

rice washing) in the Fe-fortified parboiled rice grains milled for 60 and 120 s,

respectively, in the 3 rice cultivars tested (Prom-u-thai et al. 2008)

The bioavailability of the fortified rice was also analysed by Prom-u-Thai , Galhn, Cheng ,

Fukai , Rerkasem and Huang in 2008 by in vitro studies using Caco-2 cells. The

bioavailability of Fe-fortified rice increased with the increase in Fe concentration. The

42

uptakes of the fortified Fe in parboiled rice milled for 120 s was well above those of the

unfortified raw or parboiled rice particularly from the high Fe rice line IR68144-2B-3-2-2

and popular Jasmine rice cultivar KDML 105. Fe bioavailability decreased with increase in

milling time and rinsing of Fe-fortified rice in water due to their negative effects on total Fe

concentrations in the parboiled rice grains, but uptakes were still well above that of their

unfortified raw or parboiled rice grains. Rinsing or washing the Fe-fortified and milled rice

grains decreased the bioavailability to 85 ng ferritin mg protein_1

in the YRF cultivar,

compared to about 100 ng ferritin mg protein_1

in its non-rinsed grains. Dilute acid-

extractable (DAE) Fe was linearly and positively correlated with the uptake of Fe assessed by

the in vitro digestion/Caco-2 cell, which can be used as a rapid method for optimising levels

of bioavailable Fe to be fortified in the parboiled rice by parboiled-rice mills if this Fe-

fortification technique should be adopted in south and southeast (Prom-u-Thai et al., 2009).

Another advantage of using caco-2 model system for iron bioavailability is the formation of

ferritin a molecule that stores iron at an intracellular level and this is used a direct measure of

iron uptake (Glahn et al., 1998). Radioimmunoassay can be used to measure ferritin and

therefore this eliminates the need to radio label iron in the food system. Ferritin, a protein is

controls the levels of iron available in the body. It does so by storing and releasing iron from

it in a controlled fashion and thereby able to maintain a buffering system against iron

deficiency. When the levels of iron in the blood is too low ferritin releases more iron and if

the body experiences iron overload ferritin can store the surplus iron (Glanh et al., 2008).

The structure of ferritin is such that it has a hollow sphere where iron can be stored in the Fe

(III) oxidation state. Iron is converted to mineral ferrihydrite [FeO(OH)]8[FeO(H2PO4)] and

incorporated into the inner wall of the sphere. The body requires iron in the form of Fe (II)

and therefore it has to change its form from Fe (III) before being sent for utilisation by the

body. After this conversion iron leaves the spherical structure. This implies that the stability

of ferritin is essential for iron storage and maintenance in the body. Ferritin is made up of 24

peptides assembled as a hollow spherical shell and has a molecular weight of 474,000 g/mol.

The diameter of the shell is about 80 Angstorm and the walls are 10 Angstorm thick. For the

purpose of movement of ions and molecules through ferritin small channels are formed at the

intersections of three or four peptides and these channels contribute to ferritin‘s ability to

release or capture iron. The channels can be classified into two types: four fold channels

which occur at the intersection of four peptide subunits and three fold channels which occur

43

at the three peptide intersection. The two channels have different chemical properties and also

function differently. Solubilised Fe2+ ions exit through the three-fold channel by the

property of polarity. The three fold channel of ferritin is lined with aspartate (Asp) and

glutamate (Glu) which are polar amino acids. This allows a favourable interaction between

Fe2+ and water (which is positively charged) and attracts negative poles of side chains. The

four folded channels are lined with nonpolar amino acids (leucine) and therefore it does not

interact favourably with Fe2+ ions and therefore Fe

2+ ions do not leave these channels. It is

however speculated that the electron transfer occurs from Fe (III) to Fe (II) at the four-fold

channel (Washington University, 2000).

2.11.4 Bioavailability of β-carotene in carrots

The release and absorption of carotenes from processed carrots using in vitro digestion

coupled with Caco-2 cell trans-well culture model was examined by Netzel et al.,(2011). The

absorption of carotenoids can be strongly affected by the processing conditions used to

prepare the food matrix that they are contained in. In order to determine the effect of

processing on carotenoid bioavailability, the carrots were homogenised and then the raw,

blanched and cooked carrots were exposed to an in vitro gastric and intestinal digestion

model. Final digested sample was placed on a Caco-2 cell trans-well monolayer culture to

mimic intestinal absorption. From the results it was found that cooked carrot puree had the

highest release of carotenes as they consisted mainly of single plant cell proteins followed by

blanched which consisted of plant cell clusters and raw carrot puree which consisted of larger

plant cell clusters. Absorption of carotenes through Caco-2 cells was highest from the digesta

of cooked carrot puree followed by the digesta of blanched puree. Therefore, it was

concluded that if thermal processing/mechanical homogenisation was done to disrupt plant

cell wall matrix, the in vitrobioavailability of carotenes from carrots was quite high (Netzel et

al., 2011)

2.11.5 Bioavailability of Folic Acid in Fortified Milk Products

The intestinal absorption of folic acid and 5-methyl-tetrahydro folate (5-CH3-H4folate) in

fortified milk was studied using mono-layers of human colon carcinoma (Caco-2) cells. Only

a small difference in transport rate and underlying transport mechanisms, across Caco-2 cells

was found between folic acid and 5-CH3-H4-folate. With the presence of Folate Binding

44

Protein (FBP), the absorption of folic acid and 5-CH3-H4folate was found to be lower and

dependent on the extent of binding to FBP at the luminal side of the intestinal cells (Verwei,

2004).

2.11.6 Micronutrient Interaction in vitro

In a National Fortification program implemented in Venezuela in the year 1993 to reduce iron

deficiency anaemia, the fortificants were a mixture of ferrous fumarate, vitamin A and other

vitamins. The uptake of ferrous fumarate from the mixture was studied using Caco 2-cells. It

was found that vitamin A did not enhance the absorption of iron, however when β-carotene

(6µmol/L) was used alongside compared to no β-carotene addition it was found that β-

carotene enhanced iron absorption compared to no β-carotene addition. Also β-carotene

overcame the inhibitory effect of phytates and tannic acid (potential inhibitor of iron) and

improved iron uptake. The experiment was validated for the use of Caco-2 cells to study the

in vitro absorption of iron (García-Cassal, Leets & Layrisse, 1993).

2.12 Conclusion

Based on the information gathered so far from literature significant information regarding the

necessity for fortification of vitamin A, iron and folic acid would clearly be a possible

solution to address the micronutrient deficiency issue on a global scale. Rice as a potential

medium of fortification is also convincing as the rice grain has to be processed and also is a

staple in many developing countries. Parboiling has significantly been a potential method for

fortification of nutrients into rice. The papers on high pressure technology provide potential

ideas and possibilities to fortify rice with the above mentioned micronutrients. Sufficient

evidence from literature on other foods states that bio-accessibility study of fortified nutrients

in rice could be successfully studied using Caco-2 cells. The study focussed on fortifying rice

with vitamin A, folic acid and iron using the parboiling technique the high pressure

processing as a potentially novel technique for fortification.

The aims of the current study were to assess:

1. The feasibility of fortifying rice with three micronutrients (folic acid, iron and β –

carotene) comparing two techniques- parboiling (ancient technique) and HPP (novel

technique).

45

2. The efficiency of uptake of micronutrients using the parboiling process and understanding

micronutrients‘ interaction influencing the uptake of each other.

3. The physicochemical properties of the fortified rice, giving special importance to rice

starch.

4. The Acceptability of fortified rice among rice eating consumers.

5. The bio-accessibility and absorption of the micronutrients from the fortified rice (by

parboiling and HPP methods) using in vitro digestion and caco-2 cell culture model.

46

CHAPTER 3

3. OPTIMIZATION OF FORTIFICATION OF RICE WITH FOLIC ACID, IRON AND

β-CAROTENE BY THE PARBOILING METHOD

3.1. Introduction

As mentioned in section 2.2 (page no. 9) during parboiling, a hydrothermal process, the

crystalline form of rice starch in the paddy is transformed to amorphous form as a result of

irreversible starch swelling and fusion. This process essentially modifies the physical,

chemical and organoleptic properties of rice with added economic and nutritional properties

(Gariboldi 1974, Luh and Mickus 1980, Kasasian 1982, Bhattacharya 1985 & Pillayar 1988,

1990). Some major advantages of the parboiling process are (Ali & Ojha, 1976):

1. Increased head rice yield

2. Prevention of nutrient loss during milling

3. Salvaging of wet or damaged paddy

4. Rice preparation based on consumer requirements

Parboiling process involves soaking the rice in hot water for about 10-24 hours during which

time the paddy is saturated with moisture. The soaked paddy is then steamed till the starch is

gelatinized following which the paddy is milled (Joachim, 2011). This ancient process has

been practised in many Asian countries such as India, Bangladesh, Pakistan, Myanmar,

Malaysia, Thailand, and also in developed countries such as Switzerland, USA and France

(Pillayar, 1981).

Parboiling has been an effective means of fortification for minerals such as Iron (Prom-u-thai

et al.,2007), zinc (Prom-u-thaiet al., 2009), Iodine (Tulyathan, 2007) and also for a water

soluble vitamin - folic acid (Kam et al., 2012). Simultaneous fortification of zinc and iron

into rice has also been made feasible by Prom-u-thai et al.,(2011) using the parboiling

technique. Therefore, the feasibility of fortifying rice with a provitamin of fat soluble vitamin

A vitamin (β- carotene), a water soluble vitamin (folic acid) and a mineral (iron) was

explored in this study using the parboiling method. The aim of this study was to understand

(1) if parboiling would be a plausible rice fortification method for multi-micronutrients and

optimise the fortification conditions (2) the possibility to scale-up the optimized method (3)

47

the efficiency and uptake of the micronutrients by the grain using the parboiling method and

evaluating micronutrient interactions affecting uptake efficiency and (4) studying the loss of

micronutrients at various stages of the parboiling process.

3.2. Materials and methods

3.2.1. Brown Rice Samples

Dehusked brown rice (BR) of a known long grain variety (Pusa 1121) was purchased in large

quantities from a local supermarket in Sydney and stored at 11˚C for the entire period of

study. Prior to use, the rice was cleaned from dust and broken grains using the dehuller.

3.2.2. Fortificants used for Parboiling

Food grade folic acid was purchased from DSM (Catalogue No: 5004357004), Australia.

Lucarotin® 10 CWD S/Y (Catalogue No: 50051378) which has β-carotene encapsulated in a

proteinaceous matrix was purchased from BASF Company, Germany. Sodium EDTA (III)

(Ferrazone®-

Catalogue No: 18154-32-0) forms of iron was purchased from Akzo Nobel

Functional, The Netherlands was chosen as fortificant as it imparted least colour to the rice

post-processing and also had higher bioavailability compared to ferrous sulphate, ferrous

fumarate and ferrious bisglycinate (Trinidad et al., 2002)

3.2.3. Parboiling Procedure

Graded BR was soaked in water in the ratio of 1:2 with the micronutrients at 70˚C in a water

bath for 2 hours. The mass of micronutrients added is listed in Table3.1. The detailed process

of optimised parboiling condition is schematically presented in Figure 3.1:

Table 3.1 Concentration of micronutrient addition into rice for Parboiling (per 300g of

rice)

CONDITION FOLIC ACID (mg) -CAROTENE (mg) IRON (mg)

A 150 62.5 25

B 300 125 50

C 600 250 100

D 1200 500 200

48

Figure 3.1 Schematic representation of optimized parboiling condition

Each condition was replicated and a control sample was also prepared by parboiling the rice

without any micronutrients in the soaking water.

Soaking: Rice was soaked in water

or a water solution of nutrients in

the ratio 1:2 at 70˚C for 2 hours.

After 2 hours, the soaking solution

was discarded.

Steaming: Soaked rice was

steamed at 100°C for 1 hour to

induce starch gelatinization

Drying: The steamed rice was air

dried at room temperature until the

moisture content dropped to 10±2%.

Milling: The dried rice was then

milled at 0s, 60 and 120s for bran

removal.

0s 60s 120s

49

3.2.4. Parboiling procedure for Scale-up Studies

Graded BR (2.5 kg) was soaked in the solution of micronutrients in the ratio 1:2 at 70˚C for 2

hours followed by steaming at 100˚C for 1 hour (experimental condition) and it was

compared to soaking at 70˚C for 4 hours. The soaked rice was steamed for 15 minutes at

100˚C, followed by tempering for 45 min in an air-tight container. The samples were spread

on a tray and air dried at room temperature until the moisture content dropped to 10±2% .

Each condition was replicated and a control sample was also prepared by parboiling the rice

without any micronutrients in the soaking water. The masses of nutrients added were 1.2 g

folic acid, 500 mg β-carotene, 200 mg iron in 5L of water.

3.2.5. Milling of Parboiled rice

For each sample, 100g of parboiled brown rice was milled in a laboratory scale grain mill

(Satake Test Grain Mill, Japan) for 60 and 120 seconds to yield white rice. The

corresponding degree of milling was ~6 and 11% respectively.

Condition A (Table 3.1) was chosen to further explore the efficiency of the parboiling

process. The moisture content during soaking and steaming, leaching of solids during the

soaking step, loss of micronutrients in the soaking step and the concentration of

micronutrients in the presence of each other were examined using condition A. The results

will be presented in Section3.3.9 (page no. 76). The combinations of micronutrients added

during soaking process is presented in Table 3.2. Each nutrient was added singly into rice and

in combination of two and three.

50

Table 3.2 Micronutrient mass dissolved in 600 mL of soaking solution for the parboiling

process (mg/300g of rice)

Rice treatments β-carotene Folic acid Iron

1 62.5 - -

2 - 150 -

3 - - 25

4 62.5 150 -

5 62.5 - 25

6 - 150 25

7 62.5 150 25

3.2.6. Moisture content in fortified rice and mass of solids that leached out after

soaking

The moisture content of brown rice was determined by the standard oven method (AOAC,

2012 (Method 925.09)). Throughout soaking, 20g rice in triplicates was removed from the

water bath at regular intervals of time (0, 30, 60, 90, 120 minutes) using tea infusers. ~20g

samples were placed in moisture dishes, accurately weighed and dried in a vacuum oven at

70°C for 24 h. The dishes were removed from the oven and cooled for 3 h in a desiccator.

Moisture content was expressed on a wet basis (w.b.). The same method was once again

employed to assess the mass of solids leached into the soak water before and after soaking.

51

3.2.7. Cooking of parboiled rice

For cooking the rice, 10g of each sample was washed with approximately 30 mL of water and

was cooked with 25 mL of water in a 100 mL beaker covered by a watch glass. The total time

taken for rice to cook was 24 minutes at boiling temperature of water (100°C). The degree of

cooking was qualitatively assessed by mashing a rice kernel between glass slides and

observing the absence of white belly. Cooked rice was kept for ten minutes in the cooked

state to absorb moisture and then cooled to room temperature in the beaker covered with

watch glass and then mashed and homogenized using mortar and pestle for analysis. The rice

that was milled for 120s was used for cooking as the whiter coloured rice is popular with

consumers.

3.2.8. Sample extraction and analysis for the micronutrients

The soak water in which the rice was soaked during parboiling was analysed for

micronutrients concentration before (0 mins) and after 120 mins of soaking. Milled rice,

brown rice and the rice bran were analysed for each micronutrient concentration in duplicates

to determine the concentration of the fortified micronutrients in the grain and also to

understand the loss on milling. Cooked rice was also analysed for retention of micronutrients.

3.2.8.1. Analysis of Folic acid

Folic acid analysis was done according to Pfeiffer et al., (1997) & Tamura (1998) with some

modifications. Single enzyme extraction with α-amylase (from Bacillussubtilis) purchased

from MP Biomedical LLC, Australia, Catalogue No.: 100447 (100g)) by incubation of the

rice extract for 2h at 37°C was performed in a screw capped centrifuge tube (purchased from

Greiner Bio-one; catalogue no.: 82050-346). At the end of the incubation, the enzyme was

deactivated by heating the sample in boiling water bath for 3min. Sample was cooled and

then centrifuged at 4000rpm for 15min and the supernatant was collected for analysis.

Sample purification was carried out by solid-phase extraction (SPE) on a strong anion

exchange cartridge (500 mg, 3 mL, Phenomenex, Australia). The cartridge was conditioned

with methanol (2x 2.5 mL) and water (2x 2.5mL) to activate the sorbent and remove matrix

interfering components (Patring & Jastrebova, 2007). After loading an aliquot of the sample

extract, the cartridge was washed (3x2.5mL) with water and eluted with 0.1M sodium acetate

52

containing 10% sodium chloride (UNIVAR, Australia) and 1% ascorbic acid (Sigma-Aldrich,

Sydney, Australia) under gravity. Extreme care was taken such that the cartridge did not run

dry during the loading and eluting procedure. The purified sample was filtered through

0.45μm regenerated cellulose (RC) syringe filter (Minisart RC 25, Germany) prior to HPLC

analysis.

Analyses were performed using a HPLC system (model LC AD, Shimadzu Prominence,

USA) consisting of an autosampler, a thermostable column compartment (maintaining the

column temperature at 35°C) and a photodiode array detector (monitoring at 280nm). The

HPLC system was controlled by a computer running LC Solution Shimadzu Chromatogram

Data System.

The separation of folic acid was performed by using a reversed-phase Luna C18 column,

5μm, 150x4.6mm i.d. (Phenomenex, Australia) with a C18 security guard column

(Phenomenex, Australia). Folic acid was determined by using a gradient elution program with

acetonitrile and 30mM phosphate (potassium dihydrogen phosphate purchased from

UNIVAR, Australia) buffer (pH 2.2). The flow rate was 0.8mL/min. The injection volume

was 100μl. The gradient program was as follows: 5% (v/v) acetonitrile maintained

isocratically for the first 8 min. The acetonitrile concentration was then raised linearly to 24%

within 23min then returned to 5% after 5min. The run time between injections was 40min.

Peak was identified based on the retention time and absorption spectrum acquired for the

peak at 280 nm corresponding to the external folic acid standard. HPLC calibration was done

on a daily basis to ensure data integrity.

3.2.8.2. Analysis of β-carotene

β-carotene analysis was done according to Lamberts & Delcour (2008) by saponification of

the sample followed by liquid-liquid extraction.

The extract was dried under nitrogen and then made up with 3 mL of methanol and then

passed through 0.45μm Polytetrafluoroethylene (PTFE) syringe filter (Minisart RC 25,

Germany) prior to HPLC analysis. Analyses were performed using a HPLC system (model

LC AD, Shimadzu Prominence, USA) consisting of an autosampler, a thermostable column

compartment (maintaining the column temperature of 40°C) and a photodiode array detector

(monitoring at 450nm). The HPLC system was controlled by a computer running LC Solution

Shimadzu Chromatogram Data System.

53

The detection of β-carotene was performed by reverse phase HPLC using Luna C18 column,

5μm, 150x4.6mm i.d. (Phenomenex, Australia) with a C18 security guard column

(Phenomenex, Australia). β-carotene was determined using an isocratic elution with methanol

and acetonitrile in the ratio 40:60. The run time between injections was 30min. Peak

identification was based on the retention time and absorption spectrum acquired for the peak

at 450 nm corresponding to the external standard. HPLC calibration was done on a daily basis

to ensure data integrity (Lamberts & Delcour, 2008).

3.2.8.3. Analysis of Iron

Approximately 0.5 g of accurately weighed ground rice sample was digested overnight with

10 mL of 70% nitric acid. The digested samples were heated on a heating mantle until they

produced brown fumes. When the brown fumes subsided the samples were cooled and were

treated with 2 mL of 35% hydrogen peroxide. The samples were heated again for about 5

minutes and then cooled and filtered using Whatman® qualitative filter paper (55 mm

diameter; 11 µm pore size (particle retention)) to remove particulate matter. The filtrate was

then made up to 25 mL and analysed using ICP-OES. One ppm Yttrium was added online as

internal standard to correct for the loss of analyte during sample injection (United States

Environmental Protection Act (USEPA), 1996). The ICP-OES system (Perkin Elmer, Model:

Optima DV7300 ICPOES) was run under the following conditions:

54

Table 3.3 Conditions of ICP-OES instrument for Iron analysis

Radio Frequency Power 1300 watts

Plasma Gas Flow 15 L/min

Auxiliary Gas Flow 0.5 L/min

Nebulizer Gas Flow 0.70 L/min

Sample Introduction System Burgener PEEK Mira Mist nebuliser with

cyclonic spray chamber

Pump rate 0.5 mL/min

Viewing height 15mm above load coil

View mode Axial

Number of Replicates 3

Note:The quantification was performed using the calibration curve with iron standards

diluted from certified 1000 mg/L iron standard that was simultaneously run alongside

samples to maintain integrity of the analysis. The LOD was 0.05 ppm.

3.2.9. Calculation of % concentration of micronutrients in the soak water before and

after the 2 hours soaking: % uptake and % retention of micronutrients in rice after

parboiling and cookingrespectively

This study focused on the % initial concentration of micronutrients in the soak water before

the soaking process (0 min: before soaking) and % residual concentration of micronutrients

after the soaking process (120 min: after soaking), %uptake of micronutrients from the soak

water into the rice and % retention of micronutrients in the rice after cooking. The former two

terminologies (% initial concentration and % residual concentration) were performed when

rice was soaked with the micronutrients solution at 70°C for 120 minutes. In order to

familiarize with each terminology and how they are expressed mathematically they are

described in detail below.

% initial concentration of micronutrients in the soak water here refers to the concentration of

micronutrients present in the soaking water before the soaking step at 70°C (at 0 min soaking

55

time). After 2 hours of soaking the remanent concentration of nutrients can be referred to as

residual concentration, expressed as % in the remaining soaking water.

% initial concentration of micronutrients in the soak water = (F fortificant- S before soaking)/F

fortificant x 100 Equation (1)

% residual concentration in the soak water = (S before soaking- S after soaking)/S before soaking x 100

Equation (2)

Where F fortificant is the mass of fortificant added per 100 mL of water; S before soaking is the mass

of micronutrient per 100mL of the soaking solution before soaking (mg); and S after soaking is

the mass of nutrient remainingin the soaking solution (mg/100mL).

% Uptake of the micronutrients into the rice refers to the concentration of folic acid, iron and

β-carotene present in the rice kernel after parboiling, i.e.after soaking, steaming and drying.

The % micronutrient uptake in fortified parboiled rice (uncooked) was calculated as the ratio

between the concentration of the respective micronutrients present in the fortified parboiled

rice kernel and the corresponding folic acid added to the soaking solution as the fortificant.

Hence, the % uptake was calculated as follows:

% Uptake = (M fort-rice / M fort) x 100 Equation (3)

where Mfort-rice is the analysed concentration of nutrient in the uncooked fortified parboiled

rice; and, Mfort is the initial mass of micronutrient added to the rice in the (Table 3.1) soaking

water (600 mL).

The % retention of micronutrients in the following context refers to the concentration of

nutrients present in the cooked fortified parboiled rice in relation to the concentration of

micronutrients present in the uncooked form of fortified parboiled rice. The % retention was

calculated as follows:

% Retention = (M cooked rice/ M fort-rice) x 100 Equation (4)

where Mcooked rice is the analysed micronutrient concentration in the cooked fortified parboiled

rice.

3.2.10. Statistical Analysis

The data collected was analysed using a descriptive statistic package (Statistical Package for

56

Social Scientist or SPSS (version 21.0, 2013) and the results were interpreted and presented

on tables and charts. The least significant difference (LSD) at p < 0.05 was applied to

compare the means for significant differences of the treatments.

3.3. Results and Discussion

3.3.1. Optimization of the soaking step of parboiling

Husk is a significant barrier for the water absorption process in brown rice (Thakur & Gupta

2006 & Kam et al. 2013). Thakur and Gupta further investigated the diffusion coefficient for

water absorption studies of paddy and brown rice and stated that at different temperatures, the

diffusion coefficient follows an Arrhenius-type relation and the activation energy for the

Arrhenius equation was determined using the Ea/R parameter. The activation energy for

brown rice was 1.2 times higher than the activation energy for paddy and 1.9 times that of the

husk concluding that the husk imparted effective resistance to the water percolation process

in paddy. Therefore, dehusked brown rice was found to be an effective medium for rice

fortification rather than the whole paddy itself (Thakur and Gupta, 2006).

The rate of moisture migration into the rice depends on the temperature of soaking: higher the

soaking temperature, higher the rate of moisture migration (Thakur and Gupta, 2006). For the

present study, 70°C was considered optimum. Beyond 70°C, the rice after soaking was

cooked. The maximum time the rice could withstand temperature beyond 70°C was 15

minutes. Considering the efficiency of migration for the process of fortification, 70°C for 2

hours soaking was deduced to be optimum.

Two hours soaking time is optimum for folic acid fortification by the parboiling process

(Kam et al., 2012). In this study, 2 hours soaking time was adopted. The concentration of the

micronutrients after 1 hour soaking (data not presented) was much lower than 2 hours.

Increasing the soaking time further did not significantly increase the retention of the

micronutrients (data presented in the following section). Therefore 2 hours was chosen for the

soaking time.

Steaming, the next step in the parboiling process causes irreversible changes to the structure

of the starch granules in the rice due to the application of moist heat (100˚C). The kinetic

energy of the starch increased and intermolecular hydrogen bonds are ruptured, leading to the

disruption of structural integrity of starch (Lund, 1984) and starch gelatinisation. During

57

starch gelatinization, the swollen starch may fuse with the inner bran and scutellum layers in

the endosperm, preventing the loss of vitamin during further treatments such as milling (Rao

& Bhattacharya, 1966). For steaming temperature and duration, 100°C was chosen as the

optimum and the duration of steaming was 1 hour based on the quality parameter for

parboiled rice that would render parboiled rice glassy in appearance without any white belly

(Bhattacharya, 1985). This appearance was obtained at 100°Ctemperature and therefore based

on starch gelatinization and rice acceptance, this condition was chosen.

In terms of drying, shade drying at room temperature was chosen rather than mechanical or

sun drying to achieve maximum concentration of nutrients in the fortified rice that would

otherwise not be possible as the nutrients chosen to be added to rice particularly folic acid

and β- carotene are sensitive to heat and light. But by air drying there is an expected loss due

to oxidation at room temperature (Kam et al., 2013). Hence, shade drying was adopted and

the drying took longer (4 days) than mechanical or sun- drying, but the loss of vitamin were

likely to have been minimised due to air drying. Shade drying also resulted in better rice

milling quality and head rice yield from parboiled rice (Bhattacharya & Swamy, 1967).The

drying process was continued until the moisture content dropped to 10-12% (d.b.).

3.3.2. Moisture content of parboiled rice

Normally, soaking is done to achieve quick and uniform water absorption (Wimberly, 1983).

Wimberly (1983) also reported that the grain reaches a moisture level of 30-35% in 2 to 4

hours, which was also observed in this study. The effect of soaking time on moisture content

of rice is shown in Figure 3.2. The moisture analysis was done on samples that were soaked

with micronutrients according to condition A in Table 3.1 (page no.47).

58

Figure 3.2 Moisture content of rice with different fortificants during soaking at 30-min

time interval. BC: β-carotene; Fe: iron; FA: folic acid(The error bars represent standard

errors of corresponding means from three replicates)

Figure 3.2 shows moisture uptake during the 2 hours soaking period. Since the soaking is the

first step in the parboiling process and it is the key step in the inward diffusion of

micronutrients from the soaking water into the rice, moisture analysis is an essential step in

parboiling. After 30-min soaking, the moisture content for all samples increased, from ~22-

28% to ~28-30%. On soaking for 60 min, the moisture content was approximately 31%.

There was a further increase at 90 min for samples fortified with iron; folic acid; iron and

folic acid; and folic acid and β-carotene, where the moisture content reached 32%; whereas

others slightly increased. On soaking for 120 min, rice fortified with β-carotene achieved the

highest moisture content of 37%, whereas for folic acid the moisture content increased the

most from 22% at 0 min to 34% at 120 min. Similarly, in rice fortified with β-carotene and

folic acid, the moisture content increased from 24% at 0 min to 33% at 120 min.

0

5

10

15

20

25

30

35

40

45

50

0 30 60 90 120

Mo

istu

re c

on

ten

t (%

wb

)

Time (min)

No nutrients

BC

Fe

FA

Fe+BC

Fe+FA

FA+BC

Fe+BC+FA

59

Figure 3.3 Moisture content of rice after 2 hours soaking and 1 hour of steaming at

100 °C (The error bars represent standard errors of corresponding means from three

replicates)

After steaming, the moisture content of all samples further increased to35-37% (Figure 3.3).

In particular, rice fortified with Fe and folic acid had the lowest moisture content of 35%,

while rice fortified with folic acid had the highest moisture content of 37%. Steaming adds

water through condensation and therefore, it increases the total water absorption (Wimberly,

1983).

3.3.3. Leaching of solids during the soaking step of the parboiling process

The micronutrients were dissolved in the soaking water for fortification by the parboiling

process as per condition A mentioned in Table 3.1. Some changes in the concentration of the

micronutrients before and after soaking were expected. Therefore, the soaking water was

analysed before and after soaking for the initial and residual micronutrients and also for the

diffusion of solids into and out of the rice. Solids loss (measured after parboiling) increased

in the soaking solution for all treatments (Figure 3.4). The migration of nutrients was by

inward diffusion into endosperm only. While oil and protein present natively in rice are

reported to diffuse outward during the parboiling process (based on microscopic

observations) they cannot diffuse as readily as water-soluble vitamins through cell walls.

