How Enzymes May Be Helpful for Upgrading Fish By-Products

How Enzymes May Be Helpfulfor Upgrading Fish By-ProductsEnhancement of Fat Extraction

J DumayC Barthomeuf

J-P Berge

ABSTRACT The beneficial health effects of consumption of marinefoods in general and of marine oils in particular are well recognizedFish by products constitute valuable sources of components such aslipids for human consumption and liver and roe from large cod (Gadusmorhua) are currently exploited This paper presents a new approach forextracting lipids from several cod by-products by using a pre-hydrolysisstep with large spectrum proteases in order to disrupt tissues and cellmembranes Yields extraction for total lipids phospholipids EPA andDHA are compared to those obtained by organic extraction [Article cop-ies available for a fee from The Haworth Document Delivery Service1-800-HAWORTH E-mail address ltdocdeliveryhaworthpresscomgt Website

J Dumay is affiliated with the Laboratoire de Geacutenie alimentaire Deacutepartement valo-risation des Produits IFREMER centre de Nantes BP 21105 44311 Nantes cedex 03France (E-mail jpbergeifremerfr)

C Barthomeuf is affiliated with the Laboratoire de pharmacognosie etBiotechnologies Faculteacute de pharmacie Universiteacute drsquoauvergne Place H Dunant63001 Clermond-Fd cedex France

J-P Berge is affiliated with the Laboratoire de Geacutenie alimentaire Deacutepartementvalorisation des Produits IFREMER