When the rice is boiled in excess water other solids such as starch and water soluble vitamins

are also leached out (Juliano, 1993). Bello et al., (2004) also studied the leaching of solids

from the rice during the soaking process. The results from the study conducted by Bello et

05

1015202530354045

Mois

ture

con

ten

t (%

wb

)

60

al., (2004) showed that for raw and dehulled rice the mass of solids leaching remained low

and practically constant with time during soaking. However for the white rice the mass of

solids leaching increased dramatically with time, particularly for the soaking temperature of

65°C and thereby leading to the inference that the presence of bran prevents water absorption

and therefore leaching of solids (Bello et al., 2004). Thus, in the current experiment, the mass

of solids that leached out after soaking could be from the starch, proteins and fat in the rice

and also the some endogenous water-soluble vitamin.

Figure 3.4 Total solids in rice soaking solution before and after parboiling for the

differentsoaking solutions (The bars represent standard errors of corresponding means from

three replicates)

0.000.020.040.060.080.100.120.14

Tota

l so

lid

s (g

)

0 min

120 min

61

3.3.4. Concentration of Nutrients during the soaking stage of the parboiling process

Table 3.4 shows theinitial concentration of micronutrients present in the soak water before

soaking (0 min) and theresidual concentration in the soak water after soaking (120 min)

expressed in % of concentration. The mass of micronutrients added per 300 g of rice was

based on condition A (Table 3.1).

Table 3.4 % of initial concentrations of micronutrient in soak water before and after

parboiling process and % residual concentration of micronutrients after 120 min of

soaking

Nutrient

loss Combination**

Concentration

in mg/ 300g

rice

%

micronutrients

in soak water

at 0 min

% residual

micronutrients

at 120 mins

β-carotene

β-carotene only 62.5 74±7.3 3.5±0.07

β-carotene, Folic acid 62.5,150 70±16.4 4±0.6

β-carotene, Iron 62.5,25 93±3.6 15.3±0.5

β-carotene, Folic acid,

Iron 62.5,150,25 76±17.6 5.6±0.6

Iron

Iron Only 25 99±0.2 15±0.2

Iron, Β-Carotene 25,62.5 99±0.1 0±0.1

Iron, Folic Acid 25,150 99±0.3 77±2.9

Iron, β-Carotene, Folic

Acid 25,62.5,150 98±0.3 54±1.8

Folic acid

Folic Acid Only 150 98±0.4 0.7±0.3

Folic Acid, β-Carotene 150,62.5 97±1.7 48±0.1

Folic Acid, Iron 150,25 98±0.3 37±0.2

Folic Acid, β-Carotene,

Iron 150,62.5,25 98±0.6 8±0.2

Notes:

Concentration values expressed as Mean±Standard Deviation, unit expressed in %

Rice was soaked with each of the nutrient combinations at 70°C for 2 hours for the

steeping process.

It is generally expected that the mass of micronutrients in the soaking water before the

process would be higher compared to after soaking. If this hypothesis was observed it can be

explained that it would be because of the diffusion of micronutrients along with water from

the soak water into the rice grain. The soak water is the micronutrient resource for the rice.

Therefore when there is change in the concentration of micronutrients in the soak water, it

62

can be expected that there would be change in the micronutrient concentration of the rice.

Food grade fortificants were added to the soaking water for the fortification process and each

of these fortificants had analytical purity value which was presented in the quality assurance

certificate for that fortificant. Minor discrepancies in the initial concentration of each

micronutrient in the study as presented in the above table were due to the analytical purity

and the results obtained comply with the product quality assurance certificate. However when

concentration decreases after soaking it implies that the micronutrients have penetrated into

the rice from the soak water thereby resulting in the depletion of initial concentration. Hence,

lower the concentration of micronutrients in the soak water after soaking, the higher its

concentration in the rice.

Therefore, key findings from Table 3.4 are as follows:

Initial analysed concentration for folic acid and iron were close to 100% of the

nominal mass added before soaking. Minor difference could be due to the analytical

purity of the fortificant since food grade micronutrients were used. .

For β-carotene the initial concentration was close to 95% when it was soaked along

with the other micronutrients and this concentration could be covered by the standard

deviation.

After 2 hours of soaking, higher residual iron was present in the soak water when it

was soaked with folic acid and in the mixture of fortificant. When soaked with β-

carotene there was close to 0% of iron in the soak water indicating that most of it has

been absorbed by rice. In the presence of β-carotene the uptake of iron was the highest

and hence it can be concluded that β-carotene enhances iron diffusion.

When folic acid was soaked with β-carotene and with iron there was higher residual

concentration compared to the other 2 conditions thereby indicating that in both these

conditions there is lesser penetration of folic acid than when folic acid was soaked

alone and in mixture of all the micronutrients.

Although β-carotene was present in lower concentration than folic acid and iron in the

initial concentration, after soaking much of it has diffused into the rice.

These results collectively show that the soaking step of the parboiling process is efficient in

the delivery of micronutrients into the rice. However, these results which shows difference in

absorption of micronutrients into the rice at different combinations of micronutrients (Table

3.4) were not statistically significant (p > 0.05) when the analysis of variance was carried out.

63

No correlation was found between β-carotene, Fe and folic acid concentration in soak water

before steeping (p = 0.22; 0.15; 0.46, respectively) and soak water after steeping (p = 0.27;

0.62; 0.18, respectively) in the presence of other nutrients. With p > 0.05, it is therefore

concluded that there are no mean differences between treatments in terms of concentration of

micronutrients present in the soak water.

Overall, it can be concluded that the uptake of nutrients by the rice grain during parboiling is

very effective as the residual concentration of micronutrients in the soak water after soaking

is quite low. This lower concentration of micronutrient in the soak water referred to as loss in

concentration (as previously mentioned) is described in detail below for each micronutrient.

3.3.4.1. Folic acid

As seen in Table 3.4 initially folic acid concentration is close to 100% in the soak water. The

decrease from the soak water is an indication of how much of a nutrient has diffused into the

rice. After 2 hours, there was higher decrease from the soak water. Folic acid soaked with

iron had the lowest loss in concentration followed by soaking with β-carotene implying that

much of the folic acid had not diffused into the rice. On comparing the condition of folic acid

loss to iron loss when these 2 micronutrients were soaked together iron loss was lowest as

well (Figure 3.7). This implied that folic acid uptake by the rice was limited in the presence

of another micronutrient and the uptake efficacy in rice was much higher when there was no

interference from other micronutrients.

Figure 3.5 Mean folic acid %loss in concentration in the soak solution after soaking for

120 minutes with different treatments (The bars represent standard errors of corresponding

means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)

0

20

40

60

80

100

120

FA FA+BC FA+Fe Mix

% l

oss

of

foli

c aci

d i

n t

he

soak

solu

tion

64

3.3.4.2. Vitamin A (β-carotene)

The % loss in concentration of β – carotene remaining in the soak water after the soaking step

at 70°C for 2 hours is presented in Figure 3.6. After 120 min, the concentration of

micronutrients remaining in the soak water is much less implying that the loss is due to

uptake by the rice during the soaking process. Soak water with only β-carotene and β-

carotene with folic acid had the highest loss in concentration while that with iron had the

lowest loss in concentration of residual concentration after soaking. From the results of %

loss in concentration of folic acid and β-carotene it can be said that in the presence of iron

both these micronutrients had the lowest loss after soaking indicating iron was a limiting

factor for uptake in rice during these 2 conditions. Hence the variation can be attributed to the

competition between the micronutrients to penetrate into the grain influencing the uptake

efficiency.

Figure 3.6 Mean β-carotene % loss in concentration in the soak solution after soaking

for 120 minutes with different treatments (The bars represent standard errors of

corresponding mean values from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)

.

3.3.4.3. Iron

Fe loss in concentration in the soaking water also increased after soaking, compared to that of

fresh soak water as depicted in Figure 3.7. An interesting finding in this soaking study was

that in the presence of folic acid the loss in concentration of iron in the soaking water is

lowest. This implies that there is lower uptake of iron into the rice when there is folic acid

78

80

82

84

86

88

90

92

94

96

98

BC BC+FA BC+Fe Mix

% l

oss

of

β-c

aro

ten

e in

th

e so

ak

wate

r

65

present in the soak water. However in the absence of folic acid when iron was soaked alone

and when it was soaked with β-carotene there was higher loss in the soak water and therefore

higher uptake by the rice. This could be due to the lowering of the pH of the soak water in the

presence of folic acid which significantly affected the uptake of iron by the rice. The pH of

the soak water with mixture of micronutrients was 4.71 before soaking and 5.8 after soaking.

In the case of folic acid with iron the pH of soak water before soaking was 4.92 and 5.71 after

soaking. Wu et al., (2010), reported that folic acid solubility in water increases with an

increase in temperature. And also greater solubility is observed in basic and high acidic

conditions. Therefore the increased solubility of folic acid could be a potential barrier for iron

uptake in rice.

Figure 3.7 Mean iron % loss in concentration in the soak solution after soaking for 120

minutes with different treatments (The bars represent standard errors of corresponding

means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)

3.3.5. Uptake of micronutrients in the fortified rice and effectiveness of Parboiling as a

method of fortification

The previous section focused on the micronutrients concentration in the soak water. As

mentioned the decrease of micronutrients in the soak water can be counted as gain in the rice.

Hence in this section, the results from concentration of micronutrients in rice soaked as per

conditions A, B, C and D as mentioned in Table 3.1with all three micronutrients combined

0

20

40

60

80

100

120

Fe Fe+BC Fe+FA Mix

% l

oss

of

iron

in

th

e so

ak

wate

r

66

will be presented. There was no detectable folic acid and - carotene found in the unfortified

(control) brown rice as it is not a natural source of these 2 compounds. Pusa 1121 basmati

rice (BR) variety used in this study had an innate concentration of 12±4.2 mg/kg of iron in it.

This concentration of iron in Pusa (1121) rice variety was comparable with the values

obtained by Anuradha et al., (2012) who reported Pusa 1121 contained 14-18 mg/kg of native

iron.

Figure 3.8 Concentration of micronutrients (d.w.b) in uncooked rice fortified by

parboiling at varying concentrations and milling times (0s, 60s and 120s) (Error bars

indicate the standard deviations between duplicates (A, B, C and D refers to the mass of the

micronutrients added))

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

Folic Acid

β- carotene

Iron

67

Table 3.5 Percentage uptake of micronutrients after the parboiling process in uncooked

rice showing concentration of micronutrients at different milling times at conditions A,

B, C and D.

Sample Name/milling time Folic Acid β-carotene Iron

A 0s 12.7 44a 43

b

A 60s 14.2 26ab

41.5

A 120s 4 21 43

B 0s 23.5 30 23.5

B 60s 21.7 18 16.5

B 120s 21 14 23.5

C 0s 25 11

a 19.3

C 60s 22 9ab

12.2

C 120s 21 8 11.1

D 0s 14 17

a 8.8

D 60s 11 12ab

7.5

D 120s 7 3 6.8b

Note:a, b& ab

denotes significant difference between samples

Figure 3.8 shows the concentration of micronutrients in parboiled fortified uncooked rice. At

various conditions of addition of the fortificants to the rice, folic acid showed the highest

concentration followed by β-carotene and iron. Folic acid concentration ranged between 20 to

560 µg/g rice. For β- carotene the concentration was 50 to 277 µg/g of rice and for iron the

concentration was 28 to 64 µg/g of rice. With the increase in the concentration of fortificants

added to the soaking water the concentration of micronutrients in rice increased. The

quantification of concentration was performed at 3 different milling times (0s, 60s and 120s).

Folic acid concentration was highest in the aleuronic layer of the rice which is in agreement

with an earlier study (Kam et al., 2012). In the case of β-carotene a similar trend was

observed. The aleuronic layer had the highest uptake and with the increase in the milling time

the nutrient concentration decreased. The concentration of iron, however, seemed similar in

the aleurone layer and endosperm. Fortification through parboiling increased the

concentration of iron in the outer surface of rice although,unlike folic acid and β-carotene,

there was very little penetration into the grain. This can be proven with the data from the soak

water where there was high residual concentration of iron when the fortificants were present

in the mixture (Table 3.4). The migration of micronutrients by inward diffusion into the

endosperm during the soaking stage (Lund, 1984) occurs due to the difference in the moisture

gradient between the rice kernels and the surrounding environment. During soaking there was

68

a positive uptake of the nutrients into the rice as the soaking was determined to be the main

step of fortification during the parboiling process.

According to Prom –u- Thai et al., (2008) Fe fortified rice contained 45-96% of added Fe

when milled at 60 s and 20-98% when milled at 120s. The concentration also varied

depending on the cultivar of rice that was used for fortification. In the same study solubility

of Fe by using 2 fortificants (FeSO4 and Na2EDTA-Fe) for rice fortification by parboiling

were studied. There was minimal variation to concentration of Fe due to the solubility of the

fortificants (Prom –u Thai, 2008). In the present study, iron concentration in the fortified rice

did not increase vastly after fortification even when 200 mg of iron was added per 300 g of

rice. Thus the uptake of iron during fortification was limited. The reason for this has been

discussed in the following section where high iron was retained at in the rice bran when

soaked at condition A. Soaking rice at the lowest concentration of fortificant for iron the

uptake was over 100%, perhaps because the innate iron (12±4.2 ppm) in brown basmati rice

contributed to the final concentration.

Folic acid and iron fortification of rice by the parboiling method has been well studied

previously as discussed in the above paragraphs. However, fortification of rice with β-

carotene was novel as the common forms of β-carotene fortificants include retinyl palmitate

or retinyl acetate(Ranum, 2000 and Stringer, 2000). These are fat soluble and hence pose as a

challenge for rice fortitifcation. Thus by using a water-soluble form of β-carotene this

challenge was overcome in this study. Compared to iron and folic acid this form of β-

carotene (Lucarotin®) was not as soluble initially as it can be seen from Table 3.4 where there

was less concentration of it present in the soak water before the soaking step. However after

soaking, there was less residual concentration of β-carotene in the soak water showing that

much of it had been absorbed in the rice (85% in rice condition A and 14% in rice bran).

The higher the concentration of fortificants added to the rice the lower was the % uptake. At

higher concentrations there was higher loss of fortificants and the uptake was poorer (Table

3.5). Therefore the lowest concentration of fortificants addition was better than higher

concentrations although when higher concentrations of the fortificants were added the

concentration for each micronutrient in the rice grain after milling was higher after

fortification. Addition of lower concentrations of fortificants to the soaking water can also

reduce the overall cost of the finished fortified product and could be an economical solution

for implementing fortification program.

69

3.3.6. Loss of nutrients on milling

During parboiling while soaking the rice with the nutrients, penetration of the nutrients into

the rice kernel occurred: some into the bran and some into the endosperm. Fortified rice was

milled at 0s (no milling), 60s and 120s and the concentration analysis was done for folic acid,

β-carotene and iron in the above section. With the increase in milling time there was a

decrease in the concentration of the micronutrients. Because folic acid was more concentrated

on the outer surface of the grain (Kam et al., 2012), longer milling meant more folate loss.

Experimental data obtained from analysing the concentration of fortified micronutrients in

the rice bran is presented in this section. As it can be seen from the figure 3.9 most of the iron

has been retained in the bran rather than in the endosperm of the rice. According to Doesthale

et al., (1979) the degree of milling affected endogenous iron concentration during parboiling

and that the movement of iron into the endosperm was very weak. Therefore based on the

results from present study it can be inferred that iron, although soluble in water, did not

penetrate very easily in to the grain during the parboiling process. The concentration of β-

carotene in the rice bran was very low. This once again shows that there is high uptake of β-

carotene in the fortified rice and parboiling can be an effective method for β-carotene

fortification using Lucarotin®. It is therefore apparent that the nutrients penetrate several

layers of the rice grain and are retained in the starchy endosperm. There is considerable loss

during milling. The rice bran along with the added micronutrients can be salvaged to produce

by- products for making porridge for example to maximize the utilization of the fortified

product. Lipase present in rice bran causes rancidity due to oxidation and this process is rapid

when bran is separated from rice (Saunders, 1990a). Since parboiling process can deactivate

lipase the shelf-life of rice bran can thus be improved (Silva, Sanches, Amante, 2006, Slavin,

Lampe, 1992). Therefore there is higher value-addition to the fortified rice bran due to

parboiling.

70

Figure 3.9 Concentration of micronutrients in the fortified rice bran (Condition A)

during milling(Error bars indicate the standard deviations between duplicates)

Figure 3.10 Schematic representation of mass balance (on a d.w.b.) in terms of nutrients

concentration and loss at key stages of parboiling in rice soaked with all the

micronutrient at condition A (FA- 150mg; BC- 62.5 mg; Fe – 25mg/600mL soaking

solution used on 300g rice)

From the above schematic Figure 3.10 it can be seen that the maximum loss due to the

process was observed for folic acid and there was no loss for iron. As rice has endogenous

iron present in it and the iron content of the rice was higher after the parboiling process.

0

50

100

150

200

250

60s 120s

Con

cen

trati

on

in

µg/g

Milling Time

Folic acid

β-carotene

Iron

71

There was high concentration of residual iron in the soak water after the soaking step. This

shows that the concentration for iron was close to saturation and therefore there was lack of

concentration gradient which can cause lesser uptake of iron into the grains during the

process compared to the other 2 micronutrients. As it can be seen from Figure 3.10 564% of

iron has been retained in unmilled rice showing that there is exceeding concentration of iron

in the rice itself and hence can be explained by the residual iron in the soak water. Iron

uptake was also limited in the presence of folic acid (Table 3.4-page no.61).For β-carotene

the results from the % loss in soak water implied that ~99% had been lost after soaking and

99% of it was taken up by the rice (Table 3.5- page no.67& Figure 3.9 page no. -70). This

implied that ~1% has been lost due to the parboiling process and thus adding low

concentration of β-carotene fortificant in the form of Lucarotin®was highly efficient for rice

fortification using parboiling.

3.3.7. Retention of micronutrients on cooking the fortified parboiled rice

Rice is always consumed cooked. Therefore it is essential to know the loss of micronutrients

after washing and cooking the fortified rice. This would give a clear picture of whether the

micronutrients from the fortified rice would be able to meet the RDI values. Therefore, in this

section results from the cooked fortified rice has been collated and presented.

Figure 3.11 Retention of micronutrients in cooked rice fortified by parboiling at varying

concentrations (Error bars indicate standard deviation between replicate samples; A, B, C

and D refers to the concentration of the micronutrients added to the soaking water)

Absorption method for cooking was followed and the rice milled for 120s was chosen for

cooking as it had 11% bran removal (on an average) and was therefore whiter than the 0s and

0

20

40

60

80

100

120

140

160

180

200

A B C D

Folic Acid

β-carotene

Iron

72

60s milled rice. Figure 3.11 shows the retention of each micronutrient after cooking the

parboiled rice.Nutrients were lost on cooking. Folic acid was well retained followed by iron

and then β-carotene which showed highest loss compared to uncooked rice after cooking.

73

Table 3.6 % retention of micronutrients after cooking fortified parboiled rice (milled at

120s) (concentrations of the fortificant added in mg/ 300g of rice)

Treatments Folic

Acid

(FA)

%

Initial

concentration

of FA added

β-

carotene

(BC) %

Initial

concentration

of BC added

Iron

(Fe)

%

Initial

concentration

of Fe added

A 98 150 35 62.5 90 25

B 46 300 30 125 95 50

C 35 600 27 250 81 100

D 65 1200 39 500 89 200

Table 3-6 shows the % retention of micronutrients in milled (120s) fortified rice after

cooking. For folic acid the highest retention was at the lowest concentration of fortificant mix

for all three micronutrients after cooking the rice. This means that when low mass of

fortificant was added the % retention is higher after cooking. This could minimize the

quantity of fortificant needed to be added when a food is being fortified by the principle of

diffusion as long as the actual concentration was adequate. For β-carotene highest retention

was with treatment D (500 mg of fortificant) followed by treatment A (62.5 mg) and for iron,

the highest retention was with treatment B (50 mg). It is evident that iron is more stable than

the other micronutrients being a mineral and it is least affected by the cooking process in

fortified rice compared to folic acid and β- carotene. Although ~99% of β-carotene was taken

up by the rice during fortification there was heavy loss during cooking.Degradation of β-

carotene was high when subjected to heat (Borchgrevink and Charley, 1966; Lee and

Ammerman, 1974; Teixeira Neto et al., 1981). Apart from cooking in most Asian countries,

the practice of washing the rice before cooking is common.Due to this about 20 to 100% of

the vitamins are lost depending on the volume of water used in rinsing and the length of

cooking time (Hoffpauer, 1992). In ultra-rice (extruded fortified rice) vitamin A (in the form

of retinyl palmitate) loss was measured under normal rice cooking conditions (approximately

5 min boiling followed by 20-25 min under low-heat) and it was reported that there was a

loss of approximately 26% due to cooking (Flores et al., 1994). Food folates are lost on

cooking by 50%, however folic acid is more easily absorbed and less affected than folates

(tetrahydrofolate, 10-formyltetrahydrofolate, 5,10- Methyltetrahydrofolate, 5-

methyltetrahydrofolate) upon cooking (FAO, 2002). On the whole, lower concentrations of

fortificants seemed effective in terms of uptake during parboiling and after cooking the

fortified rice thus proving the robustness of the fortification process because three

micronutrients could be fortified into rice by a single processing method.

74

3.3.8. Pilot scale studies of experimental condition in comparison to conventional

parboiling condition

The lab scale process (300g of rice) was conducted on a pilot scale (2.5 kg of rice) in order to

see if there was variation in uptake of the micronutrients during parboiling and retention after

cooking due to the scale-up of the parboiling process. If this optimized process is to be

commercialized, it would make more sense economically to produce rice in batches of

several tonnes rather than in small quantities. Therefore as a small comparison to the

industrial batch scale the pilot scale study was performed using condition D (FA- 1.2 g; BC-

500 mg and Fe- 200 mg/ 300g of rice). Condition D used highest concentrations of the

nutrients added to the rice which showed higher concentration in parboiled fortified rice

based on the results from the lab-scale studies (Figure 3.8 -page no. 66).

The uptake of the nutrients performed on a lab scale after soaking for 2 hours (UNSW

optimized condition) and 4 hours (conventional parboiling condition, IICPT, Thanjavur) is

presented in Figure 3.12. There was minimal difference between the different soaking times

for folic acid and iron. In the study conducted by Kam, Arcot & Ward (2012) it was found

that progressive increment in the degree of gelatinization coincided with the increasing folic

acid uptake for prolonged soaking durations (up to 4 hours). Therefore it was reported that

starch gelatinization may be a potential contributor to retain or bind the folic acid (fortificant)

that had diffused into the endosperm from the soaking water (Kam et al., 2012). For β-

carotene the concentration was higher during 2 hours soaking than for 4 hours soaking.

Longer soaking time resulted in poorer uptake of β-carotene. Therefore shorter soaking time

can be better for higher uptake of micronutrients during parboiling.

75

Figure 3.12 Comparison of micronutrient concentration(in d.w.b) in uncooked rice

using experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4

hours) on pilot scale (Error bars indicate standard deviation of duplicate samples)

Figure 3.13 Comparison of micronutrient retention in (in d.w.b.) cooked rice using

experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4 hours)

on pilot scale(Error bars indicate standard deviation of duplicate samples)

There was no significant difference in uptake of nutrients between the soaking times (2 hours

and 4 hours) and different milling times using single factor ANOVA with p>0.05 (p = 0.7 for

folic acid; p= 0.3 for β – carotene; p= 0.9 for iron). Folic acid was retained more followed by

β-carotene and then iron. Concentration of nutrients at 0s upto 120s milling was 415– 230

mg/kg for folic acid; 247-86 mg/kg β-carotene; 53-39 mg/kg for iron (on d.w.b.) (Figure

3.12)

0

50

100

150

200

250

300

350

400

450

500

2

hours

0s 60s 120 s 4

hours

0s 60s 120 s

Con

cen

trati

on

in

µg/g

Folic Acid

β-Carotene

Iron

0

20

40

60

80

100

120

140

160

180

200

2 hours 4 hours

Con

cen

trati

on

in

μg/g

Folic Acid

β-Carotene

Iron

76

The retention of micronutrients after cooking in the scale-up study was similar to that of the

lab scale study. There was no significant difference between the two methods (folic acid p=

0.3; β- carotene p = 0.7; iron p = 0.7) using single factor ANOVA (p≥0.05). However, slight

variations between lab scale and pilot scale studies can be attributed to individual variation in

the size and shape of rice kernels as the distribution of the nutrients varied with each rice

kernel. There was no significant difference between the two different soaking times even

after cooking as shown in Figure 3.13 (p ≥0.05). The concentration of micronutrients in the

uncooked fortified rice at pilot scale was also similar to lab-scale concentration results (p ≥

0.05). This shows that the parboiling condition is reproducible for large scale production.

3.3.9. Micronutrients interaction affecting uptake of each other during the parboiling

process

In this study the novelty was that rice was fortified by parboiling with not just one

micronutrient but with three. It is possible that these micronutrients might have a synergistic

or antagonistic effect on each other during the soaking step. This was studied further by

analysing the concentration of each of these micronutrients in the presence of each other and

individually as well. This interaction has not been systematically studied and there is limited

information in terms of micronutrient synergistic effects in food, although there is a lot of

information about micronutrient interaction through in vivo and in vitro studies (Sandström,

2001, Gibson et al., 2006, Arigony et al., 2013). Hence Section 3.3.10 focuses on the

concentration of micronutrients in rice at different combinations using treatment A (FA- 150

mg, BC- 62.5 mg and Fe- 25mg/600 mL of soaking water)

3.3.10. Nutrient concentration in dried parboiled rice after milling

3.3.10.1. Folic acid

Folic acid concentration in rice was high when it was either soaked alone or when it was

combined with both iron and β-carotene. This can also be seen from the soak water data

where there is lesser residual folic acid in these 2 conditions compared to the other two

(FA+Fe and FA+ BC).

77

Figure 3.14 Mean folic acid concentration (in d.w.b) in dried parboiled rice with

different fortifications before and after milling at 60 s and 120 s (The bars represent

standard errors of corresponding means from three replicates. BC: β-carotene; Fe: iron; FA:

folic acid)

Observations are consistent across the different fortification conditions of rice studied

(p<0.05), where concentration of folic acid declined in milled rice (Figure 3.14). In unmilled

rice, folic acid concentration ranged from 39 to 43μg per g rice. At 60 s of milling, folic acid

concentration ranged from 25 to 39 μg/g and from 24 to 26 μg/g for 120 s. The uptake of

folic acid by the rice was similar for all the conditions and as mentioned before there was loss

due to milling. During starch gelatinization, water-soluble vitamin may adhere to the

endosperm (Bhattacharya, 2006). Folic acid concentration n in the starchy endosperm (at

120s milling) is almost consistent showing comparable level of diffusion across different

treatments.

3.3.10.2. Vitamin A (β-carotene)

Rice that was soaked only with β-carotene showed the lowest uptake. When it was

soakedalong with folic acid and iron it showed highest uptake which was comparable to

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

0 60 120

Foli

c aci

d c

on

cen

trati

on

(u

g/g

)

Milling Time (s)

FA

FA+Fe

FA+BC

FA+Fe+BC

78

Table 3.5. From the soak water data (Table 3.4- page no.61) residual concentrations of β-

carotene was not highly varied for the following treatments: β-carotene, β-carotene and folic

acid and β-carotene, folic acid and iron. The reason for lower uptake when soaked alone

could be due to lack of competition during the diffusion process which occurs due to the

change in the moisture gradient between soak water and rice during the soaking step of the

parboiling process.

Figure 3.15 Mean vitamin A (β-carotene) concentration in (d.w.b) dried parboiled rice

(11%) with different treatments before and after milling at 60 s and 120 s (The bars

represent standard errors of corresponding means from replicates. BC: β-carotene; Fe: iron;

FA: folic acid)

The β-carotene concentration decreased with increase in milling time (Figure 3.15). Some

fortified parboiled rice samples showed very small carotenoid peaks when analysed using the

HPLC which was hard to be quantified (Method limit of detection: 0.07µg/mL). Studies by

Lamberts and Delcour (2008) suggested that the decrease of β-carotene in brown rice during

parboiling was due to the loss of soluble solids in the soaking water, oxidation of carotenoids

during soaking and destruction of carotenoids during steamingThe concentration results for

β-carotene in fortified rice (Figure 3.15) were comparable to those in Figure 3.4(page no. 60),

where total solids were higher when β-carotene was present in soaking water after parboiling

compared to other treatments.

3.3.10.3. Iron

Iron uptake was the highest when it was soaked along with β-carotene and this can be

compared to Table 3.4 where there is close to 0% residual Fe in the soak water after soaking.

0

20

40

60

80

100

120

140

160

180

0 60 120

β-c

aro

ten

e co

nce

ntr

ati

on

(µ

g/g

)

Milling Time (s)

BC

BC+Fe

BC+FA

BC+Fe+FA

79

When it was soaked with the other 2 micronutrients there was not much loss due to the

milling process (23 -55%) at varying degrees of milling.

Figure 3.16 Mean iron concentration in (d.w.b.) dried parboiled rice with different

fortifications before and after milling at 60 s and 120 s (The bars represent standard errors

of corresponding means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)

The concentration of Fe in all milled rice declined, except for milled rice fortified with Fe

and β-carotene which increased from 55 µg/g at 0 s to 64 µg/g of Fe at 120 s (p>0.05). In the

presence of β-carotene the diffusion of iron into the rice grain could be enhanced and

therefore the loss during milling could be lesser implying higher uptake. Studies by Layrisse

et al., (1997) indicated that physiologically vitamin A aided in iron absorption in the presence

of inhibitors by mobilizing iron reserved in haemoglobin synthesis. In the study conducted by

Gracía-Casal et al., (1998) it was concluded that β-carotene had a positive effect on iron

absorption in humans. The form of β-carotene (Lucarotin®

) used in this study also contained

other antioxidants – ascorbyl palmitate (2%) and DL- alpha- tocopherol (2%). The effect of

anti-oxidants on iron could be higher when it was soaked along with β-carotene and therefore

showed higher uptake in this case. Protection of iron in the presence of anti-oxidants was well

studied in vitro and in vivo(Pizzaro et al., 2006& Yamamoto and Niki, 1988).However the

mechanism of increased Fe absorption progressively in rice as seen in this study could

potentially be due to the same mechanism as at physiological level. This could be the reason

why Fe concentration increased with milling time only with BC as this trend is opposite to

normal trend where there is high loss of micronutrients with increase in milling time. This

result needs to be confirmed through further detailed study to understand the exact

mechanism.

0

20

40

60

80

100

120

0 s 60 s 120 s

Iro

n C

on

cen

tra

tio

n in

µg

/g

Milling time (s)

Fe

Fe+BC

Fe+FA

Fe+FA+BC

80

3.3.11. Efficacy of parboiling as a means of fortification

Fortification does not require changes in existing food patterns. Thus, in this study,

estimation of nutrient intake based on rice consumption can be made to deduce the

effectiveness of rice fortification. Average per person consumption of rice differs from

country to country. In Bangladesh, where parboiled rice is commonly eaten, the average

annual rice consumption (kilograms) per person is 160 kg (IRRI b, n.d.), therefore it is

assumed that ~440g of rice is consumed per day. The following calculations are the

estimation on nutrient retention in cooked fortified rice as a premix based on average rice

consumption per day.

For folic acid, its bioavailability in fortified foods is about 85% (NHMRC, 2006). Therefore,

it can be assumed that:

Folic acid concentration in 440 g of cooked fortified rice/ day = 20 μg/g x 440 = 8800 μg

= 8.8mg of folic acid/ 440g of rice

Theoretically folic acid bioavailability (85%) in this fortified rice would be = 0.85 x 8.8 mg =

7.48 mg = 7480 μg/ 440g of rice

However, given that the upper level of intake for dietary folate equivalents is less than 800 μg

per day for children, and 1000 μg per day for females, the folic acid concentration in

parboiled rice needs to be modified to avoid overdose of folate intake for long-term

consumption. However an overdose of folic acid does not have any adverse effect as it a

water-soluble vitamin (Butterworth & Tamura, 1989).

Retinol equivalent expressed as vitamin A -1 μg is assumed to equate to 6 μg of β-carotene

when converted in the human body and the RDI of vitamin A for an adult female is 700 μg

(ODS-NIH, 2006) which is equivalent to 4200 μg of β-carotene. From our study, the

parboiled rice had at least 17.9 μg/g of β-carotene. Assuming that the bioavailability (ranges

from 5% to 65%) of the β-carotene is similar to those in vegetables and fruits (Haskell,

2012),

β-carotene concentration in 440 g of cooked fortified rice/per day = 17.9 μg x 440 = 7876 µg/

440 g of rice

81

β-carotene bioavailability in fortified rice (5% bioavailability) = 0.05 x 7.8 mg = 0.39 mg =

390 μg/ 440 g of rice

β-carotene bioavailability in fortified rice (65% bioavailability) = 0.65 x 7.8 mg = 5.07 mg =

5070 μg/ 440 g of rice

Thus, it can be concluded that the premix fortified rice can supply about 50% of the RDI as

Vitamin A equivalent and the bioavailability of β-carotene ranges between 390- 5070 μg.

Hallberg et al (1998) estimated that only 15% of fortified iron was well absorbed due to the

use of low-bioavailability elemental iron powders and the iron inhibitors in the diet. The iron

bioavailability further decreases to 10-12% when the diet consists of little meat with more

fruits and vegetables, and whole grain cereals (Hallberg et al., , 1998). There is evidence

from Meng et al. (2005) that iron bioavailability in rice can be as low as 1% due to many

inhibitory factors in plant foods. Considering a meal in Bangladesh is made up of fortified

rice with meat and vegetables, it is therefore assumed that:

Iron concentration in 440 g of cooked fortified rice/ day = 0.045 mg/g x 440 g = 19.8 mg of

iron/day.

Iron bioavailability in fortified rice = 0.15 x 19.8 mg = 2.97mg of iron absorbed/ 440 g of rice

According to ODS-NIH (2014), RDI of Fe for men and women at different life stages is 8 mg

and 8-18 mg, respectively. Thus, the daily iron intake of an individual would be fulfilled if

the individual relies on fortified rice as the sole source of dietary iron based on RDI.

The mentioned quantity of rice consumption (440 g) is an approximate average value and the

quantity of rice consumed varies between countries and individuals and these calculated

values are based on the consumption of premix i.e. undiluted fortified rice. This premix is

intended to be diluted to be able to supply at least 50% of RDI for long-term consumption as

the rice premix provides more than 100% of RDI for folic acid and β-carotene. The

concentration of micronutrients are likely to reduce by 1/10th

keeping in mind the dilution

ratio (1:10).The data from this after diluting the fortified rice is described in detail and

presented in chapter 6 when sensory analysis was conducted.

82

Based on the above results it can be concluded that parboiling is an efficient rice fortification

processing method. However like any fortification development programme, rice fortification

is also challenging. The added advantage for rice fortification would be that it is a staple crop

that has been industrialized in terms of large scale processing and milling. Hence with the

data obtained from the scale-up process for rice fortification can be easily implemented

unlike other crops. As previously mentioned in the literature review cost for rice fortification

can be complex and involves a complex supply chain. However with the current scenario of

worldwide micronutrient deficiency, implementation of low cost rice fortification in countries

where rice is a staple can be a comprehensive approach to reduce the prevalence of vitamin

and mineral deficiency (Sydney Wealth Area Health Service, 2009). The collective results

from the current study show that parboiling could be an efficient and possibly a low cost

alternative than extrusion technique for rice fortification with multiple micronutrients.

3.4. Conclusions

The present study was systematic using multiple micronutrients for fortification of rice using

the parboiling process. The concentration of micronutrients at every stage of the parboiling

process (soaking, heating, drying and milling) was analysed and a summarized theoretical

mass balance can be provided for the process. The loss in concentration of micronutrients

from the soak water after 2 hours was close to 99% which indicated that most of the

micronutrients were used in the process. During parboiling process a significant

concentration of the micronutrients diffused into the rice and yet there was loss in particular

for the vitaminsdue to the processesnamely milling and cooking. The effectiveness of

enhancing nutrient uptake in rice through the nutrient-fortified parboiling process is far

greater than expected as more than half of the fortificants were retained in grains based on the

low concentration left in the soaking solution. However, nutrients concentration decreased

after steaming, drying and milling. The combined fortification of β-carotene and Fe in rice

has shown the effectiveness of Fe uptake in milled rice, where Fe concentration was higher at

120s than at 0s and 60s of milling. Also, some micronutrients show higher concentration in

the presence of another micronutrient, as seen in the case of FA and Fe, where both nutrient

concentrations appeared to be the highest among the fortification combinations at 120s

milling. Therefore, despite the fact that low concentrations of all three micronutrients were

seen in milled rice relative to what was added originally these studies indicate that parboiling

83

can still assist in the uptake of micronutrients to a large extent seeming it a successful

technique for fortification.

Fortification of rice with three micronutrients simultaneously using parboiling is feasible.

Lowest concentration of fortificants (Condition A) gave highest uptake during parboiled rice

and retention after cooking. The concentration of micronutrients through this study was as

follows: Folic Acid>β-carotene>iron (at different milling times) in parboiled fortified

uncooked rice with evident loss during milling. When lowest masses of fortificants were

added to rice during parboiling, it was found that approximately 4% of folic acid, and 21% of

- carotene and 43% of iron diffused into the uncooked rice during the fortification process at

120s milling. On an average about 60% of the folic acid, 33% of β-carotene and 78% of iron

was retained after cooking the fortified rice based on the concentration of micronutrients in

the uncooked rice which was taken as 100%. Retention of micronutrients after cooking was

as follows: Folic acid>iron>β-carotene. Rice bran contained significant concentration of

micronutrients when milled and therefore can be used for making by-products. The fortified

rice can be diluted with unfortified rice if used as a premix in the ratio of 1:10. The

concentration after scale-up (in uncooked and cooked rice) did not show significant

difference to that of laboratory scale (single factor ANOVA) which means that the optimized

parboiling condition in the lab is reproducible on a commercial scale.The efficiency of

parboiling as a means of processing technique for rice fortification can be seen with the %

uptake of the micronutrients in the uncooked rice and the % retention of micronutrients in the

cooked rice. Consumption of 100 g of the triple micronutrient fortified rice diluted with white

rice after cooking can provide at least half of RDI for all three micronutrients. This could

vary, depending on the country‘s rice eating pattern. Overall the novelty of this study was to

fortify rice with not just one but three micronutrients without deforming the rice kernel and

this was successfully established using the parboiling process. Fortification of β-carotene in

rice has always been a challenge due to its lipophilic nature. This was also overcome in this

study by using a water-soluble form of β-carotene.

84

CHAPTER 4

4. FEASIBILITY OF FORTIFYING RICE WITH FOLIC ACID, IRON AND β–

CAROTENE BY HIGH PRESSURE PROCESSING (HPP) METHOD

4.1 Introduction

High pressure processing is one of the emerging technologies which could be an alternative

to thermal processing technologies in foods. HPP technique offers a safe and effective

method in food processing by modifying protein structures, formation of new chemicals and

inactivating enzymes in foods (Yaldagard, Mortazavi and Tabatabaie, 2008). The major

advantages of using high pressure processing are (Knorr, 1993):

i. Inactivation of microorganisms and enzymes

ii. Modification of biopolymers

iii. Quality retention such as colour and flavour

iv. Changes in product functionality.

Food products such as jams were found to retain freshness, food colour and flavour upon

chemical and spectrophotometric analysis (Watanabe et al., 1991; Kimura et al., 1994;

Dervisi et al., 2001). Preliminary studies conducted by Kato et al., (2000) showed that rice

grains immersed in distilled water released rice allergenic protein which was dissolved in

water when the rice was treated at 100- 400 MPa.

Despite its wide and novel applications in the field of food processing it was found that there

were limited studies on rice, a staple food in many parts of the world and also a high source

of energy and protein. High pressure treatment of rice caused alterations in the cooking

properties and enzymatic digestion (Arai and Watanabe, 1994). Rice that was soaked and

treated by HPP resulted in changes in the internal rice structure, higher sugars and denatured

water soluble proteins. Rice that was cooked after HPP process also had better palatability

(Yamakrua et al., 2005).

Li, et al., (2011) investigated physicochemical properties of rice starch in water suspension

when subjected to high pressure treatment. The physical properties and structural properties

of the rice starch were studied by performing rapid visco analysis (RVA), differential

scanning calorimetry and X-ray diffraction (XRD) and also by observing the starch molecules

under polarised light microscope and electron microscope respectively. They reported that

85

HPP could be a potential non-thermal processing method to produce a new modified starch

(Li et al., 2011).

Although there are recent studies about the effect of HPP on rice, there was no evidence for

the possibility of fortification of rice with micronutrients. Therefore in this chapter the

feasibility of rice fortification with folic acid, iron and β- carotene were studied. Varying

pressure and time were tried and the most favourable fortification condition was deduced by

analysing the concentration of the micronutrients in the rice. The physicochemical properties

of the fortified rice were also studied (presented in detail in Chapter 5).

4.2 Materials and Methods

4.2.1 Preparation of rice and fortificants mixture

Brown basmati rice (Pusa 1121) was purchased commercially at a local grocery in Sydney,

Australia and milled for 120s in a laboratory scale milling equipment (Satake: Model TM05,

Japan) to produce white rice. When brown rice was treated with HPP it resulted in broken

kernels, even after soaking making it a barrier for rice fortification. Hence milled white rice

was used in this study. The milled white rice (25 ±0.5 g) was weighed into a 50 mL

centrifuge tube (purchased from BD Biosciences, Sydney Australia). To prepare the

fortificant solution folic acid (150 mg), iron (25 mg) and β- carotene (62.5 mg) were

dissolved in 600 mL of deionised water and was incubated at 37°C in a shaking water bath

for 15 minutes for increasing the solubility of the micronutrients in the water. Fortificant

solution (40 mL) was added to the rice in the centrifuge tube and the tube was packed airtight

with the fortificant- rice mixture such that there was no air in the tube. The centrifuge tube

containing rice mixed with excess fortificant solution was ultra-sonicated for 5 minutes to

remove minute air-bubbles in the water (the presence of which might cause breakage of the

centrifuge tube during HPP treatment). The tubes were then packed into triple cryo-bags and

were placed in the high pressure processing chamber for treatment. The high pressure

treatment was done in triplicates and the analysis of micronutrient concentration in the rice

post-treatment was also done in triplicates for each micronutrient. Unlike parboiling the soak

water (Section 3.2.8) was not analysed for residual micronutrients concentration as very little

soak solution was present in the tube after treatment.

86

Figure 4.1 Flow chart of sample preparation for Hydrostatic High Pressure Processing

treatment

4.2.2 High Pressure Treatment of rice samples

High pressure equipment - Mini Food Lab (Model No. S-FL-850-9-W / FPG5620TC25)

(Stansted ,United Kingdom) was used for this study. The pressurising fluid used was 80%

propylene glycol. The high pressure equipment had a sample chamber into which the sample

tubes were loaded and the pressure, time and temperature were adjusted and set to the desired

condition. The sample was loaded onto the sample chamber and then the sample chamber

was locked to the head of the HPP equipment. The head of the HPP equipment attached with

the sample chamber was then lowered into the body of the equipment which contained the

pressurising fluid and the safety lock was turned on. The time, temperature and pressure were

adjusted using the electronic display pad on the equipment. The equipment was connected to

a compressor through which compressed air was passed to the HPP equipment to create the

high pressure atmosphere. The up-ramp rate was 200 seconds and down-ramp rate was set at

5 sec. The decompression timeout was set at 400 sec. The HPP treatment conditions for the

Preparation of white rice by

milling brown rice for 120 s

Preparation of fortificant solution

with folic acid (150 mg), iron (25

mg) and β – carotene (62.5 mg)

Airtight packing of rice + fortificant

solution packed in a Cryobag.

Hydrostatic High Pressure Processing

treatment

87

rice samples were set according to Table 4.1. The temperature was maintained at room

temperature. The pressure was reviewed for 360s, 720s and 1080s for 1h, 2h and 3h

respectively. Post treatment the excess water was discarded from the tubes and the rice

samples were dried at room temperature until the moisture content dropped to 10-12%. The

rice samples were then packed into plastic bags and stored for measuring the concentration.

Table 4.1 HPP treatment conditions used for the rice fortification experiment

Pressure condition in MPa Time in hours

200 1h, 2h & 3h

400 1h, 2h & 3h

600 1h, 2h & 3h

Figure 4.2 High pressure equipment used for the experiment

4.2.3 Analysis of rice moisture content and micronutrient concentration post HPP

treatment

4.2.3.1 Analysis of moisture content in HPP processed rice

After the high pressure process treatment at 200, 400 and 600 MPa for 1, 2 and 3h for each

pressure treatment, moisture analysis was performed on the treated rice. The method adopted

to analyse moisture uptake during HPP treatment was according to AOAC method (1990).

About 10 g of the rice sample was accurately weighed in a moisture dish and the samples

were dried at 70°C to a constant weight (usually 24 hours). The moisture content was

calculated on wet basis.

88

4.2.3.2 Analysis of concentration of micronutrients post HPP treatment

Folic acid and β–carotene were analysed according to section (3.2.8.1-3.2.8.2) by HPLC. Iron

analysis was done using ICP-OES according to USEPA method (3050B) as mentioned in

section (3.2.8.3).

The final concentration was presented in µg/g of rice after correcting the moisture content

(dry basis). Since the rice that was treated at 400 and 600 MPa for 1,2 and 3h was not

physically intact and most of the rice was broken and had a white belly, measurement of

concentration was done only on rice that was treated at 200 MPa for 1,2 and 3h. However the

physicochemical analysis (section 5.2) was done for all the rice samples to understand the

change in starch structure during the processing which will be explained in detail in the

following chapter.

4.2.3.3 Calculation of % uptake and % retention of micronutrients in fortified rice

Calculations for % uptake (in the uncooked HPP treated rice) and % retention (in the cooked

HPP treated rice) of micronutrients in the fortified rice was done according to equation 3 and

4 in section 3.2.9 in Chapter 3.

4.2.3.4 Data Analysis

The uptake of micronutrients into the rice from the soak water during HPP and retention of

the three micronutrients fortified rice after cooking were analysed statistically using single-

factor ANOVA.The factor used being was the treatment time, to see whether there was a

significant difference in micronutrient concentrations in different treatments. Each

micronutrient was analysed individually for the three soaking times. The statistical data

analysis was done using XLSTAT version 2010.

4.3 Results and Discussions

4.3.1 Moisture analysis of HPP rice

The primary mode of nutrient penetration into rice kernels is by water penetration and

difference in concentration gradient inside and outside the rice kernel. The process of

diffusion in this case was aided by high pressure unlike parboiling which used heat. Water

89

molecules penetrate effectively into the peripheral portions of each starch granule in the rice

grain on HPP treatment. Water penetrates into the deep parts of rice grain after the HPP

treatment. HPP treatment injures rice grain microstructure and thereby it is able to penetrate

into the inner parts, aiding starch gelatinisation and digestion. (Yamakura et al., 2005).

Therefore, in this section, the results of the rice moisture content post-HPP treatment have

been presented.

With increase in pressure and time, there is an increase in the moisture content (Table 4.2

&Figure 4.3). Rice treated at 600 MPa for 3 hours had the maximum moisture content while

the rice treated at 200 MPa for 1 hour had the lowest moisture content. However, the key aim

of this work was to study the feasibility of fortifying rice with micronutrients using the HPP

technique. Therefore, 200 MPa (1h, 2h & 3h) treatments were considered suitable for this

study in the perspective of fortification, given that less energy will be needed to dry the

grains post HPP treatment and less structural changes to the grain will be seen which would

make the grain remain intact.

Table 4.2 Moisture content of HPP treated rice on wet basis

Treatments % Moisture (WB)

200 MPa(1hr) 30.8±0.3

400 MPa(1hr) 34.9±0.09

600 MPa(1hr) 53.7±0.4

200 MPa(2hr) 34.4±0.9

400 MPa(2hr) 37.2±9

600 MPa(2hr) 63.4±6.9

200 MPa(3hr) 42.2±1.5

400 MPa(3hr) 39.2±0.9

600 MPa(3hr) 66.0±0.5

Note: All values reported are Means ±SD of triplicate determinations

90

Figure 4.3 % Moisture content of rice on a wet basis at different treatment times and

pressures used

4.3.2 Concentration of micronutrients in fortified rice using HPP

The results of the micronutrients concentration in the fortified rice using high pressure

processing is presented in the figures (4.4 to 4.6) and tables (4.3 to 4.5). For all the three

micronutrients, the concentration in the rice decreases with an increase in time which is quite

the opposite that of the parboiling process. The highest concentration for all the three

micronutrients was seen at 1 hour treatment. Therefore, treating milled rice at 200 MPa for 1

hour would be considered the best possible condition for rice fortification.

0

10

20

30

40

50

60

70

80

0 1 2 3 4

Mo

istu

re c

on

ten

t in

% (

wet

ba

sis)

Treatment time in hours

200 Mpa

400 Mpa

600 Mpa

91

Table 4.3 Concentration of folic acid (in d.w.b) in high pressure processed rice

(uncooked and cooked) (200MPa) at varying time intervals µg/g of rice

Folic Acid 1h 2h 3h

Uncooked 165±33.6 91±32.3 112±34.5

Cooked 162±23.4 86±31.3 94±47

Note: All values reported are Means ± Standard deviation of triplicate determinations

Table 4.4 β-carotene concentration (in d.w.b.) in high pressure processed (200 MPa)

fortified rice at varying time intervals in µg/g

β-carotene in µg/g 1h 2h 3h

Uncooked 44±20.5 28±20.0 9±8.05

Cooked 8±2.8 5.6±3.8 1.19±2.1


Table 4.5 Concentration of Na-EDTA (in d.w.b.) iron in high pressure processed rice

(200 MPa) at varying time intervals in µg/g of rice

Iron 1h 2h 3h

Uncooked Rice 17±2.4 10±5.4 10±7.4

Cooked Rice 11±10.8 8±3.2 6±3.4


4.3.3 Statistical Analysis of micronutrient concentration

When the concentration data for folic acid was analysed statistically to see if there is any

difference between the treatment times, it was seen that between the groups, there was

significant difference. ANOVA results for single factor (treatment time) showed that for both

cooked and uncooked rice there was significant difference in terms of the treatment time (p>

0.05). The single factor ANOVA showed similar results once again for β–carotene indicating

there is significant difference in the cooked and uncooked samples between treatment times.

However for iron the results were different showing that there was no significant difference

between the treatment times for iron concentration. Both uncooked and uncooked samples

had p>0.05 and the F<Fcritical.

92

4.3.4 Loss of micronutrients due to longer treatment times and cooking

Folic acid standard solution was tested for degradation due to HPP at 200 MPa (1h, 2h and

3h). Folic acid did not show degradation after 3 hours HPP treatment and was stable and this

was in agreement with Nguyen et al., (2003) who modelled the stability of pure folic acid

standard subjected to HPP and deduced the stability of the compound. Folic acid was stable

for 7 h when treated at 600 MPa (60°C) and did not show degradation (Nguyen et al., 2003).

Thus the reasons for folic acid decrease in concentration with the increase in treatment time

could be attributed to the change in the starch structure due to the high pressure processing

technique. When the starch gelatinisation is incomplete the effect of amylolytic enzymes is

minimal on the starch (Nasehi & Javaheri, 2012). Activity of α-amylase was tested for release

of glucose in HPP rice treated at 200 MPa for 1h, 2h and 3h (Figure 4.4). With increase in

treatment time at constant pressure there was reduced release of glucose indicating that α-

amylase activity is being hindered due to effect of high pressure processing. Therefore it can

be said that with increase in treatment time, there are structural changes in the starch leading

to partial gelatinisation making it resistant to enzyme digestion and ultimately affecting the

concentration of micronutrients as α- amylase is a key enzyme used for starch digestion and

extraction of folic acid from the fortified rice.

Figure 4.4 Activity of α-amylase by testing glucose release from processed (fortified)

and unprocessed rice samples(Error bars represent standard error in replicates)

Longer HPP treatment times causing partial starch gelatinisation may have affected the assay

rather than the final concentration of folic acid in the fortified rice. Parboiled rice starch can

0

5

10

15

20

25

30

35

40

Glu

cose

con

cen

trati

on

in

mg/g

of

rice

Processed (fortified)and Unprocessedd fortified rice

samples

15 min

30 min

1 h

93

be hydrolysed by α-amylase better than HPP and unprocessed rice and thus the high

concentration of vitamin released from parboiled rice (Figure 3.8) compared to HPP rice.

According to Dhital (2015) parboiled rice is easily digestible than unprocessed rice and this

can reinforces the above results obtained from the current study. Changes in the starch

structure has been studied in detail as discussed in the following chapter which could provide

an insight into the variation in concentration of micronutrients due to longer processing time.

The structural changes in rice starch that possibly affect the micronutrient concentration in

HPP fortified rice is addressed in detail in Chapter 5.

In the present study, unlike the case of folic acid, β-carotene was extracted by saponification

and liquid-liquid extraction. There was no enzyme involved. However, the concentration was

much lower than folic acid and further decreased with longer treatment times. The reason for

this could be that the β – carotene fortificant was encapsulated in a soy protein matrix. Due to

high pressure processing, the destruction of the soy protein matrix (Floury et al., 2002) could

result in degradation of the β – carotene concentration. Apart from that pH was also an

important factor that affected the solubility and stability of soy protein. Torrenzan et al.,

(2002) reported that soy protein showed maximum solubility at pH < 3 or pH >6. In the

present study, the initial pH of the nutrient solution was between 4.7 - 5.03 and this further

increased to 5.8 after the treatment. This is still lower than 6 and therefore it is highly likely

that the protein precipitated out. When soy milk was treated using HPP it was reported that

the emulsifying activity and stability increased but the emulsifying capacity reduced

(Kajiyama et al., 1995). In the present study, β-carotene encapsulated in the protein matrix

was chosen over starch based matrix to improve solubility and form a stable emulsion in

water. The lower concentration of β-carotene with longer treatment time could be due to the

instability of the soy protein matrix during HPP treatment which in turn affects the stability of

β-carotene in which it was encapsulated.

Iron in the form of Na- EDTA has not been studied in detail because using HPP has become

popular only in the past couple of decades. From the results there was no significant

difference in the uptake of iron in the rice during 1h, 2h and 3h thus it can be said there is

lower diffusion of iron in general. Iron being a mineral is stable after cooking and the loss is

lesser than β-carotene after cooking (section 4.3.3). The statistical results also show no

significance after cooking. Lower retention after cooking could be due to washing prior to

cooking the fortified rice. According to Rastogi, (2013) high pressure processing increases the

94

bioavailability of iron by 4.63 to 10.93% in food when treated at 500 MPa implying that there

is added advantage of fortifying rice with iron using HPP.

Table 4.6 % uptake (in uncooked rice) and % retention (in cooked rice) of

micronutrients in the fortified rice before and after cooking at various soaking times

Micronutrients 1 h 2 h 3 h

Folic acid Uncooked fortified rice 41.2% 22.8% 28%

Cooked fortified rice 98% 94.5% 83.9%

Iron Uncooked fortified rice 20.4% 12% 12%

Cooked 64% 81% 60.5%

β- carotene Uncooked fortified rice 21% 13.3% 8%

Cooked fortified rice 18.6% 20.2% 7%

On comparing the % uptake of micronutrients from the soaking water into the rice between

parboiling and HPP process the % uptake was low for all three micronutrients during HPP

compared to the parboiling process (Table 4.6). Parboiled fortified rice had higher

concentration of iron (45 µg/g) compared to HPP treated rice as the raw material used was

brown rice (BR) in the case of the former process and innate iron was higher in BR than in

milled rice and due to diffusion parboiled rice was able to uptake higher iron than HPP where

milled rice was used. In the parboiling process diffusion into the rice was low due to the lower

difference in concentration gradient because of innate iron present in brown rice. Milled rice

had an endogenous iron concentration of ~2 mg/kg (Prom-u-Thai et al., 2008). This could

have resulted in lower uptake of NaEDTA-Fe in rice in current study as it can be seen that the

concentration is almost constant over the three treatment times showing limited migration of

iron and this phenomenon is also comparable to the parboiling process.

High pressure processing can cause disruption of the cell wall in biological matter thereby

making them unstable (Rastogi, 2013). In the current study milled rice was soaked in excess

water and subjected to gelatinisation through high pressure processing. As mentioned

previously approximately 10 mL of the excess soaked water (from initial 40 mL) was

discarded after the treatment and this soak water contained leached out starch from the milled

rice. The rate of starch leaching is higher from milled rice than in brown rice as the rice bran

contains less starch than the endosperm (Juliano, 1985). Thus the reason for lower uptake

95

could be correlated to the leaching of starch to which the micronutrients adhered to and were

present in the soak water that was discarded post HPP treatment. According to Bhattacharya

(2004) during structural changes in starch, water soluble vitamins tend to adhere to the starchy

endosperm. Folate uptake rate in rice followed 1st order kinetics; and the rates of natural rice

hydrolysis (starch gelatinization) and folate uptake are both time-dependent (Kam et al.,

2012). This could be the reason for higher folic acid uptake during the HPP treatment and

lower uptake of β-carotene after HPP treatment. β-carotene, a lipophilic compound was not

as likely to adhere to the starchy endosperm as folic acid even though it was encapsulated in a

protein matrix. This theory could explain the lower uptake of β-carotene in the fortified rice.

From the above table it can be seen that folic acid showed the lowest loss due to cooking

followed by iron and then β-carotene. This retention trend was also similar to that of the

parboiling process.

The adherence of each micronutrient or absorption into the endosperm is to be studied in more

depth to understand the reduction in concentration with time. From previous research it can be

understood that the structure of rice starch changes with high pressure treatment (Yamazaki et

al., 1998). The possibility of adsorption of the micronutrients on to the amylose or

amylopectin could result from this variation. The variation in the ratio of amylose and

amylopectin due to starch gelatinisation when subjected to high pressure treatment could be

one possible reasons for the lower concentration with longer processing time. To study more

in depth, fluorescence tagging of the micronutrients could be performed individually to

unravel this observable fact. This study was the first to identify HPP as a technique for

fortification of rice in a non-thermal environment. Since this was a preliminary study and the

main aim was to look at the feasibility of rice fortification, the most favourable fortification

condition was deduced. In the following chapter, the changes in the rice starch structure due

to the HPP treatment will be further discussed.

4.3.5 Efficacy of high pressure processing as a means of rice fortification

Overall, from the most favourable condition of HPP treatment for fortification, the

concentration of each micronutrient in cooked fortified rice is presented in the table below.

As mentioned in the previous chapter, it is estimated that about 440 g of rice is consumed in

countries where rice is the staple food (IRRI, n.d.). Consequently, based on this quantity (440

g), the concentration of folic acid, iron and β-carotene that can be provided by this fortified

premix rice has been calculated and presented in the Table 4.7. Since our target population

96

mainly includes pregnant women and pre-school children, the RDI is calculated based on

these levels prescribed by USFDA(2014).

Table 4.7Concentration of micronutrients consumed from the fortified rice based on

RDI(NIH-ODS, 2014 & NHMRC (Australia), 2006)

Micronutrient Folic acid (µg) Iron (µg) β – carotene (µg)

Concentration/g of rice 162±23.4 11±10.8 23±4.3

Concentration in premix

rice(µg/440g) 71,280 4840 10,120

Concentration in fortified

rice diluted in the ratio 1:10

with unfortified rice (µg/

440g)

712 48 101

% RDI obtained by

consuming fortified rice 178% 2.6% 12%

Note: The above calculations were made assuming ~440g of rice was consumed per day by a

person living in a country where rice was staple food (IRRI, n,d,)

Hence based on the above it can be seen that folic acid was able to provide more than 100%

of the RDI from the fortified rice. Excessive folic acid consumption has not shown any

adverse effect however, chronic effects of long-term consumption are unknown. Based on the

above calculations iron and β-carotene were only able to provide only2.6% and 12% of the

RDI respectively. As it can be seen from Table 4.4 the concentration of β-carotene retained

after cooking was reduced to almost less than half. Therefore there is a reasonable loss in this

case. On the other hand, folic acid and iron seem to be the more stable after cooking. These

results show that HPP was not able to meet the RDI requirement for iron and β-carotene.

These concentrations are only based on RDI and are susceptible to further changes when they

are actually digested by the body and how much would be bioavailable in the end.

4.4 Conclusions

High pressure processing, a novel food processing method could be a useful tool for rice

fortification. Rice treated at pressures above 200 MPa for three hours resulted in broken

kernels and therefore became unsuitable for fortification. The moisture absorption of rice

97

changed at different pressures with 600 MPa absorbing the maximum water. The water

uptake also increased with time. The best suitable time among those tested for processing was

found to be one hour and the pressure was 200 MPa for maximum concentration of all the

three micronutrients. The uptake during fortification and retention after cooking was the

highest for folic acid, followed by iron and then β- carotene in both uncooked and cooked

rice. There was significant difference between the treatment times for folic acid and β-

carotene but not for iron. By consuming this fortified rice (assuming 440 g of rice is

consumed per day in rice eating populations) the target population would be able to receive

more than 100% of the RDI for folic acid and iron but not for β- carotene. The reduction in

the concentration of the micronutrients could be because of the change in the starch structure

which is affected by HPP. This needs to be studied in detail by investigating the adsorption of

micronutrients on the rice during the high pressure processing. Overall it can be said that high

pressure processing would be a feasible novel method to fortify rice. However the

concentrations of micronutrients to be added should be adjusted to meet RDI in target

populations.

High-pressure equipment on a commercial scale costs between $500,000 to $2.5 million

dollars depending upon equipment capacity and extent of automation. Since it is a relatively

new processing technology with a narrow market, products that are pressure-processed may

cause an increase in the cost by 3 to 10 cents per pound than thermally processed products.

With two 215-litre HPP units operating under typical food processing conditions, the output

of approximately 20 million pounds per year is attainable. With increase in demand for HPP

equipment it is likely that the capital cost and operating cost will decrease (Ramaswamy,

2006).

98

CHAPTER 5

5. PHYSICO-CHEMICAL PROPERTIES OF RICE FORTIFIED BY THE

PARBOILING AND HPP TECHNIQUES

5.1. Introduction

In chapter 3 and chapter 4 the optimization of parboiling procedure to fortify rice with

micronutrients and feasibility of fortification through the HPP technology was described in

detail respectively. The current chapter will focus on the changes in the physicochemical

properties of fortified rice as a result of fortification through parboiling and high pressure

processing techniques used for fortification. For both the fortification techniques the

micronutrients were retained in the starchy endosperm of the rice i.e. for parboiled fortified

rice for which the rice was milled after the fortification process and for the HPP rice for

which milled white rice was used as the medium of fortification. Some of the

physicochemical properties of starch in the processed rice that were studied include:

Degree of gelatinization of the rice

Instrumental colorimetric analysis of the fortified rice

Rapid Visco analysis to understand the pasting properties of the rice

X-ray Diffraction to observe changes in the crystalline nature of the starch

Microscopic images of the processed rice starch under polarized light and differential

interference contrast

Magnetic resonance imaging of migration of water during the parboiling process

Both technologies implemented for rice fortification involved the principle of diffusion. In

the case of parboiling, heat and concentration gradient induced diffusion while for HPP there

was no application of heat whilst pressure played a vital role. The main purpose of this

section of the experiments was to understand how the processing had altered the rice starch.

Essentially in both these processes starch was the main component of fortificant adherence in

rice. Therefore changes in starch properties due to the two processing methods might explain

if there is any relationship that can be established between starch structural changes and the

concentration of micronutrients in the fortified rice. By assessing the physico-chemical

99

properties quantitatively, the qualitative properties of rice starch changes can be studied and

also it can give an understanding of how different the processed rice is compared to

unprocessed rice. Therefore, the rice flour from the fortified and unfortified samples were as

such analysed for the above mentioned physicochemical properties without extracting the

starch alone. The harsh process of starch extraction could lead to further alterations to the

already processed starch and therefore this step was avoided. Grinding of the rice flour and

using alkaline solutions can significantly alter the rice starch structure (Fields, 2013

(pers.comm.) & Ragheb, Abd El-Thalouth and Tawfik, (2006).

5.2. Materials and Methods

5.2.1. Optimization of method for studying gelatinization of starch in rice

Rice starch was gelatinized due to application of heat during parboiling and pressure during

HPP treatment. The degree of gelatinization is likely to vary depending on the process. Hence

the degree of gelatinization was determined through a colorimetric assay. Rice flour was

prepared by grinding the rice that were fortified by the parboiling process and by HPP using a

lab-scale Breville coffee grinder. The ground rice was then passed through a 125 µm mesh

and flour that was >125 µm was used for further experiments. Unprocessed brown and white

rice samples were also processed in the same way and used as controls. The degree of

gelatinization of rice starch was analysed according to Birch and Priestly (1973)& Baryam

(2006) based on the amylose/iodine blue value after dispersion in two different

concentrations of alkali. The principle of the assay was based on relation of gelatinization to

hydrogen bonding effect observed in the infrared absorption spectrum of rice. The colour

change was measured as absorbance using SpectraMax (Model: M2, Sunnyvale, CA) at 600

nm and the data was extracted using the software SoftMax ®Pro, Version 4.8, 2004).

The alkali used in this experiment to induce starch gelatinization was potassium hydroxide

obtained from UNIVAR, Australia. To optimize the molarity of potassium hydroxide (KOH)

which causes gelatinization in rice starch different volumes of 10 M KOH was added to

cooked and uncooked rice. In this preliminary experiment the molarity of KOH that gave

maximum absorbance was used for the assay that was conducted with the rice samples.

Commercial parboiled rice was used as a standard for cooked rice and assumed that it was

100

100% gelatinized and brown rice was used as standard for uncooked rice. Using the

optimized molarity of KOH the standard curve was plotted. The molarity that gave maximum

absorbance for uncooked rice and cooked rice was used for BR and parboiled rice

respectively to build the standard curve. The ratio of absorbance between these values was

used for calculation of degree of gelatinization of unknown samples. The optimized

concentration of KOH is presented in the Appendix 1.

Figure 5.1 Optimization of KOH molarity to be added to study the degree of rice

gelatinization

The present method is based on the principle of analysing the amylose content upon

gelatinization of starch using a colorimetric approach based on the iodine-amylose blue

complex. This method facilitates the determination of rice parboiling index and milled white

rice quality. By adopting this method, measurement of absolute starch concentration in

samples need not be considered and the degree of gelatinization measurement can be quick

and efficient. Gelatinization of rice starch was done using alkali potassium hydroxide (KOH)

and the optimum concentration was found to be (0.86) for cooked and 0.6 for uncooked rice

(presented in Appendix 1). For uncooked rice, gelatinization occurred at a lower

concentration of KOH compared to cooked rice and this was indicated by the absorbance

value.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 1 2 3 4

Ab

sorb

an

ce a

t 6

00

nm

Molarity of KOH added

Optimized absorbance for cooked and uncooked rice

Uncooked

Rice

Cooked Rice

101

5.2.2. Instrumental colorimetric evaluation of fortified rice

Rice that was fortified will have different appearance compared to unfortified and

unprocessed rice due to the addition of the fortificants which impart colour to the rice. The

colour change is evident to the human eye and it is subjective and highly varies between

individuals (Bergman et al., 2004, Champagne et al., 2010). However instrumental colour

evaluation is objective and can provide numerical values that can be used to standardize a

products‘ colour. In this study the fortified rice samples had a yellowish colour compared to

unprocessed white rice. Rice that was fortified by parboiling had a different colour compared

to the HPP rice. Thus to get an objective understanding of colour variation among the rice

samples, parboiling (Conditions A, B, C and D- Table 3.1) and HPP fortified rice (200, 400

& 600 MPa for 1h, 2h & 3h) were evaluated instrumentally for colorimetric values.

Instrumental colour evaluation was performed for the rice samples using a colorimeter

(Minolta CR-400 series). The instrument was calibrated using a white calibration tile. The

colour was measured as three parameters namely; L*, a* and b* colour space (McLaren,

1976), where L* describes lightness from black (0) to white (100); a* describes red-green

colour with positive a* values representing redness and negative values referring to

greenness; b* describes yellow-blue colour with positive values representing yellowness

whereas negative values representing blueness (Lamberts et al., 2006b).

Samples for colour evaluation were placed on a plastic petridish and covered by cling wrap.

The colorimeter was placed on the samples and operated to obtain L*, a* and b* values.

Parboiled rice that was optimized at condition A (mentioned in Chapter 3, section 3.2.3) was

diluted with commercial white rice in the ratio 1:10 as it was chosen to be the way to

commercialize. This will be referred to as diluted fortified rice and was used in Chapter 6 for

sensory evaluation of the fortified rice. Colour differences were calculated based on the

differences in L*, a* and b* between samples and reference materials. Brown rice that was

milled for 120s was used as a control for the untreated rice (ControlRaw) and parboiled rice

that was prepared with the same parboiling conditions but without fortificants added to the

soaking solution (ControlParboil) was used as control for the processed rice. The total colour

difference ΔE, was calculated as a single value that takes into account the differences

between L*, a* and b* of the samples and references.

102

ΔE =

𝐿∗𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐿∗𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒

2+ 𝑎∗

𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑎∗𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒

2+ (𝑏∗

𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑏∗𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 )2

The total colour difference (ΔE) between fortified and unfortified commercial white rice is

being denoted as ΔERaw whereas the difference between fortified and unfortified parboiled

rice is being denoted as ΔEParboil.

5.2.3. Rapid Viscoanalyser (RVA) of fortified rice flour samples

Rapid Viscosity analysis can be used to determine the degree of gelatinization of starch by

assessing the pasting properties of the rice starch. Graphs obtained from RVA can serve as a

sensory quality index for starches (Champagne, 1999). Therefore fortified rice samples were

assessed for the variation in the pasting properties due to the two different processing

techniques.

The instrument used was Model RVA 3D+ (Newport scientific). About 24.89±0.01 g of

water was weighed into a canister and 2.89±0.01g of each rice sample was transferred to the

water surface. The sample weight and volume of water added were adjusted for moisture

according to the following formula:

S= 86 × 𝐴 ÷ 100 − 𝑀

Where S= corrected sample mass (g),

A= Sample weight at 10% moisture

M= Actual Moisture content

Water that was added was also corrected based on the sample weight according to the

formula:

𝑊 = 25 − (𝐴 − 𝑆)

Where W is the corrected water mass (g).

The slurry was mixed using a paddle for 10 times to homogenize it. The paddle and the

canister were placed into the instrument and the RVA cycle was initiated by pressing down

103

the tower of the instrument when the software was ready for analysis. The total run time was

12 minutes and 30 seconds.

Table 5.1 The RVA program for analysing rice sample

Time (mm:ss) Type Value

00:00 Temperature (°C) 50

00:00 Speed (rpm) 960




11:06-13:00 Temperature (°C) 50

5.2.4. X-ray diffraction (XRD) of rice flour fortified by parboiling and HPP

Starch (composed of amylose and amylopectin) is defined by amorphous and semi-crystalline

rings. Amorphous region of starch contains amylose and less ordered amylopectin and the

semi-crystalline region comprises of repeated sequences of amorphous and crystalline

lamellae (Morrison, 1995). The crystalline nature of starch is altered due to processes during

which gelatinization occurs. Therefore the processed fortified rice samples were analysed for

crystallinity using XRD. Rice flour samples that were prepared for the gelatinization

experiment was used for XRD work as well. The instrument used for XRD analysis was

X‘Pert PRO Multi-purpose X-ray Diffraction system (MPD system-PANalytical, Australia).

During the experimentation process the voltage used was 45 kV/40 mA. The program for the

continuous scan has been described in Table 5.3

104

Table 5.2 Scan parameters for XRD measurement

Scan parameters Values

Start angle (°) 3.008

End angle (°) 60.008

Step size (°) 0.05

Time per step (sec) 0.5

Scan speed (°/sec) 0.1

No. Of steps 1140

Total scan time 9 min 33 sec

The rice flour samples were evenly distributed onto a sample plate to make a smooth surface

for analysis and this was loaded onto the sample changer which automated the process of

analysis. The XRD scan program was chosen for analysis using HighScore Plus software

(Version 3.0.5, 2012).

After the analysis, the XRD data was extracted using X‘Pert HighScore plus (version 1.3.2.6,

2010) and % crystallinity was calculated using the software Magicplot Student (Version

2.5.1, 2013).

5.2.5. Polarized light microscopy and Differential Interference Contrast (DIC)

Microscopic images of starch granules in the fortified rice

Due to gelatinization of rice starch under the influence of heat and pressure, the starch

molecules are likely to have physical and structural changes. These changes vary depending

upon how harsh the treatment is to the starch (Hibi et al., 1993). Hence fortified rice flour

was observed for physical intactness using polarized light microscope. Ground rice flour was

further ground to make smaller particle size manually using mortar and pestle for 10 minutes.

The rice flour was once again passed through a 125 µm and the flour that was < 125 µm was

stored in screw capped Nunc®

CryoTubes® (purchased from Sigma-Aldrich, Sydney

Australia). For observation under microscope a small quantity of the rice flour was mounted

onto a slide and was spread evenly with water. The excess flour was tipped off. To the

remaining flour 50% glycerol was added and the sample was covered with a cover slip. The

105

starch granules were observed under a polarized light microscope and differential

interference contrast microscopy and the images were captured.

Starch samples mounted on glass slides with 50% Glycerol were scanned using a Zeiss

Axioskop II – a brightfield transmitted light microscope with polarising filters and Nomarski

Optics for viewing under differential interference contrast. Images were captured using a

Zeiss HRc digital camera and archived using Zeiss Axiovision software. Images were

photographed under a 63x oil immersion lens.

5.2.6. Real-time Magnetic Resonance Imaging (MRI) of water migration in rice during

parboiling

MRI can be used to observe moisture distribution in rice grain in three dimensions with time

(Horigane et al., 2013). Moisture migration into the rice grain can be observed using MRI. In

this study, migration of micronutrients into the grain in real-time was observed based on the

assumption that migration of micronutrients will correlate with moisture uptake into the rice.

This is the first time that real-time MRI has been used for this purpose. For loading samples

on the MRI machine for evaluation, rice grain was soaked at the optimum parboiling

condition of 70 0C for 2 hours and rice kernels were removed at particular time intervals

(0,15,30, 45 , 60 and 120 min). One kernel from each interval (all in all 6 kernels) was

prepared on a plastic board (size) to which a layer of Blue-Tack ™ (Bostik) was attached.

Extreme care was taken so that all rice kernels aligned in a uniform plane for imaging. The

plastic board was placed in a water filled Greiner cryovial ™. MR Imaging was performed

using a 9.4T preclinical imaging system.

Bruker biospec 94/20 Avance III which was equipped with a 15mm Quadrature

Receive/Transmit RF-coil and running Paravision 5.1 To image the samples from all time

points in one image the rice kernel assembly was scanned in longitudinal and coronal

directions using a 2D gradient echo, Fast Low Angle Shot (FLASH) method. Field of View

(FoV): 10mmx14mm, Slice Thickness: 200mm Echo time (TE) =4ms, Repetition time

(Tr)was 64.2 ms, and the flip angle (FA) was 40.0 deg. It was taken care to use the shortest

possible TE for the available set-up to collect enough signals from the hard and still relatively

dry rice kernels. This enabled to effectively detect free water in cracks. The total scan time

was 26 min 17 s.

106

5.3. Results and Discussions:

5.3.1. Degree of Gelatinization of fortified rice samples

Processing methods in foods can significantly alter the starch structure and this can in turn

have impact on the postprandial response when consumed after cooking the rice (Björck,

1996). When rice is subjected to heat or pressure treatment the starch granules are disrupted

and therefore they are more susceptible to enzymatic digestion (Holm et al., 1988). During

starch retrogradation after gelatinization rice starch is converted to a crystalline form from an

amorphous form (Bhattacharya, 1985). Therefore in this section the rice that was subjected to

gelatinization due to heat and pressure was retrograded by cooling to room temperature.

Following this degree of gelatinization was assessed.

The result for the gelatinization study has been presented below:

Figure 5.2 % Gelatinization comparison between parboiled rice and HPP processed

rice. Untreated brown rice and white rice were used as controls(Error bars represent

standard errors in replicate samples)

In the present study, milled white basmati rice has 34% amylose and therefore classified as

very high amylose content rice according to the classification by Juliano (1992).

Gelatinization in the current study occurs due to application of heat and pressure on the rice

kernels. Depending upon the starch type, the pressure and heat required for gelatinization

varies. Wheat starch for instance begins to gelatinize below 300 MPa and complete

gelatinization occurs at 600 MPa (Douzals, Marechal, Coquilee & Gervais, 1996). Potato

starch on the other hand begins to gelatinize only at 600 MPa (Bauer & Knorr, 2005).

Douzals, Cornet, Gervais and Coquille (1988) found out that the release of amylose in

0.00

20.00

40.00

60.00

80.00

100.00

120.00

% Gelatinization

107

pressure induced gelatinization was lower than with the heat-induced treatment. Experimental

results from the current study showed that gelatinization induced my HPP resulted in the

lower swelling and release of amylose compared to heat treatment. This can be attributed to

the lowering of % amylose during HPP treatment at 200 MPa for 3 hours. By increasing the

pressure (400 and 600 MPa) and time (3 h) more amylose is released and this can be seen in

the results (Figure 5.2).Hence it can be said that pressure influences the level of amylose

released and thereby impacting the degree of gelatinization. This could explain the variation

in degree of gelatinization in pressure treated rice and heat treated rice as the swelling of

starch did not completely solubilize the amylose therefore leading to lower release due to

pressure treatment (Hermansson & Svegmark, 1996). The disintegration of the crystalline

region of starch was not completed by pressure due to the side-by-side dissociation and helix

unwinding of amylopectin units being suppressed by van der Waals‘ forces and stabilization

of hydrogen bonds in normal rice starch (Buckow et al., 2007). Hence, the gelatinization of

rice starch induced by pressure depends on the intensity of pressure, duration of the

treatment, size of the starch granules and ratio of amylose/amylopectin.

Parboiled rice showed maximum absorbance and the degree of gelatinization was close to

100% showing complete starch gelatinization due this process. The degree of gelatinization is

responsible for various attributes of parboiled rice (Marshall et al., 1994). Gelatinization

causes amylose molecules to leach out of the micellar network and diffuse into the

surrounding aqueous media (Hermansson & Segmark, 1996). The degree of gelatinization

has also shown to have effect on the colour of parboiled rice. Different soaking time affects

the relative darkness of the milled parboiled rice. However, translucency is also another

factor affected by parboiling and with longer soaking treatments rice quality improved in

terms of translucency (Miah et al., 2002). The rate of starch gelatinization is highly

temperature dependent (Priestly 1976 & Bakshi and Singh, 1980). For complete starch

gelatinization in paddy it took about 60 minutes at 1100

C and 40 minutes at 1200C. For

brown rice to achieve about 80% of gelatinization it took about 50 minutes at 700C and 20

minutes at 800C but it took 240 min at 70

0C and 80 min at 80

0C for paddy to attain same

degree of gelatinization. Thus it can be seen that paddy requires more time than brown rice. It

was also deduced that brown rice requires less resistance to water movement than paddy

(Bakshi and Singh 1980). These properties make BR a better medium for fortification than

paddy.

108

Table 5.3 Activation energies for gelatinization in Rough rice (paddy) and brown rice

(Bakshi & Singh, 1980)

Temperature range °C Ea Cal/g-mole

Rough rice (paddy) Brown rice

50-85 18534 24672

85-120 10474 9586

From the above table it can be seen that the activation energy for paddy (rough rice) is lower

than brown rice for temperatures below 850C. On the other hand, at temperatures above 85

0C

the activation energy for brown rice is lower than paddy. This type of reaction is common in

the case of catalytic reactions (Bakshi and Singh 1980). Sttaterfield (1970) observed that the

activation energy for diffusion-limited reaction is about one-half of the activation energy

observed in the case of just the reaction only. In parboiling for temperatures below 850C, the

proportion of rice components when soaked is a limiting factor while for temperatures above

850C diffusion of water in rice is a limiting factor. This explains the results obtained in the

present study when soaked at 700C using the parboiling method the added nutrients to brown

rice may have contributed to be a limiting factor for uptake of micronutrients. This can be

further explained by comparing the iron concentration in the fortified rice after parboiling

process. The uptake of iron is almost constant at different conditions (A, B, C and D (Table

3.1)) during parboiling showing that brown rice could have restricted the uptake of iron

during the soaking step.

Generally, both pressure and heat treatment have significant effects on the degree of

gelatinization. However, the magnitude of difference for gelatinization varied between the

treatments. Heat gelatinized rice produced more translucent and acceptable rice compared to

the pressure treatment. Thus variation in degree of gelatinization can be attributed as a major

factor for concentration of micronutrients in the rice as it varied between the two methods for

each micronutrient.

5.3.2. Colorimetric analysis of fortified processed rice

Evaluation of colour is an important aspect of food products as it influences the consumer‘s

behaviour towards purchasing and consuming a particular food (Brewer and McKeith, 1999,

VanHurley, 2007). According to Champange et al., (2010) white rice consumers expect the

cooked and uncooked rice to be white in colour with minimal discolouration. However

109

parboiled rice consumers can accept some colour change in the rice consumed such as a tinge

of yellow or brown (Tomlins et al., 2007). Instrumental colour evaluation of fortified rice in

comparison to commercial white rice is presented in the table below.

110

Table 5.4 Color parameters L* (Lightness), a* (Redness) and b* (Yellowness) of

fortified rice milled at different milling times at various concentrations

Sample L* a* b*

A 0s 61.9 a,b,

2.7c,d

13.3 e,f

A 60s 64.6 a,b,

0.9c,d

16.2 e,f,n

A120s 66.4 a,b,g,i

-0.7c,d,k

16.7 e,f

B 0s 65.0 a,b

2.5c,d

17.4 e,f

B 60s 66.7 a,b

2.3c,d

18.6 e,f

B120s 69.5 a,b,h,i

0.9c,d,h,k

17.4 e,f,o

C 0s 60.0 a,b,

2.9c,d

18.4 e,f

C 60s 65.3 a,b,

1.0c,d

18.2 e,f

C 120s 69.5 a,b,h,i

0.97c,d,h,k

17.7 e,f,n

D 0s 57.3 a,b,h

3.2c,d

19.0 e,f

D 60s 65.5 a,b,

1.9c,d

20.2 e,f

D 120s 64.4 a,b,

1.5 c,d,h,k

22.0 e,f,n

Control parboiled

rice (unfortified) 65.9

a,b,h,j 0.25

c 15.2

Commercial

Parboiled rice 64.5 0.19

15.9

Commercial white

rice 71.6 -0.6

9.9

Diluted Fortified

rice 70.6

l -0.54

,l 10.2

m

Note:Values followed by different letters in the columns indicate significantly different

means at p < 0.05.

Based on the L*, a* and b* values obtained from the colorimeter the colour difference was

calculated based on the formula mentioned in section 5.2.2 (page no.101) to see the

difference in mean colour between parboiled rice and raw rice. Commercial white rice and

commercial parboiled rice were used as reference for respective samples.

111

Table 5.5 Colour difference between fortified rice and commercial white rice

(Controlraw) and parboiled rice (Controlparboil)

Sample ΔE parboil ΔE raw

Diluted Fortified rice 8.4 1.0

In house parboiled unfortified

rice milled at 0s 1.6 7.8

Premix fortified rice -0s (A) 3.8 12.3

Premix fortified rice 60s (A) 3.3 9.5

Premix fortified rice 120s (A) 0.8 6.2

Premix fortified rice 0s (B) 5.3 15.9



Premix fortified rice 0s © 5.9 14.8



Premix fortified rice 0s (D) 8.4 17.4



Table 5.6 Colour parameters L* (Lightness), a* (Redness) and b* (Yellowness) of HPP

fortified rice at different pressure and time treatment

Pressure L a B ∆E raw

200 1h 69.1 a,b

-1.1 12.8 c,d

3.8

200 2h 64.4 a,b

-0.8 12.7 c,d

7.7

200 3h 70.9 a,b

-1.4 12.6 c,d

2.8

400 1h 67.9 a,b

-1.0 13.0 c,d

4.9

400 2h 51.6 a,b

-1.3 10.9 c,d

20.0

400 3h 67.4 a,b

-1.6 11.2 c,d

4.5

600 1h 58.3 a,b

-1.2 10.1 c,d

13.4

600 2h 52.8 a,b

-1.1 9.0 c,d

18.9

600 3h 59.0 a,b

-1.1 10.6 c,d

12.6

Note: Values with different alphabets indicate significantly different means at p <

0.05.

5.3.2.1. Total colour difference for parboiled rice and HPP rice– against uncooked

commercial white rice (ControlRaw) and parboiled rice (ControlParboil)

Rice eating populations commonly consume milled rice (Bhattacharya, 2004) and therefore it

can be set as a standard for fortified rice. Instrumental colour evaluation can be an objective

way to see the colour difference between the fortified rice and white rice for consumer

acceptance.

112

For the parboiled rice, the total colour difference ΔE increased with concentration of the

fortificant added and also with the milling time. ΔE was comparable statistically at different

fortificant concentration levels to the commercial fortified rice. ΔEparboil value was the lowest

for the in-house parboiled fortified rice- A120s (0.8) and ΔERaw was lowest for the diluted

fortified rice (1.0) making them comparable in colour to commercial parboiled rice and

commercial white rice respectively. Based on the fact that the commercial parboiled rice has

already been widely accepted by consumers it can be said that the product can be marketed

and acceptable locally and internationally. Also the variation between white rice and fortified

rice was much higher compared to parboiled rice making it distinctly different in terms of

colour. The major contributors for colour change was deduced to be L* and b* when highest

mass of fortificant was added and this has been reflected in the ΔE that was calculated in the

present study (Kam et al., 2013).

For HPP treated rice, commercial white rice was used as a benchmark. The results show that

the L*, a* and b* values were comparable to commercial white rice. Similar to results from

parboiled rice, the major contributors for variation in colours were L* and b* while a* was

more or less the same and also there was no significant difference statistically as well

between treatments for a*.The lowest ΔERaw value was obtained for 200 MPa 3 hours

treatment and highest was for 600 MPa 2 hours treatment. Fortified premix rice (condition A)

milled at 120s and the diluted fortified rice (milled at 120s) had the lowest ΔEparboil and ΔERaw

value respectively. This shows that the optimized parboiled fortified rice had the lowest

difference in colour compared to its corresponding reference.

Overall, ΔEparboil was lower than ΔEraw indicating that the colour difference between fortified

rice and parboiled rice was lesser than when compared with uncooked commercial white rice.

When the fortified parboiled rice was diluted with white rice the ΔEraw was the lowest

implyingthat there is minimal variation from the commercial white rice and therefore it is

suitable for commercialization at this dilution (1:10). The likelihood of product acceptance

would increase if there is no foreign pigmentation in rice (Hurrell, 1997). From the ΔEparboil

value it can be seen that fortification during parboiling does not create a huge magnitude of

colour change. However, colour change is evident due to fortification using both processing

methods. ΔEraw for HPP rice treated at 200 MPa (1h, 2h and 3h) was relatively lower than

parboiled rice as parboiling introduces enzymatic and non-enzymatic reactions and also

113

diffusion of pigments from the rice bran into the rice endosperm (Bhattacharya, 2004;

Lamberts et al., 2006a; Lamberts et al., 2006b). However on higher (400 & 600 MPa) and

longer (2h & 3h) pressure treatments HPP rice ΔEraw was high due to low redness value(a*)

making it less acceptable.

5.3.3. RVA pasting curves

Treating starch in excess water or applying heat and moisture treatment is known to cause

variation in the pasting properties and lower the viscosity of the pasting starch (Knutson

1990; Gunaratne and Hoover 2002; Singh et al 2005; Vermeylen et al 2006). In this study

rice has been treated by heat and moisture treatment and also subjected to high pressure.

Therefore, the two processes are expected to have an impact on the starch pasting properties.

It was tried to deduce if fortification had any impact on the rice pasting properties by

comparing the fortified rice to commercial white rice and parboiled rice which were used as

control.

The pasting curve data for the processed rice starch has been presented in Table 5.9 below. It

can be seen that with an increase in the pressure treatment, there is a decrease in the viscosity

(peak viscosity and final viscosity). However, more or less all the peak times were the same

for all the samples. The highest peak and final viscosity was observed for unprocessed white

rice. This shows that high pressure treatment and parboiling modifies the starch structure and

in agreement with previous studies from literature which show that similar results were

obtained for modified starches and high pressure processed starches (Stolt et al., 2001 & Pei-

Ling, 2011). The process of fortification did not have any impact on the pasting properties

unlike the processing. The reasons for lower peak and trough viscosities were explained by

Pei-ling et al., (2011) by three steps. The first reason was that HPP treatment weakened the

resistance to shear and therefore withholding the starch granules from breakdown prior to

arrival at peak viscosity. The second reason was that continued shear lead to sustained

fragmentation of the ruptured granules. Finally, HPP affects molecular weight polymers

including crystallinity melting, amorphous hydration and gelatinization of starch granules

(Blaszcak et al., 2005).

The peak time value for unprocessed and processed starch was almost the same i.e. at 7

minutes. The pasting temperature on the other hand increased slightly between treatments. It

was more or less similar for 200 MPa treatment and the control rice samples. However there

was a slight increase for the treatment at 400 and 600 MPa indicating that pasting

114

temperature was higher for HPP modified starches than native starches. The breakdown value

indicates the end point of gelatinization due to the melting of starch crystals or complex

viscosity increase. It was due to the disintegration of gelatinized and swollen starch granules

(Hermanson & Sevgmark, 1996). In the present study, most of the breakdown values were

negative except for 600 MPa treated rice and parboiled rice indicating that these 2 types of

rice showed higher degree of pre-gelatinization and this can be correlated with the degree of

gelatinization data as well. Setback value extracted from RVA implies the starch thaw

stability. Lower setback values mean lower tendency to retrograde which is commonly

observed in pre-gelatinized and modified starches (Deffenbaugh & Walker, 1989). In this

study lowest setback value was observed for the commercial parboiled rice and the highest

for the unprocessed white rice.

115

Table 5.7 RVA data for fortified rice sample processed by parboiling and HPP

techniques

Note: RVU stands for rapid viscosity unit. Each viscosity values are Mean±SD of

replicateanalysis.

RVA graph for parboiled rice (Figure 5.6) showed dead peak implying that it has been

completely gelatinized. Commercial parboiled rice also showed RVA pattern similar to in-

house parboiled rice. Therefore the RVA results are comparable to the degree of

gelatinization in the present study which was close to 100% for in-house parboiled rice. In

this current study, glucose released from parboiled rice was found to be high after digesting

Sample

Peak

viscosity

(RVU)

Trough

viscosity

(RVU)

Breakdown

(min:sec)

Final

Viscosity

(RVU)

Setback

Viscosity

(RVU)

Peak

Time

(mins:

sec)

Pasting

Temperature

(°C)

200 MPa 1 hour 1188±2.8 1190.5±7.7 0.00 2618±8.4 1427.5±9.1 6.93 92.65

200 MPa 2

hours 1035±14.1 1028.5±0.7 -4.00 2416.5±0.7 1388.5±3.5 7 93.3

200 MPa 3

hours 1195.5± 1199±1.4 3.00 2611±7.0 1412±4.2 7 92.6

400 MPa 1 hour 771±24.0 788.5±3.5 -3.00 1791±2.8 1002±4.2 7.00 94.85

400 MPa 2

hours 899±14.1 910±14.1 -11.00 1913.5±7.7 1001±2.8 7.00 95.00

400 MPa 3

hours 860±23.0 871.5±9.1 -1.00 1627±4.2 756±12.7 7.00 93.4

600 MPa 1 hour 912.5±6.3 911±9.8 4.00 1960.5±40.3 1064±24.0 7.00 94.25

600 MPa 2

hours 538±36.7 505±8.4 1.00 934±19.7 429±9.8 7.00 94.95

600 MPa 3

hours 442±57.9 486±8.8 3.00 1152±8.4 658.5±23.3 7.00 94.95

In-house

parboiled Rice 215.5±0.7 210.5±10.6 -3.00 428±2.8 206±7.0 7.00 Nil

Unprocessed

White Rice

1297.5±12.

0 1304.5±6.3 -3.00 3252±31.1 1972±5.6 7.00 92.45

Unprocessed

brown Rice

1458.5±43.

1 1418.5±24.7 -8.00 2613.5±6.3 1166.5±33.2 6.87 93.35

Cooked Rice 250±9.8 251.5±12.0 -3.00 456±5.6 200.5±3.5 7.00 Nil

Commercial

Parboiled Rice 155.5±19.0 170±2.8 1.00 226±8.4 60.5±3.5 6.93 Nil

116

with α-amylase compared to HPP rice and therefore completely gelatinized starch is more

susceptible to hydrolysis of starch than partially gelatinized starch. By comparing the

concentration of folic acid and β-carotene in the parboiled fortified rice and HPP rice (for

both the treatments condition A from Table 3.1 (page no.47) was the initial mass of added

micronutrients) it was found that parboiled rice had higher concentration of the 2 vitamin

(folic acid: 367 µg/g & β-carotene: 235 µg/g) than HPP rice (folic acid: 164 µg/g & β-

carotene: 43 µg/g). This could be due to the higher degree of gelatinization due to which folic

acid and β-carotene were able to be released better from the rice matrix in parboiled rice than

HPP rice during extraction. This can also be correlated with HPP results where increase in

treatment time resulted in lower concentration of folic acid and β-carotene in the rice (Table

4.4 (page no.91), 4.7 (page no.91)) due partial gelatinization and formation of resistant starch.

Hence the degree of gelatinization of rice starch during the processing can be correlated with

the variation in concentration of the micronutrients in the fortified rice.


pressure process

117


pressure process


pressure processing

118

Figure 5.6 RVA graph for untreated rice and parboiled rice

119

5.3.4. X-ray Diffraction (XRD) of fortified rice flour samples showing crystalline

patterns

The results from previous section illustrate that crystalline nature of starch is changed due to

processing affecting the birefringence of starch. In this section the crystalline nature and

degree of crystallinity of starch samples that were processed and unprocessed were examined

using XRD. Although parboiled rice was well studied for crystalline properties using XRD

not many studies have looked at the processing of the whole rice kernels under high pressure

and then studying its physiochemical properties. In this study the influence of heat and

pressure on the native crystal pattern of rice starch was explored.

Crystals of native starch show A-type crystals (Katz, 1928). This can be seen in Figure 5.7for

the unprocessed control rice. There is not much change in the 2Θ value for unprocessed and

parboiled rice. The crystal structure also seems not altered. However there was a decrease in

the intensity of counts for the same 2Θ value for parboiled rice. In this study, it can be

observed that for commercial parboiled rice, there is minor change in type of crystal but it is

incomplete. This type of crystal is alike with V-type of crystal formed by the amylose-lipid

interaction under severe parboiling conditions (Dercyke et al., 2005). Change in native starch

structure can be correlated with the degree of gelatinization as discussed in the previous

sections.

120

Figure 5.7 XRD pattern for parboiled and commercial white, brown and parboiled rice

Figure 5.8 XRD pattern for HPP treated rice at 200 MPa for 1,2 and 3h

NPB- Non-parboiled

PB- Parboiled

121

Figure 5.9 XRD pattern for HPP treated rice at 400 MPa for 1,2 and 3h

Figure 5.10 XRD pattern for HPP treated rice at 600 MPa for 1, 2 and 3h

122

Li et al., (2011) studied physicochemical properties of purified rice starch suspended in

excess water and subjected to high pressure processing. The results obtained in the present

study were in agreement with the study by Li et al., (2011). In the present study when the rice

flour was treated at 600 MPa for 2 hours it was seen that there was evident change in the

predominant crystal type, i.e. from A-type to B-type. These findings were also comparable to

the study conducted by Li et al., (2011). This shows that not only purified rice starch but also

rice flour exhibits same XRD pattern after processing and also the addition of micronutrients

did not impact the crystalline pattern of rice starch. It is also in agreement with the data

obtained from microscopic images and pasting properties. Pressure treatment at 600 MPa

resulted in complete disintegration of starches and partial gelatinization. The threshold

pressure for conversion of crystal type from A-type to B-type existed in the range between

480- 600 MPa (Li et al., 2011). For B- type starches water fills up the channel in the cell unit

of the crystallite and stabilizes the structure(Katopo et al. 2002). But for A-type starches

amylopectin branches are more scattered and therefore more flexible allowing rearrangement

of double helices and thereby generating a channel that includes water molecules under

pressure (Jane et al., 1997). As a result crystal structure changes from A-type to B-type and

this requires a threshold pressure treatment of 600 MPa and 2 hours approximately.

Table 5.8 % Crystallinity (extracted from XRD) of rice sample treated by high pressure

processing and parboiling

Sample % Crystallinity

Brown rice 46.6±2.1

White rice 37.8±2.6

200 MPa (1h) 38.5±2.1

200 MPa (2h) 35.3±1.3

200 MPa (3h) 36.5±1.7

400 MPa (1h) 30.8±3.2

400 MPa (2h) 30.5±1.2

400 MPa (3h) 24.2±2.5

600 MPa (1h) 23.8±3.8

600 MPa (2h) 22.6±2.2

600 MPa (3h) 17.1±2.5

Parboiled (0s) 23±2.1

Parboiled (60s) 24.9±1.6

Parboiled (120s) 20.8±1.4

Notes: Crystallinity values are Mean±SD of duplicate analysis.

Bracketed alphanumerical values represent treatment time in hours for HPP and milling time

in seconds for parboiled rice.

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From the above table, native starch had a % crystallinity of 38.5%. Typical native A-type

crystals from cereals exhibit strong peaks at 15.04°, 16.84°, 17.96° and 23.03° 2Θ and

weaker peaks at 20.04°, 26.3° and 30.26° 2Θ. This pattern can be observed from the XRD

graphs for all the rice flour samples. With an increase in the pressure treatment, there was a

decrease in the % crystallinity. Parboiling treatment also resulted in lower crystallinity

compared to native starches although there was no change in the type of the rice crystal.

Basmati rice (Pusa 1121) has not been previously studied for XRD properties after being

subjected to HPP or parboiling treatment. The results from XRD can be correlated with the

degree of gelatinization and RVA results. Degree of gelatinization is inversely proportional to

the % crystallinity whereas RVA results (peak, final and setback viscosity) are directly

proportional to the % crystallinity. The degree of milling of parboiled rice did not

significantly affect the % crystallinity and also the parboiling treatment was not severe to

impose change in crystal structure.

Collectively the data from degree of gelatinization, RVA, microscopy and XRD denote that

physico-chemical properties are affected by the swelling property of amylose in the rice. The

rice variety used in this study had high amylose (34%) and hence the variation in the starch

properties can be linked to the micronutrients concentration in the rice. Although the addition

of micronutrients (folic acid in particular) did not have any influence on the starch alteration,

its uptake to the fortification medium (rice) can be associated with the amylose modification

due to processing.

5.3.5. Microscopic images of starch granules processed by parboiling and HPP

treatment

In the above sections physicochemical properties of starch granules were explored by

quantitative techniques. In this section, the change in the rice starch was examined visually

using polarized light microscopy and differential interference contrast microscopy (DIC).

Parboiled rice has been previously well studied under polarized light microscopy (Juliano,

1985; Pillaiyar, 1988) however there are limited studies which used DIC to observe starch

changes under microscope. DIC has the advantage of capturing images in striking colour

(optical contrast) with a 3-dimensional shadowed-like appearance and at outstanding

resolution (John Innes Centre, n.d.). Starch granules from the fortified processed rice

observed under polarized light microscopy and DIC are presented in the current section.

124

a . b.

Figure 5.11a & b: Starch images for rice treated at 200 MPa for 1 hour (a. under

differential interference contrast; b. under cross-polarized light)

a. b.

Figure 5.12a & b: Starch images for rice treated at 200 MPa for 2 hours (a. under


a. b.



125

a. b .



a. b.

Figure 5.15a & b: Starch images for rice treated at 400 MPa for 2 hours (a. under cross-

polarized light under; b. differential interference contrast)

a. b.



126

a. b.



a. b.

Figure 5.18a & b: Damaged starch images for rice treated at 600 MPa for 2 hours (a.

under differential interference contrast; b. under cross-polarized light)

a. b.

Figure 5.19a & b: Healthy starch grain images for rice treated at 600 MPa for 2 hours

(a. under differential interference contrast; b. under cross-polarized light)

127

a. b.

Figure 5.20a & b: Partially integrated starch Starch images for rice treated at 600 MPa

for 3 hours (a. under cross-polarized light under; b. differential interference contrast)

a. b.

Figure 5.21 Figure 5.17 a & b: Completely damaged starch Starch images for rice

treated at 600 MPa for 3 hours (a. under cross-polarized light under; b. differential

interference contrast)

a. b.

Figure 5.22a & b: Starch images for parboiled rice milled at 0s (a. under differential

interference contrast; b. under cross-polarized light)

128

a. b.

Figure 5.23a & b: Starch images for parboiled rice milled at 60s (a. under differential

interference contrast; b. under cross-polarized light)

a. b.

Figure 5.24 a & b: Starch images from in-house parboiled rice milled at 120s a. shows

birefringence in some starch granules under DIC while in b. it can be seen that there

has been a damaged starch granule

a. b.

Figure 5.25a & b: Starch images from comemrical parboiled rice showing

birefriengence (a. under DIC and b. cross polarized light)

129

a. b.

Figure 5.26 a & b: Starch images from untreated rice showing birefriengence under a.

DIC and b. cross polarized light

Birefringence (Maltese-cross –pattern) is caused by the radial orientation of crystallites in

native starch granules under polarized light microscope (Yuryev, Wasserman, Andreev, &

Tolstoguzov, 2002). From the images it can be seen that there is some visible birefringence

and Maltese cross in most samples. For samples treated at 200 MPa at 1hour, 2 hours and 3

hours Maltese cross and birefringence is visible for all the samples with minor damage with

longer treatment times. For rice samples treated at 400 MPa losses in birefringence can be

observed with increase in treatment time. Rice samples treated at 600 MPa there was evident

loss of birefringence with increase in treatment time. The damage was higher when the rice

was treated at 600 MPa for 2 hours and 3 hours. Birefringence was completely lost for some

intact starch granules while the granular structure and the crystalline region are deformed.

Method of grinding can also play a role in damaging starch granules (Li et al., 2014).

Damage to starch due to grinding does not follow any linear relationship. When isolated

starch granules from different botanical sources were milled cryogenically, it showed non-

linear relationships in damage level, disruption of double helices and crystalline structure and

degradation of starch (Dhital et al., 2011 & Stark& Yin, 1986). When parboiled, the starch

granules stop exhibiting birefringence under polarized light, and the orderly polyhedral

structure of the compound granules is converted into a consistent mass (Juliano, 1985;

Pillaiyar, 1988). Loss of birefringence in in-house parboiled samples shows that in house

parboiling treatment caused more damage compared to rice starch than commercial parboiled

sample which retained partial birefringence. This shows that heat and moisture treatment can

cause higher degree of distortion to the crystalline region of the starch thereby causing loss in

birefringence.

130

HPP modification of non-crystalline granule tapioca starch slurry in water occurred at 600

MPa for 30 min treatment. Swelling and disintegration of waxy corn starch was observed at

600 MPa treatments. Variation in swelling was also observed at different concentrations of

the starch-water slurry (Pei-Ling et al., 2012). Similar results were observed in the present

study as well. High pressure was a much milder treatment than heating in terms of

gelatinization (Muhr & Blanshard, 1982). Cooking results in swelling of starch grains and

making them lose definitions in polarization cross and other signs of gelatinization. In

general, cooking affects microscopic features of each starch species in a unique way. Watson

& Dikeman (1977) observed different layers of rice – hull, aleurone layer and starchy

endosperm before and after parboiling using scanning electron microscope (SEM). It was

found that parboiling showed complete gelatinization of endosperm and it was hard to

distinguish between the parenchymal cell of the hull and aleurone layer after parboiling.

Birefringence is associated with the semi-crystalline nature of starch which is made up of

orderly sequences of amylose and amylopectin. Swelling of amylose followed by

amylopectin followed by change in sequence of starch can thus be accounted for

birefringence loss. Hydrolysisof starch amylases can be aided by gelatinization (Blish et al.,

1938). For in-house parboiled rice in the present study most of the starch granules lost the

crystalline structure. Also the starch was almost completely gelatinized in contrast to HPP

processed rice which showed partial gelatinization (5.3.1). This implies that α-amylase that

was used to breakdown starch to release folic acid from the fortified rice would be able to

hydrolyse parboiled rice starch better than HPP rice. This confirms the results where there

was decrease in concentration of folic acid with increase in pressure treatment time (section

4.3.2) as being due to the formation of resistant starch during HPP treatment which was not

susceptible to hydrolysis.

Combining the results from sections 5.3.2, 5.3.3 and 5.3.5 it can be suggested that the starchy

endosperm where the fortified micronutrients are adhered to the rice is altered due to the

processes. Correlating the microscopic images to XRD, the experimental data shows that

HPP caused variation to native starch crystal and parboiling retained native starch crystal

pattern although it was a harsher treatment. Therefore it can be said that the two processes

alter the arrangement of amylose and amylopectin uniquely and this variation in the sequence

during processing can be attributed to the variation in micronutrient concentration in fortified

rice.

131

5.3.6. Real-time Magnetic Resonance Imaging of rice during parboiling process

Migration of water during the soaking stage of parboiling was investigated using MRI. Water

migration into the kernel would likely indicate how much of the nutrients are likely to be

absorbed into the grain as they are dissolved in the solution for the fortification process. Real-

time MRI was used for the first time in this study to see if water migration can provide

insight into absorption of the nutrients into the grain.

Figure 5.27 Coronal section images of rice after parboiling process at different time

intervals at the optimized condition

0 mins

30 mins

45 mins

60 mins

120 mins

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Figure 5.28 Sagittal section of soaked rice at different time intervals during the

optimized parboiling conditions

a. b.

Figure 5.29 a&b: Migration of water with and without micronutrients where A- is with

Fe and B – is without any micronutrients

Figure 5.27 shows the variation in the water molecules measured as resonance among rice

kernels soaked at different times. The top image of the rice kernel shows the reference kernel

which was dry rice, not parboiled prior to imaging. It can be seen that – due to the low proton

0 min

A 60 mins

B 60 mins

A 90 mins

B 90 mins

A 120 mins

B 120 mins

0 mins

A 15 mins

B 15 mins

A 30 mins

B 30 mins

A 45 min

B 45 mins

0 mins

30 mins

45 mins

60 mins

120 mins

133

abundance- this reference shows essentially very low signal which could be due to the

minimal content of moisture in dry rice. With increase in soaking time, more water starts to

diffuse into rice kernels, which can be measured as additional signal from MRI. From the

MRI images the cracks in the rice kernel are visible. With the progress in time the centre of

the rice kernel can be seen as brighter which is presumably due to increased uptake of water

and softening of the kernel core. The sagittal section shows a similar trend (Figure 5.28). It

can also be observed that the innermost core of the rice looks brightest compared to other

layers. This contrast is evident especially during the last 30 minutes which means that there

are some modifications and changes occurring during this step altering the starchy

endosperm. The previous sectionsdetermined the qualitative changes in rice starch structure

and this alteration could be narrowed down to occur during this period of the soaking time.

This essentially reinforces that this method measures a combination of water content and

structural features of starch. However, pure diffusion of water during the later stages could

not be obtained as a result of changes in starch degradation. But adopting a lower soaking

temperature it would be possible to unravel water diffusion more effectively where starch

decomposition does not initialize sooner or by concentrating more on the earlier stages of

diffusion. However, in both cases it is not really pure variation in water but will contain the

structural features as well.

Figure 5.29 shows that there is not much signal variation with and without iron (25 mg) in the

soaking water. Very likely this is due the low level of iron concentration in the soaking water

which is below the limit where significant image alterations can be detected in the MRI. Also

the diffusion of water molecules into the rice kernel is independent of the micronutrients

present in the soaking water so that iron content might not be increased by the same quantity

as water. In other words, irrespective of the concentration of the of micronutrients the

moisture distribution remains the same in rice after the soaking step of parboiling. This can

be substantiated with the moisture data presented in Chapter 3 section 3.3.2 (Figure 3.2-page

no. 58) Signal intensity of the rice increases with time as seen in the MRI by about 35% on an

average after 120 minutes soaking and the trend is similar for rice in the presence and

absence of micronutrients. However, for methodological reasons the signal intensity of a

gradient echo method does not depend solely on water distribution in the sample but is also

influenced by local sample structures. This is why MRI imaging signal cannot be directly

translated to moisture content and it is not possible to actually quantify the water content

from MRI methods.

134

From the experimental data for uncooked parboiled rice (section 3.5.5) it can be said that the

maximum concentrations of the micronutrients were on the outer surface of the rice kernel

rather than in the centre. Inference from the MRI data showed that there is a nominal degree

of structural changes observed in the rice core indicating that the core was softened due to the

process. This can be seen by the signal which was higher on the inside of the rice kernel

rather than the outside. It can be speculated that there is evident movement of water during

the parboiling process but the intensity of movement of the micronutrients were much less

compared to the water. This could be the reason for higher concentration of micronutrients on

the outer layer rather than in the rice core. Similar results were obtained by Horigane et al.,

(2006) where moisture absorption was slower on the periphery of the white rice grain

especially on the dorsal side from the apex of the grain despite the closest contact with water

when rice grain was soaked at room temperature. Cracks observed on the transverse section

of the rice kernel were formed on the ventral side and can be seen in the 30 min soaking

image (Figure 5.27). The central line has the highest intensity of signal and is proposed to be

the main route for water infiltration which then passes on to the cracks on grain surface. Rice

aleurone layer thickness varies depending on the rice variety however it is thicker on the

dorsal side than on the ventral side (del Rosario et al., 1968).After 30 minutes soaking, the

cracks become obscure. Generally, water migrates from the pericarp vascular bundle into the

embryo (Horigane et al., 2001). Water distribution was highest in the cracks and regions

along the central line than other parts of endosperm (Horigane et al., 2006).

In brown grains of Koshikari and Yamadanishiki varieties, water penetration was inhibited by

the embryo, pericarp and seed coat due to which the rate of uptake was much lower than

milled rice. In case of BR, infiltration started from the embryo and the surface of the rice

grain. However it was observed that the final moisture distribution was much more

homogenous in milled rice although water tended to be more retained around the central line

of the softer tissue than other parts of the endosperm. The core endosperm of rice contains

amyloplasts with thin walled starch granules (Horigane et al., 2006). The outermost cell

layers i.e. the sub-alourone layer contained high protein and lipids and lesser amyloplasts and

compound starch granules than inner endosperm (Marshall & Wadswoth, 1994). This could

be a reason for uptake of water molecules around the central core which is more hydrophilic

than other parts which contain relatively more hydrophobic compounds.

135

5.4. Conclusions

Qualitative changes in rice starch structure due to parboiling and high pressure processing

was collectively studied in this chapter by analysing the degree of gelatinization, evaluating

pasting properties of starch using RVA and crystalline alterations using XRD. Microscopic

examination of starch due to processing was also investigated and migration of water during

parboiling was also explored using MRI.

Parboiled rice showed close to 100% gelatinization of starch while for the HPP

treatment it was proportional to the increase in pressure.

Pasting properties of HPP treated rice exhibited partial gelatinization of starch but not

complete gelatinization as in the case of parboiled rice where there were dead peaks

present.

Parboiling treatment did not result in change in starch crystal structure however for

600 MPa treatments after 1 hour, starch crystallinity changed from A-type to B-type.

Treatment of rice starch at 600 MPa and parboiled rice starch showed complete

distortion of starch granules compared to other pressure treatments (200 and 400

MPa). Loss in birefringence and Maltese cross was evident from polarized light

microscope and differential interference contrast microscope.

Moisture distribution for parboiled rice kernels examined by MRI was more

concentrated on the rice core- endosperm than in other parts. Gradual uptake of water

during the parboiling process could be seen using this imaging method. The

movement of water into rice was independent of the micronutrients in the soaking

water.

To summarize, starch properties are significantly altered due to the processing methods

and they vary between the two processing methods. The degree of alteration varied

between different HPP treatment and parboiling process. HPP resulted in partial

modification of rice starch while parboiling caused complete gelatinization. Different

parameters studied to gain an insight into starch structural changes showed that the

variation in the amylose swelling and leaching is speculated to be associated with the

dissimilarity in vitamin uptake during the fortification by both the processes. There is

evident matrix effect that influences the release of vitamin from the starch matrix and

therefore there is variation in concentration of vitamin in rice after parboiling and HPP.

Although 400 MPa and 600 MPa treatments were not suitable for implementing rice

fortification, the qualities of rice starch make them suitable for other products where they

136

can replace pre-gelatinized starch. Whilst the findings in this chapter relate to the

modification in rice starch in terms of fortification another perspective that could affect

the starch properties due to processing would be the digestibility. Alterations in the rice

starch due to processing can result in the formation of resistant starch and thereby

influence the taste and digestion of the rice in vivo after consumption. The acceptability

of the fortified rice among consumers and the bio-accessibility of the fortified rice would

therefore be addressed in the following chapters (6 & 7).

137

CHAPTER 6

6. CONSUMER ACCEPTANCE STUDY OF FORTIFIED PARBOILED RICE

6.1 Introduction

In the previous chapters optimisation of fortification process through two different techniques

(parboiling and HPP) were discussed and the changes that occurred to rice starch due to

processing was also addressed. The success of a fortified rice product would depend on

consumer acceptance of the product, when cooked. Acceptance of cooked rice is a vital

attribute especially to consumers for whom rice is a staple food. Rice preparation methods

and degree of cooking is very subjective and varies highly depending on the region of the

world. For instance, the Japanese prefer cooked rice that is soft and sticky and of short-grain

variety such as Japonica. Consumers from the U.S. like medium and long grain rice that is

firm and not sticky. In the Asian countries such as Thailand, Pakistan and India fragrant rices

are favoured (Lyon, 1999).

With quality attributes on one hand, nutritional value of rice also plays an important role in

rice consumption worldwide. Brown rice is considered healthy in the U.S. (Marshall and

Wadsworth, 1994) despite better palatability and digestibility of milled rice compared to

brown rice (Misaki and Yasumatsu, 1985). Hence nutritional value of rice is also a significant

factor in consumer purchase and acceptance.

Acceptability of fortified rice can be a challenge as it is highly likely that the addition of

extraneous nutrients could alter the native rice taste. Several studies have been performed by

researchers all over the world to understand acceptance of fortified rice. Lee et al., (1995)

evaluated the sensory qualities of calcium fortified rice. The data from their study proved that

there is significant decrease in tenderness and adhesiveness when the rice had additional

calcium. Iron-fortified extruded rice was evaluated for acceptance using a triangle test and it

was deduced that when ferric pyrophosphate was used as a fortificant, the tested rice closely

resembled that of the control rice which was natural unfortified white rice (non –extruded) in

both cooked and uncooked forms (Moritteo et al., 2005). Folic acid fortified rice using the

parboiling method was also investigated for consumer acceptance by Kam et al., (2012).

The aim of this study was to analyse consumer acceptance of parboiled fortified rice. Rice

fortified using the HPP technique could not be used for sensory studies as the pressurising

138

fluid used was 80% propylene glycol which seeped into the rice during the HPP treatment in

small quantities. Due to safety reasons this study could not be conducted. The sensory study

for parboiled fortified rice was conducted in two phases i.e.by investigating if people would

buy the rice from the supermarket and if they liked the taste of the fortified rice. This was

conducted by performing descriptive sensory tests and the data was analysed statistically for

interpretation of results.


6.2.1 Preparation of fortified parboiled rice

Brown rice was prepared as described in section 3.2.1. For the two phases of the sensory

evaluation study, folic acid (150 mg), β- carotene (62.5 mg) and iron (25 mg) were dissolved

in the soaking water. To this, 300g of brown basmati rice that was purchased commercially

was added to the fortificant solution in rice-water ratio of 1:2. The rice was soaked at 70°C

for 2 hours and then it was steamed at 100°C for 1 hour. Following this the rice was dried at

room temperature until the moisture content dropped to 12% (wet weight basis). The

parboiled brown rice was milled in a food-grade lab scale mill (Satake Test grain Mill, Japan)

for 120s such that the bran removal was about 10-11% (Section 3.2.5). The parboiled rice

was then diluted in the ratio 1:10 with unfortified commercial white rice since the parboiled

rice was prepared as a premix and intended to be mixed with white rice for better acceptance

among consumers.

6.2.2 Micronutrient analysis of the fortified rice

The fortified parboiled rice (cooked and uncooked) was analysed for concentration of folic

acid, iron and β – carotene as discussed in sections 3.2.8.1 – 3.2.8.3. Both concentrated

premix and the diluted rice were tested for concentration of micronutrients in uncooked rice

and retention after cooking. Folic acid and β – carotene were analysed using HPLC while

iron analysis was performed using ICP-OES.

6.2.2 Sensory evaluation of parboiled fortified rice: An Overview

A schematic representation of the sensory evaluation performed for the parboiled fortified

rice is presented in Figure 6.1. Phase 1 of the sensory study was performed on uncooked rice

to understand if consumers were willing to purchase the fortified diluted rice if made

commercially available. In Phase 2 of the sensory study the parboiled fortified rice that was

diluted with the unfortified white rice was cooked and evaluated by consumers for

acceptability. Methodology for the two studies that were conducted is explained in detail in

the following sections.

139

Figure 6.1 Schematic representation of fortified parboiled rice prepared for Consumer

Acceptance Study 1 and Study- 2

Brown rice

(300 g)

Soaked in micronutrients

solution in the ratio 1:2 at

700C for 2 hours

Excess water drained off and

steamed at 1000C for 1 hour.

Dried in the shade until

moisture content <12%

Milled for 120s

Study 1: Visual Consumer

Acceptance Study of uncooked

fortified parboiled rice

Fortificant (mg/300g rice) added to

soaking water: FA- 150mg; BC- 62.5

mg; Fe- 25 mg.

Samples:

1. Commercial parboiled rice

2. Commercial White rice

3. Fortified diluted rice

4. Unfortified parboiled rice

Study 2: Consumer Acceptance Study

of cooked fortified rice after diluting.

Fortificant (mg/300g rice) added to

soaking water: FA- 150mg; BC- 62.5

mg; Fe- 25 mg.

Samples:

1. Fortified diluted rice

2. Commercial White rice

140

6.2.3 Study 1: Visual consumer acceptance of uncooked fortified parboiled rice

6.2.3.1 Samples preparation

For the first phase of the sensory evaluation four rice samples were presented to 49 untrained

participants/consumers. The samples used were as follows: commercial parboiled unfortified

rice, unfortified commercial white rice, diluted parboiled fortified rice with unfortified white

rice and unfortified parboiled rice. Commercial parboiled rice and commercial white rice

were used as controls. Parboiled rice has a characteristic yellow colour and colour is a major

influential factor for rice purchase from a consumer‘s perspective. The study was performed

with uncooked rice to determine if the rice would be purchased from the supermarket if it

were available commercially (Tomlins et al., 2007). Therefore, as a preliminary step to

understand consumers‘ choice about purchasing fortified parboiled rice from the market,

phase 1 of the sensory evaluation study was performed.

6.2.3.2 Survey Methodology

The four uncooked rice samples as mentioned in the above section were evaluated by 49

untrained consumers who were students and staff of the University of New South Wales,

Sydney, Australia. About 20g of each rice sample packed in transparent Ziploc® pouches

were presented to the consumers with a randomized 3-digit code. A survey questionnaire was

also provided along with the samples for the consumers to fill up. The questionnaire was

divided into 2 parts: the first part included questions about the demography and rice

consumption pattern of the consumers; while Part II evaluated their opinion, likes and

purchase intent of each rice type. Table illustrating the items that were presented in the

consumer questionnaire along with the likert scale is in Appendix (2&3).

For the study to evaluate the purchase intent of fortified rice, a five point purchase intent

scale was used based on Mcdaniel and Gates, (1998) & Kam et al., (2013) i.e. ―Definitely

would not buy‖, ―Probably would not buy‖, ―Undecided Probably would buy‖ and

―Definitely would buy‖ For simpler calculations and convenience of the study ‗Definitely

would not buy‘ and ‗probably would not buy‘ were categorised under ―Not buy‖ (1 and 2).

On the other hand ―Probably would buy‖ and: definitely would buy‖ were categorised into

―Would buy‖ (4 and 5) (Moskowitz, 2004 & Kam et al., 2013).

141

6.2.4 Study 2: Sensory evaluation (tasting) of cooked fortified rice after mixing with

unfortified white rice

6.2.4.1 Sample Preparation

For the second phase of the study fortified rice was prepared by adding folic acid: 150 mg; β-

carotene: 62.5 mg; iron: 25 mg/ 300g of brown rice. The parboiling conditions followed are

described in section 3.2.3. Two samples were provided to the consumers for the sensory

study which included commercial unfortified white rice and fortified rice mixed with

unfortified white rice in the ratio1:10. The two rice samples were cooked and presented to the

consumers for sensory evaluation labelled with a random 3-digit number code.

Preparation of uncooked fortified rice with unfortified white rice was essential to monitor and

ensure that the added nutrient intake from the rice were within the safe upper limits of daily

intake. It has been estimated that in rice eating countries such as India and Bangladesh

approximately 440g of rice is usually consumed on a daily basis (IRRI, n.d.). In Table

6.1(page no. 144) the concentration of folic acid, β-carotene and iron that was retained in the

cooked rice has been presented in µg/440 g of rice. The fortified premix rice and unfortified

white rice were mixed in the ratio of 1:10 in a laboratory food roller mixer (Thermoline

Scientific, ten roller mixer, Model 210 RM) on a lab scale. The mixing ratio was based on the

daily requirement for target populations. The mixing ratio (1:10) was chosen such that it

would be able to provide at least half of RDI for each micronutrient i.e. 400 µg of folic acid,

18 mg of iron and 700 µg of vitamin A i.e. 44 g of fortified rice in 396 g of unfortified rice.

The mixing was done to derive an appropriate concentration of the micronutrients added as

well as to dilute the colour intensity of the fortified rice to increase consumer acceptance. It

also masked any unique aroma of the parboiled rice. Mixing with white rice also ensured that

the consumers will choose the fortified diluted rice over normal white rice.

6.2.4.2 Cooking method

The mixed rice (250 g) was washed thrice in approximately 500 mL of water and drained.

The rice was cooked in an electric cooker (Breville, BRC200, Australia) in a rice: water ratio

of 1: 1.75 for 19 minutes at boiling temperature. It worth noting that there is variation in the

cooking time of the rice where fortified parboiled rice took 24 minutes to cook at room

temperature while when mixed with white rice the cooking time reduced 19 minutes (Section

142

3.2.7). The rice was kept for about 10 minutes in the cooked state to absorb moisture and then

it was kept in the rice cooker at a temperature of 60±1 °C until served.

6.2.4.3 Survey Methodology

The cooked rice was served in a plastic container (approximately 20g) to the consumers and

the samples were labelled with a randomised 3-digit code. Untrained consumers (No: 54)

were recruited and most of them were staff and students from the University of New South

Wales. The sensory evaluation was performed in a partitioned booth under uniform light

conditions and the subjects were not informed about the nature of the study.

The survey questionnaire for the second phase of the study was also similar to the first phase

of the study (Appendix 4). The former part of the questionnaire included demographic

questions about the consumers and about the rice consumption pattern. The latter part of the

questionnaire evaluated their opinion about the cooked rice based on 5 different attributes.

The intensity of each attribute was evaluated on a 5-point just-about-right (JAR) scale. For

convenience sake the 5-point scale was categorized into ―Not enough‖ (1 and 2), ―Just-about

right‖ (3) and ―Too much‖ (4 and 5) (Popper and Gibes, 2004 & Kam et al., 2013).

6.2.5 Data Analysis

6.2.5.1 Study 1: Visual Consumer acceptance of uncooked fortified parboiled rice

To understand and interpret the data obtained from the results of the sensory evaluation

study, statistical tests were performed. For interpreting the results obtained regarding the

perception and liking of attributes in Appendix 2 (question 2-5), one way analysis of variance

ANOVA was used to compare the difference between the samples (including the control).

The average of the means was compared using Tukey‘s (HSD) means comparison test (p<

0.05). To compare the variability among different attributes between general consumers and

consumers who were familiar with parboiled rice, independent samples t-test was performed.

Chi-squared (χ2) statistical test was performed for the purchase intent questions in Appendix

2 (questions 6-7) to compare the response distribution between ―Not buy‖, ―Undecided‖ and

―Would buy‖ between the samples and control and also for the response between general

consumers and consumers who were familiar with parboiled rice. To analyse the effect of

143

additional health benefits associated with the consumers on rice purchase intent it was further

analysed using paired t- test.

6.2.5.2 Study 2: Consumer acceptance of cooked fortified rice after mixing

One way ANOVA was performed for questions regarding the liking of attributes in Appendix

4 (Questions 1, 3,6,8,11,14 and 15) to study the mean differences between samples including

control. Tukey‘s test was performed to compare the means (p< 0.05). Chi square (χ2)

statistical test was performed to compare purchase intent between samples and control

(Question 16-17 (Appendix 4) in Table 6.5). To understand the consumers overall liking (9-

point hedonic scale) in Study 2, penalty analysis was performed. One - way ANOVA and

Tukey‘s test was performed to evaluate significant difference between groups.

Homogeneity of variance within the data was measured on a 10- point intensity scale, 9-

point hedonic scale and 5- point likert scale. Levene‘s test was performed. The data which

were all above p <0.05 showed equal variance across samples. All statistical analysis was

performed using XLSTAT (version 2010).

6.3 Results and Discussion

6.3.1 Analysis of micronutrients in the fortified rice before and after dilution

The parboiled fortified rice with 3 micronutrients is intended to be a premix. Consuming the

fortified rice without dilution would result in overdose of each micronutrient on a daily basis

based on RDI. Therefore it is essential to dilute the concentration of the micronutrients for

consumption. The ratio for dilution was chosen to be 1:10 in order to meet at least half of the

RDI (according to NIH-ODS, 2014 & NHMRC, 2006) for each micronutrient and also to

account for the loss due to milling (120s) and cooking. Concentration of the micronutrients

in the fortified rice (premix and diluted rice – cooked and uncooked) are presented in Figure

6.2. Assuming that approximately 440g of cooked rice is consumed on a daily basis by rice

eating populations, folic acid in the fortified diluted rice was able to meet more than 100 % of

RDI- close to the upper limit and for β-carotene about half of RDI and just about the RDI for

iron (Table 6.1). Therefore, this dilution ratio was considered reasonable based on the

concentration of the added nutrients of the cooked fortified rice diluted with white rice. In the

following sections, the results from the sensory evaluation have been presented and discussed

to understand if this dilution factor of the premix influences the acceptance of the fortified

rice in terms of purchase intent and taste.

144

Table 6.1 Dietary Reference Intake for the selected micronutrients (Vitamin A, folic

acid and Iron) (Dietary reference intake: Elements and Minerals, (NIH-ODS, 2014 &

NHMRC, 2006))

Micronutrients

Concentration in

440g of cooked

diluted fortified

rice

Adequate Intake Upper limit

Vitamin A (retinol

equivalents, precursors

of carotenoids)

334.4 µg

(β-carotene)

Infants (1-3 years old–

pre-schoolers) 300 µg/day 600 µg/day

Male (19-50 years old) 900 µg/day 3000 µg/day

Female (19 – 50 years

old) 700 µg/day 3000 µg/day

Folic acid 1100 µg


pre-schoolers) 150 µg/day 300 µg/day

Male (19-50 years old) 400 µg/day 1000 µg/day


old) 400 µg/day 1000 µg/day

Iron 19.8 mg


pre-schoolers) 7 mg/day 40 mg/day

Male (19-50 years old) 8 mg/day 45 mg/day


old) 18 mg/day 45 mg/day

145

Figure 6.2 Concentration of micronutrients (in d.w.b.) in fortified premix (cooked and uncooked) and diluted fortified rice (cooked and

uncooked)

0100020003000400050006000700080009000

Diluted

Uncooked

Diluted

Cooked

Premix

Uncooked

Premix

CookedCon

cen

trati

on

in

µg/1

00g

Folic acid concentration in fortified rice

012345678

Con

cen

trati

on

in

mg/1

00g

Iron concentration in fortified rice

154

76

211194

0

50

100

150

200

250

Diluted

Uncooked

Diluted

Cooked

Premix

Uncooked

Premix

Cooked Con

cen

trati

on

in

µg/1

00g

β-carotene concentration in fortified rice

146

6.3.2 Study 1: Visual Consumer Acceptance of uncooked fortified parboiled rice

6.3.2.1 Demographics and rice eating pattern of consumers

For part 1 of the consumer acceptance study a total of 49 people were recruited comprising

comprised of 39% men and 61% women. Majority of the participants were Asian (71.4%)

and the rest were from Australia or other countries mentioned in Table 6.2. Majority of the

people belonged to the age group of 20-30 years (75.5%) and 72% of this cohort consumed

rice on a daily basis or at least 2-3 times a week. Hence, this population seemed suitable for

this study as they were representative of the rice-eating populations. Among various types of

rice, white rice was consumed by 94% of the selected subjects which indicated that these

consumers were also representative of white-rice eating populations. It is also to be noted that

none of the recruited consumers ate parboiled rice though about half of the cohort (52%)

were aware of the availability of parboiled rice.

147

Table 6.2 Socio-demographic characteristics of the participants of consumer acceptance

Study 1 and Study 2

Demographic variable Percentage (%)

(Study 1 n=49) (Study 2 n= 54)

Gender

Men 39 26

Women 61 74

Age

<19 2 1.8

20-30 75.5 81.4

31-40 18.3 12.9

41-50 0 1.8

51-60 2 1.8

Nationality

Australian 20.8 24

Chinese 22.9 20.3

Indonesian 27 16.6

Indian 2.08 7.4

Malaysian 4.16 12.9

Others (Iranian, Bangladesh, British, Singapore,

Thailand, Samoan, New Zealand, Filipino) 22.9 14.6

Employment Status

Casual 20.4 42.5

Full time 30.6 35.1

Part time 10.2 5.5

Unemployed 36.7 16.6

148

Table 6.3 Rice consumption pattern of participants of the consumer acceptance study 1

and study 2

Item Percentage (%)

Study 1 (n= 49) Study 2 (n=54)

Type of rice consumed usually

White rice 93.7 72.2

Brown rice 6.2 18.5

Red/Purple/Black rice

7.4

Parboiled rice

1.8

Consumption frequency

Daily 72.9 72.2

At least once a week 20.3 18.5

At least once a month 2 3.7

Rarely 4.1 5.5

Methods to cook rice

Rice cooker 100 88.8

Rapid cooking (Cooking in excess water)

3.7

Absorption cooking ( cook in a pot)

7.4

Heard about parboiled rice?

Yes 52 75.9

No 47.9 24

6.3.2.2 Degree of visual acceptance of fortified diluted uncooked rice

Figure 6.3 shows the hedonic preferences of rice samples for visual acceptance attributes

(commercial parboiled rice, commercial white rice, fortified diluted rice and unfortified

parboiled rice). The sample of interest was fortified diluted rice and the aim of phase 1 of

consumer acceptance study was to investigate if the participants of the study showed any

preference based on colour and appearance of the fortified diluted rice and their purchase

intent.

149

Figure 6.3 Preference of visual attributes investigated in Study 1 tested by participants (n=49) presented on a hedonic scale

Note: Uniformity represents the appearance based on the colour of the rice samples.

02468

10

Commercial

parboiled rice

Commercial

white rice

Fortified

diluted rice

Unfortified

Parboiled

Uniformity in appearance

02468

10

Commercial

parboiled

rice

Commercial

white rice

Fortified

Diluted rice

Unfortified

Parboiled

Pre

fere

nce

on

a h

ed

on

ic

scal

e

Colour preference

0

2

4

6

8

10

Commercial

parboiled

rice

Commercial

white rice

Fortified

Diluted Rice

Unfortified

Parboiled

Pre

fere

nce

in h

ed

on

ic s

cale

Colour Intensity

0

2

4

6

8

10

Commercial

parboiled rice

Commercial

white rice

Fortified

diluted rice

Unfortified

Parboiled

Overall acceptance of

appearance

150

As it can be seen from the Figure 6.3, uniformity of the samples based on colour for fortified

diluted rice had the lowest score indicating that there is evident heterogeneity in the sample.

The interesting observation regarding uniformity was that commercial parboiled rice gained

the highest score compared to commercial white rice which was the most common type of

rice consumed by the participants of the study. Parboiled rice from this study was also

accepted to be uniform compared to the fortified diluted rice. This shows that consumers

generally prefer uniformity in colour and they are willing to purchase despite the colouration

as long as they are uniform.

In terms of colour intensity, unfortified parboiled rice was considered to have the highest

intensity compared to the other samples. Colour intensity of unfortified white rice was similar

to the fortified diluted rice. Hence it can be interpreted that unfortified white rice was able to

lessen the intensity of colour of the parboiled rice and could be chosen as an appropriate

diluent for the premix. When it came to colour preference clearly most consumers preferred

commercial white rice over the other samples and the least preference was observed for the

fortified diluted rice. Commercial parboiled rice was preferred second to commercial white

rice indicating that despite many of the participants not being familiar with parboiled rice; it

was preferred and is possible for consumers to accept it even if not mixed with white rice. For

the overall acceptance fortified dilute rice was least preferred among the 4 samples. Therefore

it can be said that appearance is an important factor for consumer acceptance and since the

fortified rice was diluted with white rice there was a major impact on the uniformity of the

sample. Although the concentration of micronutrients in the fortified rice diluted with white

rice met the RDI requirements (Table 6.1) the dilution factor influenced the consumer

acceptance behaviour. The fortified premix diluted with commercial parboiled rice is likely to

be preferred over dilution with white rice as consumer acceptance study indicates that

commercial parboiled rice was the second preference after white rice for consumers.

6.3.2.3 Evaluation of purchase intent of uncooked diluted fortified rice with and without

nutritional information

As mentioned in the questionnaire (Appendix 2) two consecutive purchase intent questions

were asked of the fortified rice to understand if people would buy the fortified parboiled rice

diluted with unfortified white rice when sold commercially in supermarkets. Results from the

present study showed that 67% of people were willing to buy commercial white rice if it were

fortified with micronutrients. However, only 24.5% were willing to buy the fortified diluted

151

rice. Moreover t-tests revealed that the purchase intent for fortified parboiled rice was

significantly different from the commercial white rice. When subsequently asked about

purchase intent of the rice that might offer better nutritional value the likelihood of purchase

of the fortified rice increased. About 67% of the recruited population said that they would

probably buy the diluted fortified rice. But, 88% of the people still preferred to buy

commercial white rice if it were fortified. This result was unfavourable in terms of success of

the fortification study. One of the reasons for this was that consumers could detect fortified

rice in the diluted mixture as a speck or discolouration indicating a quality defect that may be

a factor in rejecting the lot. This was the reason for low purchase intent compared to

commercial white rice as reported by the prospective consumers who participated in the

study.

When Chi square (χ2) test was performed for the response distribution of purchase intent

before and after mentioning the health benefits of the rice it was found that there were

significant differences before mentioning the health benefit between the control white rice

and the diluted fortified rice (p= 0.002; p≤0.05). However, after mentioning about the

fortification there was no significant difference between the control sample and the diluted

fortified rice (p =0.9; p≤0.05). Moreover the results from the Chi square test (χ2) when

analysing the response distribution for purchase intent for each rice sample before and after

indicating that they were fortified, did not show any significant difference for commercial

parboiled and white rice but there was significant difference for diluted fortified rice and

unfortified parboiled rice. Hence it can be interpreted that based on the response distribution

after notification of ―health claim‖ there was an increase in the number of people who

preferred to buy the diluted fortified rice. Overall the results indicate that consumers were

willing to buy the diluted fortified rice if they got prior information about the health claim.

152

A) B)

Figure 6.4 The distribution of purchase intent responses (%) in Consumer Acceptance

Study - STUDY 1 tested by participants (n=49). A) Before the notification of health

claim; B) After the notification of health claim

6.3.2.4 Evaluation of purchase intent of uncooked fortified rice- consumers who were

familiar with parboiled rice

The perception of people who consumed parboiled rice varies compared to those who do not

consume parboiled rice as they are more familiar with the distinct colour and flavour of

parboiled rice (Prom-u-Thai et al., 2009b). The specific target population in this study would

be people who consume parboiled rice as there is a specific market for it. To gain a deeper

perspective on purchase intent among people who had previous knowledge of parboiled rice a

further study was conducted (n=26).

0102030405060708090

Per

cen

tag

e o

f re

spo

nse

Purchase Intent before notification of

health claim

Commercial

parboiled rice

Commercial

white rice

Fortified

diluted rice

Unfortified

Parboiled

0102030405060708090

100

Per

cen

tag

e o

f re

spo

nse

Purchase Intent after notification of

health claim

Commercial

parboiled rice

Commercial

white rice

Fortified

diluted rice

Unfortified

Parboiled

153

Table 6.4 Mean perceptions of appearance for uncooked rice sample- diluted fortified

rice and p-values (p≤0.05) of t-test on comparing the diluted fortified rice sample with

Control – commercial white rice (Tested by consumers in the Consumer Acceptance

Study (colour intensity, degree of liking of colour, uniformity of colour, overall

appearance))

Colour

intensity

p -value

for t-test

Colour

preference

p-value

for t-test Uniformity

p-value

for t-

test

Overall

acceptance

p-value

for t-

test

General

consumers 5.1±1.0

0.5

4.6±1.6

0.7

3.6±1.6

0.19

4.6±1.1

0.5 Consumers

familiar

with

parboiled

rice

4.9±1.6 4.7±1.1 4.4±2.1 4.3±1.6

Note: General consumers (n=22) and those who were familiar with parboiled rice (n=27))

The mean liking scores for the appearance attributes (colour intensity, degree of liking of

colour, colour uniformity and degree of overall liking) are presented for the diluted fortified

rice in Table 6.4. The overall mean for the two groups – general consumers and consumers

familiar with parboiled rice was not very different. However on performing t-test for

individual parameters, except for colour preference there was no significant difference for the

other three attributes between the two groups. This shows that there is not much difference in

the way of perception of the fortified rice when people had prior experience or knowledge

about parboiled rice for individual parameters.

Moreover, when the response distribution was analysed statistically using Chi square test (χ2)

there was no significant difference between the general consumers and consumers who were

familiar with parboiled rice in terms of their purchase intent before and after the health claim

for the diluted fortified rice (p=0.9). This was further investigated by performing paired t-test

for investigating the additional health claim between the 2 groups. When the sample of

interest – diluted fortified rice was compared with the control – commercial white rice and

paired t-test was performed to understand additional health claim benefits for consumers who

were familiar with parboiled rice and general consumers there was significant difference for

both groups (p= 0.03- general consumers; p=0.04-people familiar with parboiled rice) before

154

and after mentioning the health claim indicating that both these groups of consumers are

more likely to purchase the fortified diluted rice compared to commercial white rice. For

other samples there was no significant difference between the two groups and also within and

between samples. In this study therefore it can be said that the willingness to buy was

independent of people‘s prior experience with parboiled rice. However the willingness to buy

diluted fortified rice increased after mentioning the health claim.

Study 2: Consumer acceptance of cooked fortified rice after mixing

6.3.2.5 Consumer demographics and rice eating preferences

For Study 2, a total of 54 participants were recruited and this comprised of 26% men and

74% women. The demographics of consumers recruited for Study 1 and 2 were similar.

Majority of the participants belonged to the age group of 20-30 years (81%). From the

demographic data 68.5% were of Asian origin and the others identified themselves as

Australian, British, Samoan etc. Over 70% of these people consumed rice on a daily basis or

at least 2-3 times a week. Hence, this cohort was also considered to be representative of rice -

consuming populations.

6.3.2.6 Degree of liking of cooked rice attributes

Table 6.5 presents average liking of the rice sensory attributes (appearance, colour, odour,

texture, taste and aftertaste) after cooking. Overall there was no significant difference

between the control white rice sample and the test sample (diluted fortified parboiled) for any

attribute. Unlike for the uncooked rice, the cooked rice appeared to be uniform in colour.

Also it has to be noted that for each attribute the average for the test sample i.e. the diluted

fortified rice was slightly higher than the control white rice indicating that the fortified rice

after cooking was better liked than the control white rice by the taste panel. In terms of

tasting, none of the panel members commented on any specific difference or adverse taste

such as being ―metallic‖ which is highly likely in iron fortified foods. Hence, it can be said

that in terms of key sensory attributes, both the fortified rice and control white rice appeared

and tasted similar and therefore, it is highly likely to be accepted by the wider community

after cooking.

155

Table 6.5 Mean degree of liking score for sensory attributes (appearance, colour, odour,

texture, taste, aftertaste and overall liking) of rice samples tested by consumers in Phase

2 of the Consumer Acceptance Study (n=54)

Attributes Diluted fortified rice Control White rice

Appearance 6.5±1.1 6.4±1.1

Colour 6.2±1.2 6.4±1.3

Odour 6.2±1.3 6±1.2

Texture 6±1.2a,b

5.6±1.2a,b

Taste 6.2±1.2a,b

5.7±1.3a,b

Aftertaste 5.7±1 5.4±1

Overall 6.2±1.2 6±1.2

Note: Values indicate Mean ± standard deviation for the sensory attributes

Values followed by (a,b) indicate significant difference of means at p ≤0.05.

6.3.2.7 Consumers’ perception of attributes JAR (Just about right)

For an attribute to be acceptable and nominal it is generally expected that at least 70% of the

response from the consumers lie in the JAR region (Meullenet et al., 2007). From figure 6.5 it

can be said that about 70% of the attributes evaluated in the sensory study is prevalent in the

JAR area. Colour change that was evident in uncooked rice when fortified rice was diluted

with white rice was not evident when the rice was cooked and hence not considered as a

foreign particle. The acceptability of the product thus increased. According to Hurrell (1997),

iron-fortified rice was disliked by many consumers since the rice was black in colour and it

was considered as specks from the bulk of rice. This scenario was not apparent in the given

study. Also there was no significant difference in colour between the control white rice and

the fortified rice in this study which clearly showed that after cooking, colour was no longer a

concern for consumer acceptability.

When the texture of the control and fortified rice was compared, there was no significant

difference once again between the two samples (p ≤ 0.05). Hardness and stickiness are the

two parameters that define texture of rice. For hardness there was no significant difference

between the two groups however for stickiness there was significant difference indicating that

the fortified rice did affect the overall texture of the diluted fortified rice. Normally parboiled

rice is firmer and harder in texture compared to white rice (Bhattacharya, 2004). This

156

phenomenon was not observed when the fortified parboiled rice was mixed with white rice in

the ratio 1:10. Thus the texture of the fortified rice was comparable and quite similar to

commercial white rice.

For taste intensity and after-taste, there was no significant difference statistically between the

two samples. However for taste preference there was significant difference. This indicated

that due to the presence of parboiled rice there is alteration in the normal rice taste which can

be perceived by consumers. Similarly for odour the overall preference did not show

significant difference, however there was significant difference for odour intensity indicating

that parboiled rice imparts particular flavour even after dilution with white rice which

seemingly influenced consumer perception. Since the attributes fell in the JAR region and

also overall fortified rice was preferred over white rice it can be said that fortified rice is

acceptable by consumers although there were minor differences when compared with

commercial white rice.

157

Figure 6.5 Bar graph showing consumer preference of cooked fortified rice compared

with unfortified rice

0

20

40

60

80

Not Enough JAR Too Much

% F

req

uen

cyUniformity

Fortified

Rice

Unfortified

Rice0

20406080

% F

req

uen

cy Taste

Fortified Rice

Unfortified

Rice

020406080

% F

req

uen

cy Aroma

Fortified Rice

Unfortified

Rice

0204060

% F

req

uen

cy

Stickiness

Fortified

Rice

Unfortified

Rice

020406080

% F

req

uen

cy

Hardness

Fortified rice

Unfortified

rice

0

50

100

% F

req

uen

cy

Colour Intensity

Fortified Rice

Unfortified

Rice

5.95

6

6.05

6.1

6.15

6.2

6.25

Fortified Rice Unfortified rice

Pre

fere

nce

on

a H

edon

ic

scale

of

10

Overall Liking

158

6.3.2.8 Evaluation of purchase intent of cooked rice

The purchase intent of fortified rice was classified into ―Not buy‖, ―Undecided‖ and ―Would

buy. From Figure 6.6 it can be seen that the distribution of the response skewed towards

‗would buy‘ for both fortified and unfortified rice. ―Willingness to buy‖ showed no

significant difference when fortified rice was compared with white rice when t-test was

performed. Similar results were obtained for the chi square (χ2) test for response distribution

as well and there was no significant difference. Experimental data from Figure 6.6 shows that

consumers preferred to buy fortified rice compared to unfortified rice and this was higher

after mentioning the health claim. Fortified rice was equally accepted as unfortified rice in

terms of purchase intent. It has to be noted that fortified rice was mixed with white rice

instead of parboiled rice and hence there is a possibility that it can be less preferred because

of the white rice background. Despite this, the data from the sensory study shows that it is

apparent that people would prefer to buy the fortified rice. From the demographic survey in

this study about 75% of the people were aware of parboiled rice. So if the fortified rice was

mixed with unfortified parboiled rice, this population would have accepted the rice anyway.

As parboiling of rice is quite common in many rice eating countries, such populations will

not have difficulty in accepting fortified parboiled rice and frequent consumption will

guarantee adequate intake of the added nutrients.

159

a.

b.

Figure 6.6 (a & b) Graph showing purchase intent of fortified rice before and after

mentioning about fortification

0

10

20

30

40

50

60

70

80

Purchase Intent (before

mentioning fortification

Purchase Intent (after

mentioning fortification)

% F

req

uen

cy

Purchase Intent for Unfortified rice

Would not Buy

Undecided

Would buy

0

20

40

60

80

100

Purchase Intent (before

mentioning fortification

Purchase Intent (after

mentioning fortification)

% F

req

uen

cy

Purchase Intent for Fortified rice

Would not Buy

Undecided

Would buy

160

6.4 Conclusions

Retention analysis of micronutrients in fortified cooked rice showed that the dilution ratio of

1:10 was optimum and was able to meet half of the RDI for β-carotene (700 µg), just about the

RDI for iron (18 mg) and more than 100% of RDI for folic acid (400 µg) (NIH-ODS (USA),

2014, NHMRC (Australia), 2006).

Visual acceptance of uncooked fortified rice was performed in Study 1. People who were

familiar and unfamiliar with parboiled rice both accepted diluted fortified rice. However,

commercial white rice was the most acceptable among all. The colour intensity and uniformity in

appearance were the two factors where the diluted fortified rice was scored down due to

variation in rice kernel caused by the fortification process. On the other hand, commercial and

unfortified parboiled rice was also widely accepted by the consumers indicating that people are

willing to purchase parboiled rice and hence the process can be successful as a method of

fortification. By declaring the health claim of the fortified rice, the willingness to buy increased.

However this aspect did not influence the decisions of people who were familiar with parboiled

rice. The primary reason for this would be the non-uniformity in the parboiled rice. To overcome

this issue innovative packaging that would be able to mask this variation could be implemented

as this could possibly overcome the issue of non-uniformity in rice samples and influence the

purchase intent decision in a positive way between general consumers and people who were

familiar with parboiled rice to a higher extent.

In Study 2, the cooked rice was assessed for sensory acceptance of taste and it was deduced that

after cooking there was no variation between commercial white rice and fortified diluted rice.

However, there was some slight differences observed with odour intensity and stickiness

compared to white rice. But these were covered by the JAR responses, which indicated that the

fortified rice had better or comparable taste attributes to the white rice. Also another notable

finding was that the fortified diluted rice had overall better acceptance compared to the

commercial white rice indicating that parboiling itself caused favourable taste on cooking

thereby increasing consumer acceptance. The ultimate success of any fortified new product

would be acceptability by consumers followed by bioavailability after consumption. Therefore,

from the data shown in chapter 3 and the sensory study, it is reasonable to study the

bioavailability of fortified rice at the optimised condition (70°C and 2 hours soaking). By

161

performing bioavailability studies (discussed in the following chapter-7), essential information

on how much of the fortified micronutrient would actually be bio-available can be estimated.

162

CHAPTER 7

7. SHORT TERM RELATIVE BIO-ACCESSIBILITY AND ABSORPTION OF FOLIC

ACID, IRON AND β – CAROTENE IN FORTIFIED PARBOILED AND HIGH

PRESSURE PROCESSED RICE: CACO-2 CELL STUDY

7.1 Introduction

Understanding the bioavailability of the micronutrients fortified in the rice by the two different

processing methods will be the ultimate mark of the effectiveness of the fortification and thereby

giving an estimate of the absorption by humans. Bioavailability studies are expensive although

they can provide relative estimates of actual absorption in humans. This study focussed on

obtaining bio-accessibility data using Caco-2 cell lines. Caco-2 cells are adenocarcinoma cells

from the human colon. Well isolated and differentiated primary cell lines were utilized for this

experiment. These cells when grown in an appropriate medium can proliferate and differentiate

into enterocytes (epithelial cells of the small intestine) (Said, 2011; Simon- Assmann, Truck,

Sidhoum- Jenny, Gradwohl & Kedinger, 2007). Cell lines such as T-84 and HT-29 based on

literature have been found to be suitable for in vitro studies. However, caco-2 cells were the most

commonly used for drug interaction and absorption studies (D‘Souza, Shertzer, Menon &

Paultetti, 2003).

When the Caco-2 cell lines become confluent they form a polarized monolayer resembling the

brush border segment of the intestine with tight junctions and therefore can be convenient to

study drug metabolism in vitro (Travelin, 2002; Simon-Assmann, Truck, Sidhoum- Jenny,

Gradwohl & Kedinger, 2007). Netzel et al., (2011) studied release and absorption of carotenes

from processed carrots; O‘Callaghan & O‘Brien, (2010) studied bio-accessibility, cellular uptake

and trans-epithelial transport of α-tocopherol and retinol from a range of supplemented foods;

Ohrvik, Talkvist & Witthoft, (2010) investigated folate bio-accessibility at an in vitro level in

breads; Rodriguez-Amaya, (2010) reviewed in vitro assessment of bioavailability and

antioxidant activity of food carotenoids; Sy, Gleize, Dangles, Landrier,Veyrat & Borel, (2012);

Verwei, Van Den Berg, Havenaar & Groten, (2005) analysed the effect of folate-binding protein

on intestinal transport of folic acid and 5-methyltetrahydrofolate across Caco-2 cells. A validated

system for in vitro absorption to predict in vitro permeability and absorption using caco-2 cells

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has been well designed for experimental purposes by Delie & Rubas, (1997); Fernandez-Gracia,

Carvajal- Lerida & Perez- Galvez, (2009) and Simon-Assmann, Truck, Sidhoum-Jenny, Grawohl

& Kedinger, (2007). This chapter has been divided into the following sub-sections: reagents

required for cell culture maintenance, procedure for resuscitation of the cell line, sub-culturing of

the cells, in vitro digestion model, optimization of the in vitro digestion and absorption model for

folic acid, iron and β – carotene from the fortified rice from parboiling and HPP processes. The

aim of this chapter was thus was to study the bio-accessibility of micronutrients (folic acid, iron

and β-carotene) from the fortified rice processed by parboiling and HPP technology using in

vitro techniques. The results from the transport study will also be presented which may provide

future insight into bioavailability studies of nutrients from foods.


7.2.1 Reagents for Cell Culture Resuscitation and Maintenance

Human colon carcinoma cell lines namely, Caco-2 cells were a kind gift from the Lowy Cancer

Centre (The University of New South Wales). The viable cells were used between passage (sub-

culturing of viable cell population) numbers 30 to 50. The growth medium for the cells was

composed of Dulbeccos modified eagle medium (DMEM), foetal bovine serum (FBS), penicillin

(5000 u/mL), streptomycin (5000 µg/mL), non-essential amino acids and GlutaMAX™ and

these were purchased from Invitrogen (Sydney, NSW, Australia). The growth media composition

was as follows: 435 mL of DMEM, 10 % (v/v) FBS, 5 % (v/v) non-essential amino acids, 5 %

(v/v) Penicillin/Streptomycin and 5 % (v/v) Glutamax, and stored in the fridge for usage.

7.2.2 Reagents for Cell Viability Assay

Lucifer yellow (LY) which was non-toxic to the cells were obtained from Sigma-Aldrich

(Sydney, Australia). The cyto-toxicity assay was performed in 96-well microtitre plates

(Nunc™) obtained from Thermo Fischer (Sydney, Australia) and the integrity of the caco-2

monolayer assessed using LY was analysed using a black titre plate supplied by BD Biosciences

(Sydney, Australia). Cell viability and toxicity assays were performed using CellTiter 96 ®

a non-

radioactive cell proliferation assay (MTT) purchased from Promega (cat no. G5421). The assay

was performed using MTS ([3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

(4sulfophenyl) -2H-tetrazolium) and PMS (phenazine methosulfate (PMS), respectively). The

164

assay solutions – MTS and PMS were thawed and warmed in a 37 0C water bath and the PMS

solution was then added to the MTS solution and 1200 µL of this solution was used for the cyto-

toxicity assay.

7.2.3 Reagents for in vitrodigestion

Fortified rice samples were digested using enzymes that would be secreted in the human system

to mimic in vivo digestion. The enzymes used were α-amylase, hog bile extract, porcine

pancreatin purchased from MP Biomedicals (Solon, OH). Proteolytic enzyme – pepsin (15.2

mg/mL) was prepared by dissolving in 0.1 M Hydrochloric acid (Univar, Ajax-Finechem,

Sydney, Australia). Post-gastric enzymes pancreatin (2 mg/mL) combined with hog bile extracts

(11.7 mg/mL) were dissolved in 0.1 M NaHCO3 (Univar, Ajax-Finechem, Sydney, Australia)

for in vitro digestion. The enzyme mixture was vortexed and placed in a 37 °C water bath for 15

minutes. Enzyme α-amylase (20 mg/mL) was prepared by vortexing in Milli-Q water and

centrifuged at 10,000 g at room temperature for 10 minutes (Chandra- Hioe et al., 2013).

7.2.4 Transport Study

Transport study was performed using 24-well transwell plates which consisted of 12 permeable

wells (12 mm insert diameter) with polycarbonate membranes which were purchased from

Corning (Sydney, Australia). Trans-epithelial electrical resistance (TEER) which was used to

measure integrity of the monolayer of caco-2 cells was purchased from Millipore (Billerica,

MA). Hank‘s Balanced Salt Solution (purchased from Life Technologies, Sydney Australia) was

added to the outer well of the transwell plates to maintain humidity for the cell growth during

incubation period.

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Figure 7.1 Schematic representation of Transwell permeable support with Caco-2 cells

grown on the membrane

7.2.5 Cell Culture & Maintainence Protocol

7.2.5.1 Procedure for resuscitation of frozen Caco-2 cells

Frozen Caco-2 cells presented in an ampoule (1mL) was removed from liquid nitrogen and

rapidly thawed in a 37°C water bath as this avoids osmotic damage to cells and improves cell

viability (Gull et al., 2009). The premixed growth media (~ 10 mL) was warmed in 37°C water

bath and added to the thawed cells and the cells were centrifuged to remove the dimethyl

sulfoxide (DMSO, Sigma-Aldarich, Australia). Cells were grown in 75 cm2

flasks (EasyFlasks™

,NUNC, Thermoscientific, Denmark) until it was 70% confluent. After the cells have reached the

desired confluence level (based on microscopic observation) they were split for them to be viable

until the assay was performed.

7.2.5.2 Sub-culturing of Caco-2 cells

Sub-culturing of the Caco-2 cells is essential for maintaining the cells in the optimal growing

phase and it was performed twice a week. This was performed by removing the old growth

media followed by washing of the cells with about 10 mL of Dulbecco‘s Phosphate-buffered

saline (DPBS) with no Calcium and Magnesium. This was done to remove serum which contains

Trypsin inhibitors and also other cell debris. Following this about 3 mL of trypsin (0.25%)-

EDTA was added to detach the cells from the 75 cm2

flasksurface and the cells were incubated at

37°C with 5% CO2 for about 3 minutes. Then about 10 mL of the growth media was added and

the cells were centrifuged (Boeco U-320, Hamburg, Germany) at 2000 rpm for 5 minutes. After

166

centrifugation the supernatant was discarded and the cells were re-suspended in 10 mL growth

medium. The cells were split in the ratio of 1:10 in a T75 flask and incubated at 37°C with 5%

CO2 thereby allowing sub-culturing approximately in 4 days.

7.2.5.3 Cell Density

Cell density was calculated per mL of the growth media using a haemocytometer by visualizing

and counting the cells under a microscope. Trypan blue was the dye used to distinguish the cells

in an exclusion assay. The principle of the assay is that Trypan blue has the property to dye non-

viable cells when their cell walls are not intact. On the other hand viable cells are excluded from

the dye and thereby distinguished from the non-viable cells (Stoddart, 2011). It is essential to

keep a count of the cell density as it is vital for cell sub-culturing, freezing down, and seeding on

the transwell membrane for the transport study. During cell passage process 1 mL of the

dispersed cells was retained separately for calculating cell density. The cells were then diluted

with Trypan blue in the ratio of 1:5 or 1:6 depending on the number of cells that could be

observed under the microscope. The dyed cells were then mounted on the haemocytometer with

the cover slip on it. The cells were fixed between the cover slip and the haemocytometer

inscription by capillary effect and were then counted immediately after loading them as the dye

is toxic to the cells and could lead to death if delayed. Figure 7.2 is a diagrammatic

representation of the chambers in a haemocytometer (Louis & Siegel, 2011). The cell density

was henceforth calculated depending on the number of viable cells according to the following

formula:

Cell density/ mL = Total no. of cells from the 4 quadrants x dilution factor x 104

4

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Figure 7.2 Diagrammatic representation of the chamber of a haemocytometer (Louis &

Siegel, 2011)

7.2.5.4 Freezing down Cells

For long-term storage of the viable cells and maintaining the inventory of the caco-2 cells they

were frozen down in liquid nitrogen. To do this the re-suspended cells from an early passage

number (24- 30) were centrifuged to remove the growth medium and the pellet was re-suspended

in cold FBS (90%) supplemented with DMSO (10%) and mixed gently. The cells (containing 3 x

106 / mL) were then aliquoted into a cryo-tube (Thermo Fisher, Sydney Australia). The cells

were frozen gradually by freezing them first in a -20ºC freezer and then freezing them overnight

at -85ºC and then the tubes were submerged in liquid nitrogen stored in a cryogenic vessel.

Under this condition the cells are ever-lasting as long as the liquid nitrogen is replenished until

the cells are frozen.

7.2.6 Sample Preparation

7.2.6.1 In-Vitro Digestion Protocol

The protocol for performing in vitro digestion was followed according to Netzel et al., (2011)

with some modifications. Fortificant mix (150 mg of folic acid, 62.5 mg of β-carotene, 25 mg of

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iron / 600 mL of distilled water) and cooked rice in duplicates as mentioned previously in section

(3.2.7) for analysis of folic acid, iron and β – carotene was used. To the cooked rice about 5 ±

0.01 g 2.5 mL of lukewarm de-ionized water was added along with 5 mL of α- amylase (20

mg/mL, pH 6.56) into 50 mL centrifuge tubes. The tubes were vortexed and placed in a 37˚C

shaking water bath for 5 minutes. The action of α- amylase was to mimic the hydrolysis of starch

in the mouth. Following this the pH of the samples was adjusted to 2.1-2.2 with 0.1 M HCl. The

gastric digestion was initiated with pepsin (250 µL) in the shaking water bath at 370C for 1h.

Following this the pepsin was inactivated by increasing the pH to 5.7 with 0.1 M NaHCO3 (pH

8.43) and 12 mM calcium chloride (pH 6.42). The mixture was incubated in the water bath at

37˚C for 30 minutes. The mixture was then treated with pancreatin and bile extract (1 mL) for 2h

in the 37˚C water bath after adjusting the pH to 6.8- 7 (gastric pH) with a mixture of 0.1 M

NaHCO3 and 12 mM CaCl2 (pH 8.07). The resulting mixture will be referred to as intestinal

digesta from this point of the document. The digesta was centrifuged at 10000 g for 20 min (at

4˚C) and the supernatant was collected and stored at -80˚C until further analysis.

As a negative control nil digesta was prepared and as a positive control the fortificants in

solution were also prepared and digested using the above protocol. In addition, for analysis of β –

carotene 0.5 mL of soy bean oil, vegetable oil and canola oil were added individually to the

digesta in order to improve the efficiency of the digesta to extract β – carotene. The efficiency of

extraction of β – carotene was determined between the three oils.

7.2.6.2 Cell Viability Assessment

It is important to understand the cell viability because non-viable or lysed cells could detach

from the filter at the washing stage which is mentioned below resulting in an increased filter

exposure during the transport study thereby affecting the movement of the substance from the

apical compartment to the basolateral compartment leading to a false positive result. In order to

ensure that the cells utilized in the transport absorption model are healthy it is vital to analyse the

cell viability (Stoddart, 2011). A 96 well titre plate was used and the caco-2 cells were seeded on

the wells of the plate with a cell density of 1 x 105

cells/well. The plates were incubated at 37°C

for 1 week to allow the cells to attach and differentiate. After the mentioned incubation time the

cell culture medium was removed and 100 µL of the digesta (prepared as mentioned in section

7.3.3.1) was added to the well. As a positive control Hanks Balanced Salt Solution (HBSS) was

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added to the wells with confluent cells and as a negative control H202 was added to the wells

without any cells in them.

The rice digesta, nil digesta and the fortificant digesta were all assessed for toxicity. The positive

control was HBSS added to the cells and the negative control was HBSS without the cells and

H202 added to the cells. To assess the cytotoxicity caco-2 cells were cultured in a 96 well plate

with a cell density of 1 x 105 cells per well. The outermost border wells contained HBSS in order

to maintain moisture for the cells (Addepalli & Osborne, 2011). The plate with the seeded cells

was incubated at 37˚C in a humidified atmosphere with 5% CO2 for a period of one week.

After the incubation period Lucifer yellow (LY) prepared on the day of analysis was used to test

cell toxicity. A standard LY with a concentration of 1 mM was prepared by dissolving 4.6 mg in

10 mL of HBSS. The stock solution was stored at -20˚C for future use. The stock solution was

diluted further at x 6 dilution (0.025-1 mM). The working solution was stored in the dark while

the 96- well plates were prepared. For the assay the growth media from the wells were removed

and the cells were washed with 100 µL of HBSS. Following this LY (100 µL) was added to the

wells in increasing concentrations in triplicates along with the sample namely rice digesta, nil

digesta and fortificant digesta. The plates were incubated at 370C and 5% CO2 for 1-2 hours. As

a positive and negative control, HBSS (100 µL) was added into the wells with cells and without

cells. Post incubation cell viability was tested using a commercial CellTiter 96® non-radioactive

cell proliferation assay (MTT) which was was used to assess the cell viability in response to the

in vitro digesta using a colorimetric method. The protocol was followed as provided by the

supplier. This colorimetric assay consisted of MTS and PMS. The aliquot of MTS/PMS was

thawed and 20 µL of this was added into each well and mixed thoroughly by pipetting. The

plates were again incubated at 37°C for 2 hours for colour development. The absorbance was

measured at 485 nm and the data was recorded using an ELISA plate reader (Spectra Max M2,

Molecular device, Australia).

7.2.7 Transport & Permeability Study

According to Addepalli & Osborne (2011) the cells have to be split at least thrice before being

seeded on to the 24-well Transwell membrane plate. The cell density was then determined using

the haemocytometer as mentioned in section 7.2.2.3 and then the cells were diluted such that the

final cell density of 4 x 105 cells/mL was achieved. The Transwells were then seeded with 200

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µL of the cells which contained 8 x 104 cells and cultured in a 5% CO2 atmosphere at 37ºC for

21 days. For the cell viability and toxicity test LY transport study was conducted in parallel and

for this purpose 4 x 104 cell density was utilized. The basolateral chamber of the Transwell

contained 600 µl of the growth media (Figure 7.1). The first and the last row (which did not

contain the cell or the inserts) of the Transwell contained 1 mL of sterile DPBS for maintaining

humidity within the plate (Addepalli & Osborne, 2011). The growth media in the apical and

basolateral chamber was replaced systematically twice a week to restock the nutrients and

maintain optimum pH for cell growth.

To begin, the transport study was conducted on the 21st day after seeding the Transwell plate.

Firstly the (Trans Epithelial Electrical Resistance) TEER was measured and recorded using a

Millicell voltohm-meter to assess the integrity of the cell monolayer (Verwei, Arkbåge, Groten,

Witthöft, VandenBerg, & Havenaar, 2005). The TEER measurement was done using an

electrode probe which had two ends. The longer end was immersed in the basolateral side of the

Transwell and the shorter electrode was immersed in the apical side with precaution such that the

probe does not damage the integrity of the monolayer. The baseline for the TEER was based

upon the reading obtained from the HBSS without any cells. Wells which gave TEER above 500

Ω was used for the transport study.

For the transport study the growth media was removed from both the apical and the basolateral

chambers. Following this the chambers were washed with sterile HBSS twice and then 200 µL

and 600 µL of HBSS was added to the apical and basolateral chambers respectively and the

plates were incubated at 370C with 5%CO2 for 4 hours prior to starving the cells and rinsed off

any remaining growth media from the chambers. After the incubation time the HBSS was

removed from the chambers and to the apical chamber 200 uL of LY, nil digesta, rice digesta and

the fortificant digesta were added and to the basolateral chamber 600 µL of HBSS were added.

The plates were then incubated at 37°C with 5% CO2 in a humidified incubator for 1 hour for

folic acid uptake, 16 hours for b- carotene and 22 h for iron uptake. The application of LY to the

Tranwell was to confirm the integrity of the cells monolayer.

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The LY standards (0.1 mM ) was prepared fresh on the day of the analysis and 200 µL of the

LY was applied to the apical chamber in triplicates and the plates were incubated for 2 hours.

After the incubation period the TEER was measured and the samples were collected from the

apical and the basolateral chambers and analysed using a spectrophotometer on the same day. A

standard curve of LY was constructed with solutions prepared in the concentration range of 0-

250 µM. The standards and the samples were added to a 96 well microtitre plate. The sample

obtained from the apical chamber was diluted in the ratio 1: 20 with HBSS in order to fall within

the calibration range and the basolateral chamber was not diluted because if the membrane

retained its integrity the LY should not be able to pass through the Transwell membrane. The

absorption was then measured using fluorescence spectrometer with an excitation wavelength of

430 nm and emission wavelength of 535 nm.

After the transport study the samples were collected from the apical and the basolateral chambers

and stored in a -80ºC until further analysis. To the apical and the basolateral chambers 200 µL

and 600 µL of growth media was added respectively and the TEER was measured right away.

Then the cells were removed from the Transwell insert by applying warm Trypsin and incubating

at 37ºC and 5% CO2 for 20 minutes. The cells were then collected from the apical chamber and

the trypsin was removed by centrifugation. The cells were then washed and suspended in HBSS

and stored at -80ºC until further analysis. For the transport studies, caco-2 cell lines with passage

number of 25-40 were used throughout the experimental period.

7.2.7.1 Sample Preparation for Quantitative Analysis

For quantification of the micronutrients from the transport study 6 wells from the apical and the

basolateral chambers were pooled to obtain a single sample for folic acid. For β- carotene and

iron 12 wells of samples from the apical and the basolateral chambers were pooled to obtain a

single sample. The cells were lysed using 1 M KOH with 2% sodium ascorbate (w/v) (Ohrvik et

al., 2010).

7.2.7.2 Folic acid analysis

The samples (output from the transport studies obtained from the apical and basolateral

chambers and the cell lysate) were purified using SAX SPE cartridge. The purified extract was

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analysed using HPLC and the method was followed according to section (3.2.8.1). The digesta

which was prepared according to the in vitro model was also analysed for folic acid using the

above procedure.

7.2.7.3 β – carotene analysis

The samples and the cell lysates were prepared according to Netzel et al., (2011) with some

modifications. The samples from the transport study and the digesta were spiked with β- apo

carotenoid (purchased from Sigma-Aldrich –Catalogue No: 10810-1G) and then extracted with

equal volumes of hexane: acetone (1:1) with 0.5 mL of 10% sodium chloride to remove the

emulsion. The hexane layer was collected and washed with water until it ran clear without any

visible solid particles and emulsion. The combined extracts were dried under N2 and dissolved in

methanol for analysis using HPLC as mentioned in section (3.2.8.2- page no. 52).

7.2.7.4 Iron analysis

Iron analysis for the sample from the apical chamber and digesta was analysed using ICP-OES

and the extraction procedure was the same as in section (3.2.8.3 page no.53). For the sample

collected from the basolateral chamber of the cell culture assay , iron analysis was performed

using a Ferritin assay kit (obtained from MP Biomedicals, Diagnostics Division, Orangeburg

NY) as the absorbed iron is converted to a protein (ferritin) by the human body and the same was

observed in the caco-2 cells as well. The protocol was strictly followed according to the kit

manual provided by the manufacturer.

The principle of the assay was based on solid-phase enzyme linked immunosorbent assay. The

microwell plates are coated with rabbit anti-ferritin for solid phase-immobilization and antibody-

enzyme (horseradish peroxidase) was monoclonal anti-ferritin obtained from mouse. When the

samples containing ferritin are added to the wells, the analyte is sandwiched between the solid-

phase and the enzyme-linked antibodies. After 45 minutes incubation the wells are washed with

water to remove unbound labelled antibodies. A solution of 3,3‘,5,5‘- Tetramethylbenzidine

(TMB) was added to the wells and incubated for 20 minutes for colour development. A stop

solution of 1M HCl was added to stop the reaction and the resulting colour was measured with a

spectrophotometer at 450 nm. The concentration of ferritin in the sample directly corresponds to

the color intensity of the sample. To quantify the ferritin present by the current analysis standards

with concentrations between 0- 1000 ng/mL (provided in the kit) was used. The samples and

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standards were analysed in triplicates. The sample from the basolateral chamber and the cells

were also analysed for ionisable iron using ICP-OES as mentioned above.

7.2.7.5 Calculations

The bio-accessibility study indicated the % uptake of three micronutrients in the fortified rice

and the transport was measured based on the nutrients passing through the apical chamber into

the basolateral chamber- across the human intestinal wall. Bio-accessibility refers to the portion

of micronutrients released from the food matrix and made available for the cell uptake after

digestion and it is applicable only for in vitro procedures (Hendren et al., 2002). It can be

calculated according to the following equation:

Bio-accessibility % = M digesta/ M cooked rice x 100% (Equation 5)

where M digesta and M cooked rice are the concentrations of the micronutrients (folic acid, iron and β

– carotene) present in the digesta and cooked rice respectively.

The transport of each micronutrient is referred to as the concentration of each micronutrient

detected in the basolateral chamber of the Transwell plate at the end of the designated transport

incubation time in the context of the digesta. The transport % can be calculated according to the

following equation:

Transport % = M basolateral/ M digesta x 100% (Equation 6)

where M basolateral is the concentration of micronutrients present in the basolateral compartment.

7.3 Results and Discussion

7.3.1 Cytotoxicity and cell viability

In order to perform in vitro transport study the cells that are to be seeded onto the insert of the

Transwell must be assessed for viability and also the cells should be non-toxic to the digesta that

will be used for the study. If the digesta is toxic it will lead to cell death and thereby invalidate

the transport study. Stoddart (2011) stated that cell viability implies the number of living cells in

a sample. Caco-2 cell viability was assessed according to section 7.2.3.2. The cells are

considered viable after application of digesta when there is intense color change and the

absorbance value increases progressively with time indicating that the digesta is not toxic to the

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cells. The principle behind assessing the viability of cells is that the living cells synthesise an

enzyme that can convert MTS into an aqueous and soluble formazan product. Due to this there is

a transformation in the colour from pale yellow of MTS to dark yellow or brown colour in the

presence of viable cells. The number of viable cells is directly proportional to the intensity of

colour change caused by the formation of formazon (Stoddart, 2011).

Since it was a colorimetric assay, HBSS was chosen as a positive control and wells without cells

and those with cells and had added H202 in it were chosen as negative control for colour change.

The positive control (HBSS) showed color change over time while there was no substantial

colour change in the negative control (H202). This is because H202 was toxic to the cells and

hence did not produce the enzyme that utilizes the substrate to create colour change. The other

samples such as LY, fortified rice digesta and nil digesta showed dark colour change but did not

reach OD 1 after 6 hours incubation. However, after overnight incubation there was significant

colour change where the OD was measured to be over 1 indicating that the cells are viable in the

presence of the digesta and hence the digesta is non-toxic to the cells. Therefore the digesta

produced according to the above mentioned protocol (section 7.2.3.1) was used for transport

study.

Table 7.1 Absorbance values for the digesta samples and controls in the cell proliferation

assay after 2 hours incubation

Sample/Control Absorbance at 485 nm

Unfortified Rice 0.61 ± 0.164

Fortified Parboiled Rice 0.56±0.058

Fortified HPP processed rice 0.49±0.013

Nil digesta 0.44±0.090

Fortificant 0.30±0.187

Positive Control: HBSS 0.75±0.044

Negative Control: H2O2 0.13±0.018

Note: Mean absorbance values ±standard deviation of triplicate analysis

7.3.2 In vitro-bio-accessibility of micronutrients from fortified rice

The integrity of the caco-2 monolayer after 21 days of growing it in vitro was assessed by adding

0.1 mM of LY to the apical chamber. After the designated inbcubation time (2 hours) there was

no LY present in the basolateral chamber of the transwell indicating that the junctions between

175

the caco-2 cells were intact and therefore it can be used for performing the transport study. The

TEER values were recorded and for further experiments wells with TEER ≥ 1000Ω was used for

transport study. In the following sections, bio-accessibility of the micronutrients from the

fortified rice and transport data will be presented and discussed.

Figure 7.3 Confocal microscopy image of caco-2 cells showing tight junctions (Chandra-

Hioe et al., 2013)

Table 7.2 Concentration of folic acid, iron and β-carotene (µmol) in fortificant solutions

(control), fortified parboiled rice and HPP rice

Digesta Folic acid β-carotene Iron

Fortificant 0.9±0.053 0.0009±0.0004 0.034±0.01

Parboiled rice 0.32±0.28 0.00042±0.00005 0.25±0.19

HPP rice 0.16±0.003 0.0005±0.0004 0.103±0.19

Note: Value represent Mean±Standard deviation of replicates.

After performing in-vitro digestion the folic acid concentration in the digesta was measured in

the fortificant standard solutions, fortified parboiled rice and fortified high pressure processed

rice. The fortificant standards with 150 mg of folic acid/ 600mL of water was used as a control

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and had a measured concentration of 400 µg/ mL of folic acid; parboiled fortified rice had a

measured concentration of 20 µg/ g of cooked rice on wet basis and HPP processed rice had 160

µg/g of folic acid. The concentrations of folic acid in the 3 samples after in vitro digestion are

presented in the above Table 7.2 and there was a significant difference observed in the

concentration of folic acid in the 2 different samples after in vitro digestion. This shows that the

release of folic acid after in vitro digestion varied with the food matrix.

From calculating the % bio-accessibility from the control folic acid standard solutions and the

rice it was found that the fortified rice (96%) was more bio-accessible compared to the fortificant

solution (80%). Similar observations were made by Kam et al., (2013) where folic acid fortified

rice had higher bio-accessibility compared to standard folic acid solution. However in the case of

high pressure processed rice the % bio-accessibility was much lower (46%) compared to fortified

parboiled rice. The reason for this could be attributed to the formation of resistant starch during

HPP making the starch less susceptible to in vitro enzymatic digestion leading to lower folic acid

bio-accessibility from HPP rice. This was likely due to the addition of 0.1 M HCl and 0.1 M

NaHC03 at gastric and intestinal stage of digestion which was favourable in the case of rice as the

folic acid was entrapped in a solid matrix compared to the standard solution. The rice matrix

would have provided a buffering environment for the folic acid making it more stable and less

susceptible to degradation. This also suggests that folic acid is stable in the gastric environment

when released from the rice and it is also easily released from the matrix. Folate bio-accessiblity

was better in wholemeal breads (above 75%) and breakfast foods (about 94%) compared to

fortified milk (60%) (Öhrvik et al.,2010 and Verwei et al., 2003).

Similar results were also observed for β-carotene (12% from parboiled rice 2% from HPP rice

and 0.1% from control) as well. From the digesta concentration it was calculated that iron bio-

accessibility was 38% from the fortificant alone; 31% from fortified parboiled rice and 42% from

high pressure processed rice. Fortified rice had higher concentration of iron in the digesta (Table

7.2). Vitamin A can affect iron metabolism at several stages and these include erythropoiesis and

release of iron from ferritin stores. Vitamin A and β-carotene enhanced iron absorption from

fortified maize and wheat and rice meals (Garcia –Casal et al., 1996 & Garcia –Casal, 2006).

The same group also reported that non- pro-vitaminA carotenoids such as lycopene, lutein and

zeaxanthin also increase iron absorption 2-3 folds when added to maize and wheat – bread meals.

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Davidson et al., (2003) added vitamin A to iron-fortified maize gruels and fed to vitamin A

deficient Ivorian children and reported that vitamin A significantly decreased iron absorption.

This shows that the relationship between β-carotene and iron is complex and is subject to the

methodology adopted. However, this area of micronutrient interaction needs to be highlighted as

many fruits and vegetable are good sources of both iron and β-carotene.

Table 7.3 Concentration of folic acid, β-carotene and iron present in the apical chamber of

the transwell in µg/monolayer

Apical Basolateral

Folic acid

Fortificant (control) 0.15±0.01a 2.8±0.2

a,b

Parboiled rice 4.3±0.98 a,b

0.42±0.85a,b

HPP rice 0.17±0.01a,b

0.42±0.8a

β – carotene

Fortificant (control) 0.053±0.002a 0.005± 0.002

a

Parboiled rice 0.026±0.003a,b

0.0065± 0.001a

HPP rice 0.001±0.003 a,b

0.0009±0.0002a

Iron

Fortificant (control) 0.015±a 19±1.2

a*

Parboiled rice 0.03±0.01a 26±0.75

a*

HPP rice 0.02±0.005a 24±1.5

a*

*Note: iron was quantified as ferritin in the basolateral chamber. Different superscripts within

the column indicate significant differences (p≤0.05)

7.3.3 Transport Study

7.3.3.1 Folic acid

For the transport study the digesta was added onto the apical chamber of the transwell plate and

the initial concentration of the apical chamber was the same as that of the digesta before the

transport study. After1 hour transport for folic acid, the concentration of folic acid in the apical

chamber was lower in the fortificant (control) than in the rice digesta and in the basolateral

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chamber, folic acid concentration from the fortificant (control) was higher than from that of the

parboiled rice digesta. For HPP rice after 1 hour transport, there was less concentration of folic

acid remaining in the apical chamber and much of it had been transported to the basolateral

chamber. This indicated that there was significant transportation of folic acid from the apical

chamber to the basolateral chamber. Overall from Table 7.3, it can be said that from the

fortificant control 12% of folic acid was transported and 33% and 9.4% for parboiled and HPP

rice respectively. The molecular structure of folic acid transported from the apical chamber to the

basolateral chamber was unchanged (Verwei, 2004). However if folate polyglutamates were

present they would be retained in the cells until they are converted into folate polygulatamtes in

the cytoplasm or mitochondria (Verwei, 2005) indicating that folic acid is more readily

bioavailable then folates. The concentration of folic acid detected in the basolateral chamber can

be considered as the concentration of folic acid that would be absorbed and it can also be

reported that the % transport is higher for the rice than the control folic acid solution.

7.3.3.2 β-carotene

In the case of β-carotene there was higher concentration remaining in the apical chamber rather

than in the basolateral chamber which had lower concentration after 16 hours of transport study.

This implies that there is less β-carotene transported across compared to folic acid. However, an

interesting observation was that when the cells were analysed for β-carotene concentration in

HPP processed rice and the fortificant 0.007 µg/ monolayer of β-carotene was present and ready

to be converted into chylomicrons and transported. This scenario was not observed in the case of

folic acid indicating that most of the transport had already occurred during the incubation time.

For transportation study of β-carotene (in vitro) 16 hours was deduced optimum incubation time

as after the designated incubation time the digesta became toxic to the cells hence the results

after this time could not be taken into account. Overall it was found that 4% of β-carotene was

transported from the fortificant and 23% and 13% was transported from the parboiled rice and

HPP rice respectively.

Very low concentrations of β-carotene were present in the digesta of the fortificant (control) and

in the rice. β-carotene bioavailability from fortified rice is very novel and it has not been

explored very well. However in other foods such as cassava, maize etc this has been well studied

using the caco-2 model. Micellization of β-carotene and other carotenoids into chylomicrons in

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cassava was studied by Thakkar et al., (2007) who reported that β-carotene in vitro digestion and

uptake by caco-2 cells was directly proportional to the content of β-carotene in different Cassava

cultivars. According to Failla et al., (2011) bio-accessibility of β-carotene from fortified cassava

is greater than in wild type roots. Information from literature thus suggests that assessing β-

carotene using caco-2 models is well established but not explored for fortified rice and this is the

first time it has been explored. Experimental data from the present study implies that there is

significant potential of having high bio-accessibilityof β-carotene compared to unfortified foods.

Courraud et al., (2013) studied bio-accessibility of β-carotene in various foods with and without

matrix effects. It was found that pure β-carotene stability decreased over time and in vitro

digestion stages and showed loss of approximately 60% at the end of the intestinal phase. Carrot

juice on the other hand showed greater than 100% of β-carotene at the end of the intestinal phase

of digestion. Compared to cooked spinach and raw spinach, pure form of β-carotene showed

lowest uptake post in vitro digestion. This shows that there is significant effect of matrix in the

bio-accessibility of foods. In this study although there is very low transport of β-carotene from

the fortificant there is higher concentration that has migrated from the parboiled rice compared to

high pressure processed rice. This could be because of the lower retention of β-carotene in the

cooked HPP rice than in the parboiled rice. The change in the structure of the soy protein in

which β-carotene is encapsulated could be a barrier for micellization. This could also be a

possible reason for lower transport of β-carotene compared to the other 2 micronutrients. Overall

it can be said that β-carotene from parboiled rice showed better bio-accessibility and transport

compared to HPP rice.

7.3.3.3 Iron

When iron transportation across the caco-2 monolayer was studied, it was encapsulated in the

form of ferritin protein and was quantified as such in the basolateral chamber and in the cells.

This phenomenon was definitive from the fortified rice digesta as when nil digesta and

unfortified rice was assessed by the transport study there was negligible concentration of ferritin

that was not detectable by the ELISA method. Hence ferritin was positively from the fortified

rice digesta. From Table 7.3 it can be seen that highest concentration of ferritin was from the

parboiled rice followed by HPP rice and the level were not very different. However from the

fortificant there were lower concentrations of ferritin present in the basolateral chamber. This

shows that there is once again effect of matrix as in the case of folic acid which enhances the

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concentration of iron transported from the rice rather than from the pure fortificant. As

mentioned previously in the study by Gracia et al., (2002) that β-carotene enhancing iron

absorption in the fortified rice could also be another reason for better iron bioavailability from

the rice rather than the fortificant. However it is worth noting that here was no significant

difference in the concentration of iron in the basolateral chamber for fortificant, parboiled and

HPP fortified rice.

Based on the normal range for serum ferritin (15-200 ng/mL, Iron Disorders Institute (2009)) and

the calculation of ferritin in the basolateral chamber, it can be said that with the given fortified

rice, there is high likelihood of showing improvement in the serum ferritin level of people who

consume it. A comparisson with the bioavailability study performed by Prom-u-thai (2009) (82

ng of ferritin/mg protein) using caco-2 cells shows that, the levels of ferritin was much lower in

this study. This can be attributed to the higher concentrations of ferrous sulphate and Na2EDTA-

Fe that was used as a fortificant (150mL of Fe solution with concentration range between 5.5 –

23.3 g per was added per 150 g of rice) in their study. In the present study however, 3

micronutrients were used for fortification and there was higher loss expected due to the

competition between the nutrients during the uptake process. It is also important to note that

prior to cooking the rice was washed for analysis. This could also add to the loss of the iron. In

another study conducted by Prom-u-thai et al., (2006), Fe bioavailability in the presence of anti-

nutrients such as phytates and phenolic acids which are potential iron absorption inhibitors was

assessed. In this study, it was concluded that in polished grains due to bran removal there was

high loss of Fe from raw rice and also substantial proportion of anti-nutrients such as phytates

and extractable phenols which enhanced the bio-availability of Fe in spite of the low

concentration. Despite all these factors significant concentrations of ferritin can be absorbed by

the body from the fortified rice and this indicates the success of the product in terms of bio-

accessibility.

Iron deficiency anaemia occurs in three stages namely: Iron depletion, iron deficiency and iron

deficiency anaemia. In the iron depletion stage levels of iron in the haemoglobin is normal but

the iron reserve would deplete soon with no obvious symptoms occurring at this stage. When the

body experiences iron deficiency, the stored iron and blood-borne iron in the body is low and

haemoglobin levels are lower than normal and the patient may experience symptoms such as

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tiredness. In the ultimate state of iron deficiency the patient becomes anaemic with very low

levels of haemoglobin in the blood where there is not enough oxygen to be delivered to the cells

and the patient looks pale, experiences breathlessness, dizziness and fatigue. This also causes

reduced immune functions, impaired growth and cognitive functions as symptoms (de Souza

Querioz & Torres, 2000). By consuming the fortified rice by parboiling or high pressure

processing, there is a high possibility that the iron stores would be accumulated in the body and

hence prevent the causes and symptoms of anaemia when the rice is eaten for a prolonged time.

This can also address symptoms of iron deficiency and there by prevent anaemia. Incidence of

iron deficiency anaemia can be reduced by consuming about 440 g of diluted fortified rice per

day on an average. The given concentration of ferritin in the basolateral chamber is only for 5 g

of rice which will increase when 440 g of rice is consumed on an average and therefore be able

to counter balance symptoms of IDA.

7.3.4 Improvement in the nutritional status after consumption of fortified rice

As mentioned in the previous chapter, the mixing ratio of fortified rice to commercial white rice

was 1:10 assuming that consumers would consume 440 g of cooked rice per day. For the in vitro

bio-accessibility study premix rice was used. From the above results it can be said that parboiling

process is overall much more promising than HPP treatment in terms of efficacy of nutrients

retention after cooking and also bio-accessibility. In the end for a consumer it matters as to how

much of each micronutrient is absorbed from the portion of the rice that would be consumed. In

this study, 5 g of cooked rice containing 68 µg of folic acid; 10 µg of β-carotene and 0.3 mg of

iron were used to prepare the digesta (final volume 50mL). From this 0.2 mL was added onto the

apical chamber of the transwell. This represents 1/250 times of the total digesta (50 mL). Hence

in the following paragraphs theoretical calculations of micronutrient absorption from the fortified

parboiled rice for each micronutrient is to be discussed.

In the case of the rice that was diluted in the ratio 1:10 and assuming 440g of cooked rice

would be consumed, 44g of fortified premix rice is present in it. The cooked rice would

therefore have a concentration of 13.6 µg of folic acid/g of cooked rice or 600 µg per

440g of cooked rice. Based on the current experiment the % transported from the digesta

to the basolateral chamber was 33%. Thus it can be said that theoretically 197 µg of folic

acid would be absorbed by the body from the given fortified rice. This value is very close

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to the upper limit of expected fortification level proposed by the current study. The

normal serum folate range is 2.7- 17 ng/mL (National Institute of Health, 2014). With the

concentration of folic acid absorbed from the fortified rice, it is highly likely that serum

levels after consumption may increase favourably.

For β-carotene it can be said that in the diluted fortified rice (in the ratio 1:10) there

would be 88 µg/ 440 g of rice. With the given 23% of transport from the digesta to the

basolateral chamber, 20 µg (3.3 Vitamin A equivalent) would be absorbed by the body

and metabolised. The normal serum range for Vitamin A is 50-200 µg/dL (National

Institute of Health, 2014). Therapeutic doses of vitamin A (1000-9000 IU/kg) have

shown to induce oxidative stress in sub-mitochondrial particles isolated from cerebral

cortex and cerebellum of adult rats (deOliveria and Moreira, 2007). Long-term

therapeutic dose of vitamin A can thus be harmful. Hence by administering lower levels

of vitamin A in the form ofβ-carotene through fortified foods after a high dose of

supplementation of vitamin A can help to maintain retinol balance in the body in

deficient populations.

In the case of iron the diluted fortified rice (cooked) would contain 2.64 mg/ 440g of rice

and if 31% of this transported 0.8 mg would be absorbed by the body. This can be

extrapolated to a ferritin level of 69 ng/mL was present in the basolateral chamber.

Normal serum ferritin range for men is 12-300 ng/mL and 12-150 ng/mL for women

(National Institute of Health, 2014). With the given concentration of ferritin from the

fortified rice it is possible to improve serum ferritin status with prolonged consumption of

the fortified rice.

In this study, caco-2 cells were used as an alternative to human bioavailability trials. The results

obtained from this study were an estimation of the in vitro uptake and transport in the short term.

However to understand the real picture, human trials are to be performed that could serve as a

comparison to the in vitro studies. Nevertheless, from the results it can be seen that bio-

accessibility of the micronutrients in the fortified rice through the parboiling methods and HPP

method was feasible and overall the micronutrients from the parboiled rice was better absorbed.

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7.4 Conclusions:

Overall, for the three micronutrients, the study suggests that the fortified rice had high potential

for improving the nutritional status of folic acid, iron and β-carotene. As mentioned previously in

chapter 3, rice does not have measurable concentrations of the three nutrients present naturally.

However there are minor concentrations of native non-haeme iron in the rice. From the fortified

rice there were higher concentrations of these micronutrients bio-accessible compared to the

unfortified rice. Therefore the results imply that fortification by parboiling and HPP is both

feasible but parboiled fortified rice is better option in terms of acceptability and bioavailability.

Rice eating population can highly benefit from this fortified rice and their nutritional status is

likely to improve even better if they are regularly consumed parboiled rice. These populations

can experience an improvement in their Folic acid, iron and β-carotene status and also be

benefitted if they have limited access to other food rich in these micronutrients.

The digesta from the fortified rice was shown to be non-toxic to the caco-2 cells based on the

cytotoxicity results. Overall the % transport and bio-accessibility was better for fortified rice

compared to the fortificant control solution except for iron transport. Parboiled rice which has

shown better bio-accessibility can deliver expected nutritional benefits in target populations. The

process of adding β-carotene along with iron in a fortified food itself has the possibility of

increasing blood haemoglobin concentration due to the synergistic relationship between β-

carotene and iron. This study however was performed in vitro using adenocarcinoma cell model.

Caco-2 in vitro model is not a replica of a human study although was able to give an insight on

absorption of the micronutrients from the triple-fortified rice.

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CHAPTER 8

8. Conclusion and Future work

Micronutrient deficiencies due to vitamin A, folic acid and iron are remarkably high and

prevalent worldwide. In developing countries more than 40% of women are anaemic and about

25% of children suffer from subclinical vitamin A deficiency (Micronutrient Initiative, 2001).

There are many programmes in place by governments and non-governmental organisations

(NGO) to address this issue. In terms of a long term solution reduction in poverty would be a

possible solution. However this is unlikely to happen in the near future. Food fortification could

provide an alternative in the short to medium term. Deficiency diseases are prevalent in rice

eating populations. Several technologies such as dusting, coating, extrusion and biofortification

have been employed to overcome this problem in the past. Parboiling: although an old technique

has recently been used for micronutrient fortification. High pressure processing on the other hand

is a novel technique for food preservation and has been explored for rice fortification in this

study. Both are post-harvest technologies and have their pros and cons. In many developing

countries parboiled rice is commonly consumed by rice-eating populations and thus there is no

need for capital investment. However in the case of high pressure processing there is necessity

for capital investment but this technology can save energy compared to parboiling process. HPP

process involves only 1 hour treatment and the energy is expended as pressure whereas

parboiling involves heating for 2 hours followed by steaming for an hour and the expenditure of

energy is through heat. Thus in this project the process of developing a method to fortify rice

with multiple micronutrients using 2 different technologies was explored in order to address the

public health concerns in the rice eating populations. Based on the results and discussions

presented in the earlier chapters some conclusions can be drawn.

8.1 Efficiency of parboiling as a technology to fortify rice:

Based on the results from Chapter 3 it can be concluded that parboiling is a feasible method for

fortification of rice with three micronutrients (folic acid, iron and β-carotene). The optimized

soaking (70°C for 2 hours) condition clearly would be able to maximize the uptake of the

micronutrients rather than shorter or longer soaking times. Based on the steaming time, the best

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favourable condition was 100°C for 1 hour for starch gelatinization and absence of white belly in

the rice which is an important quality parameter for purchase. By adapting to air drying at room

temperature, loss of micronutrients can be minimized compared to heat drying methods. The

retention of micronutrients (as measured after cooking) in the rice was according to the following

trend: folic acid> β-carotene>iron in the uncooked rice and folic acid>iron>β-carotene in the

cooked rice. Scale up of the conditions from 300 g to 2.5 kg showed concentrations similar to lab

scale for all the micronutrients in both uncooked and cooked parboiled rice. Also the

concentration of micronutrients in rice treated by the conventional parboiling condition was

similar to the lab scale condition implying that 2 hours is sufficient for maximum uptake of

micronutrients and by utilizing this optimized condition energy could be saved.

Table 8.1 shows the cost analysis performed by Roy et al., (2005) which showed that all the

processes were economical but the small-boiler was a clear choice as it had the shortest payback

period (the time period required to recoup the initial investment) compared to the others although

it consumes higher energy compared to the other methods. Keeping in mind the uncertainty of

investment and limitation of business capital as well, small-boilers were found to be the most

cost and energy effective process according to this study.

Table 8.1 Parboiling process cost analysis (Roy, Shimizu, Shiina and Kimura, 2005)

Process Vessel or

boiler Hopper

Soaking

tank Tube-well

Initial

Investment

Equipment $USD $USD $USD $USD $USD

Vessel (0.5- 1

t/batch) 5.32 - 42.55 21.28 397.47

Small- boiler

(2-4 t/batch) 42.55 42.55 148.94 21.28 1281.87

Medium-

boiler (5-10

t/Batch)

1170.21 63.83 638.29 212.77 5101.09

Parboiling is a well established process and the cost analysis has been performed in the above

table to get a glimpse of the financial aspect of the process. From the above table it can be said

that processing rice in bigger batches would be more economical than small scale processing

although it had a shorter payback period. The cost can be further reduced by adding more than

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one micronutrient. Also compared to hot extrusion technique, parboiling seems much more cost

efficient. With added cost of nutrients in the process the overall cost is likely to add up slightly

higher (USAID, 2008).

An approximate estimate of the cost of the rice-premix is around $1-2/kg, and the total cost for

rice fortification is from US$10/MT to US$20/MT.It can be deduced that the annual investment

is US$0.36-0.73/year for an individual consuming 100 g/day of fortified rice and $1.09-2.20/year

for an individual consuming 300 g/day. The process can be made even more effective if the rice

processing and fortification were centralized and the mills involved had production capacities

larger than 5 MT/hour (i.e., around 15,000 MT/year). Small mills have the disadvantage of

creating logistic issues and also increase the overall cost of the program (USAID, 2008).

8.1.1 Feasibility of rice fortification with multiple micronutrients using HPP technology

High pressure processing has been used for extending shelf-life of various food products and is a

popular processing method for over a decade. However the application of the technology in the

light of food fortification has never been explored. In this study, the feasibility of using this

technique as a potential method for rice fortification was explored. It can be concluded that rice

can be fortified using the HPP technology and the best condition for fortification was 200 MPa

treatment for 1 hour. Prolonged pressure treatment resulted in broken kernels and as opposed to

parboiling technique brown rice could not be used in this process as milling of the rice after the

process resulted in minimal salvaging of the kernels. After HPP treatment and cooking the rice

the retention of the micronutrients were as follows: folic acid>iron>β-carotene.

In comparison to thermal processing methods such as pasteurization and heat sterilization high

pressure processing is much more cost effective (Wang, 2009). Therefore, in terms of energy

efficiency HPP could be a possible technology that can be used for rice fortification. However it

involves a huge capital investment which is not needed in the case of parboiling.

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8.1.2 Physicochemical properties of the rice starch due to the two different processing

methods

Parboiled rice and high pressure processed rice have been previously been extensively studied

for alterations in starch physiochemical properties due to processing. However in the present

study, the starch in which the micronutrients were embedded in the fortified rice was studied to

understand how the 2 processing techniques altered native starch which was the primary novelty

of this section of work. Essentially, the physicochemical properties of the rice starch processed

by parboiling and high pressure processing was assessed for degree of gelatinization, pasting

properties, microscopic imaging of starch, crystallinity using XRD. Apart from these analytical

parameters, diffusion of micronutrients during the parboiling process was imaged in real-time

using MRI.

Collectively the results from this section of work demonstrate that both the processing methods

altered the rice starch properties significantly with the modifications pertaining to be unique to

the respective processing method. Addition of micronutrients did not significantly alter the rice

properties making the variations in rice starch dependent on the processing method. There is

evident correlation between release of micronutrients from fortified rice and the modified starch

matrix. Formation of partially gelatinized starch during HPP could hinder release of vitamins

from the matrix while parboiled rice which was fully gelatinized released more vitamins from the

embedded matrix. The variations in colour due to the processing could be of vital importance in

consumer preference and the changes in the rice starch could affect the starch digesting property.

Rice treatment at 400 and 600 MPa were not suitable for fortification as it resulted in broken

kernels, although rice starch processed at these pressures could be of use in other products as a

functional ingredient.

8.1.3 Acceptability of the fortified parboiled rice by consumers

A successful fortification does not stop with achieving higher concentrations of the

micronutrients in the grain. It has to be accepted and preferred as a choice for consumption by

consumers. In this case, rice a staple crop in Asian countries was the fortification vehicle.

Consumer acceptance study was performed to investigate if people are willing to buy the

uncooked fortified rice when commercialized and also if any changes to the taste of the fortified

rice was acceptable to the consumers. Sensory studies were performed in 2 stages: Study 1-

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Visual consumer acceptance; Study 2- Sensory evaluation of the cooked fortified rice. Due to

technical issues as mentioned in Chapter 6, HPP rice could not be tested for sensory evaluation.

Fortified parboiled rice diluted with white rice in the ratio 1:10 was pre presented to consumers

for consumer acceptance study. The dilution ratio was adjusted such that the fortified product

would be able to deliver 50% of the recommended dietary intakes of the nutrients added.

The results from Phase 1 of the sensory study showed that diluted fortified rice was not well

accepted in the uncooked form and the key factor that affected the acceptance was the non-

uniformity in rice samples. Familiarity with parboiled rice did not influence the acceptability of

the rice. Nevertheless after cooking, the fortified rice was well accepted and the non-uniformity

was neither recognized by the panel nor did it affect the acceptability of the product. In both the

studies, the purchase intent of the rice increased after mentioning about the health claim which

denotes that appropriate labelling would increase the purchase intent and consumer preference of

the fortified rice. There was slight variation in the flavour and texture when fortified rice was

diluted with white rice; nonetheless using the hedonic scale, fortified rice was preferred over

commercial white rice by consumers.

8.1.4 Bio-accessibility of the micronutrients from the fortified rice

The next step after consumer acceptance would be to understand if the fortification has

significant impact on the target population by trying to address the insufficiency of the

micronutrients and thereby improving the nutritional status of the affected populations. In this

study, caco-2 cells in vitro model was used to estimate the bio-accessibility of the micronutrients

from the fortified rice. As mentioned in Chapter 6, folic acid was able to deliver more than100%

of RDI requirement through the fortified rice, close to 50% of RDI for β-carotene and 100% of

RDI for iron. The results from the in vitro studies show that there is possibility that blood level

of folic acid, vitamin A and iron are likely to fall in the acceptable range of sufficiency if target

population consumes the triple-fortified rice over a long period of time. In vitro digestion, bio-

accessibility and transport studies were done using the premix rice and not the diluted fortified

rice. Further studies using the diluted fortified rice should be tested on humans to get a good

indication of absorption. The fortified rice diluted with white rice was not used in the caco-2 cell

study as a limitation of the analytical methodology used namely HPLC-PDA which could not

detect concentrations at ng levels especially for β-carotene. An LC-MS method for carotenoids

189

should be developed for analysis in the diluted fortified rice digesta to address the low level

quantification issue of the compound.

8.1.4.1 Limitations of the in vitro bio-accessibility and transport of micronutrients using

caco-2 cells

There are several limitations due to the in vitro approach of analysing micronutrients from the

fortified rice and also due to the usage of caco-2 cells per se that was used in this study. They are

listed below:

In this study 3 micronutrients were added in the fortified rice. Therefore the micronutrient

interaction between the micronutrients could be explored which could provide answers

for increased or decreased transportation due to the effect of the micronutrients.

The in vitro static digestion performed in this study cannot exactly resemble or replicate

the functions of human (in vivo) digestive system, as the process of digestion in vivo

involves shear, mixing, hydration, changes in conditions over time and peristalsis

(Fernandez-Garcia, Carvajal-Lerida, & Perez-Galvez, 2009). These processes can alter

the absorption of micronutrients at intestinal level in vivo and the results of which can be

different from the in vitro studies.

It has to be noted that the in vitro approach is time consuming. It requires 21 days for the

cells to differentiate fully until the stage where it can be used for transport study (Tavelin,

2002).

The cells are also highly susceptible to contamination if not well taken care of. They can

get contaminated with fungal colonies and it is also expensive to purchase the materials

and maintain the lab at suitable conditions that are favourable for cell growth.

Caco-2 cells do not have protective mucus layers; therefore the pH at the cell surface

rapidly balances the pH of the applied samples (Galkin, Pakkanen, & Vuorela, 2008).

This condition could significantly impact the transport of micronutrients across the

membrane.

8.2 Recommendations for future work

Between the two fortification methods, it is likely that parboiling could be more feasible for

implementation compared to HPP due to the fact that it is relatively novel and further studies are

190

still required to produce fortified rice on a larger scale using HPP. Therefore based on the results

from the current study, there are certain recommendations for future work.

When cooking the fortified rice, washing step can be reduced or avoided if the rice is

processed in a hygienic facility. This could reduce the loss of the micronutrients

especially for β-carotene as its loss was highest after cooking in the fortified rice after

both the processing methods. One possible solution could be to mention ―washing not

required prior to cooking‖ on the label of the packaged rice.

A suitable nutrition education program for the implementation of the fortification

program such that the target population would profit from the consumption of the rice. In

order to do this, the government along with non-governmental organisations such as

GAIN or Micronutrients Initiative could assist in putting together an effective approach

to educate people.

Based on the results from the scale-up study the optimized fortification process using

parboiling is reproducible. Therefore, it can be produced at an industrial scale by which

there would be lesser cost involved in processing and also the overall cost of the finished

rice product would not be very high. It is important that the fortified rice is available

without a substantial increase in the price. As mentioned in Chapter 7, in vitro studies

that were conducted using caco-2 cell was only an estimate of likely absorption in

humans and therefore human trials should be performed to understand the effectiveness

of the fortified rice in reality.

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CHAPTER 9

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10. SUPPLEMENTARY SECTION

Appendix 1 Optimization of the volume of KOH added to rice for gelatinization study

Volume of KOH added (10 M) Absorbance for Uncooked

Rice

Absorbance for Cooked

Rice

0 0.063 0.063

0.25 0.309 0.403

0.5 0.532 0.836

0.75 0.433 0.714

1 0.398 0.759

1.25* 0.514 0.867*

1.5 0.51 0.73

1.75 0.584 0.699

2 0.507 0.744

2.25 0.559 0.703

2.5 0.57 0.718

2.75* 0.6* 0.716

3 0.576 0.739

3.25 0.521 0.668

Note: *Values highlighted were the optimized concentration of KOH used for optimization of

degree of gelatinization experiment.

217

Appendix 2 Questionnaire used in Study I of the consumer acceptance study

Part I- Demographic and rice consumption pattern questions

A Gender

B Age

C Nationality

D Employment Status

E How often do you consumer rice?

F What type of rice do you usually consume?

H Have you heard of parboiled rice previously?

Part II- Consumer acceptance study- Study 1 questionnaire

1. How would you describe the COLOUR? (In

TWO or THREE WORDS)

Open question

2. How INTENSE is the COLOUR of the

rice? (Circle one number only)

10- point scale

3. How much do you like the COLOUR of the

rice? (Circle one number only)

9- point scale

4. How UNIFORM is the colour of the rice

sample? (Circle one number only)

10- point scale

3. How much do you like the OVERALL

APPEARANCE of the rice? (Circle one

number only)

9- point scale

6. How WILLING are you to buy this rice?

(Circle one number only)

5- point likert

7. If you are told that the rice is fortified with

vitamins and minerals which gives higher

nutritional value, how WILLING are you to

buy this rice? (Circle one number only)

5- point likert

218

Appendix 3 Sensory analysis liking scale for intensity, Just about right (JAR) and purchase intent

questions

Liking (Studies 1 &2) Intensity (Study 1) JAR (Study 2) Purchase intent

(Study 1 &2)

1 Dislike Extremely 1 Not at all 1 Not nearly enough 1 Definitely

would not buy

2 Dislike Very much 2 2 2 Probably

would not buy

3 Dislike 3 3 3 Undecided

4 Dislike moderately 4 4 4 Probably

would buy

5 Neither like nor

dislike

5 5 Much too intense 5 Definitely

would buy

6 Like moderately 6

7 Like 7

8 Like very much 8

9 Like extremely 9

10 Extremely 10 Too intense

219

Appendix 4 Questionnaire used in Study 2 (Demographics and consumers acceptance)

Part I – Demographic and consumers rice preference questions

A Gender

B Age

C Nationality

D Employment Status

E How often do you consume rice?

F What type of rice do you usually consume?

G How do you normally cook rice?

H Have you heard of parboiled rice previously?

Part II – Consumer Preference Questionnaire

A. VISUAL APPEARANCE

1. How much do you like the APPEARANCE of this rice? 9- point hedonic

2. What do you think about the UNIFORMITY of this rice? Just- about right (JAR)

3. How much do you like the COLOUR of this rice? 9- point hedonic

4. How would you describe the COLOUR? (In TWO or THREE

WORDS eg: white, yellow, off-white etc.)

Open question

5. What do you think about the COLOUR INTENSITY of this

rice?

JAR

B. SMELL

6. How much do you like the ODOUR of this rice? 9- point hedonic

7. How INTENSE is the rice ODOUR? JAR

C. TEXTURE

8. How much do you like the TEXTURE of this rice? 9- point hedonic

9. How HARD is this rice? JAR

10. How STICKY is this rice? JAR

D. TASTE

11. How much do you like the TASTE of this rice? 9- point hedonic

12. How would you describe the taste of this rice? Open question

13. How INTENSE is the TASTE of this rice? JAR

14. How much do you like the AFTERTASTE of this rice? 9-point hedonic

E. OVERALL LIKING

15. How much do you like this rice OVERALL? 9-point hedonic

16. Would you BUY this rice? 5- point likert

17. If you are told that the rice is fortified with folic acid which

gives higher nutritional value, how WILLING are you to buy

this rice?

5-point likert

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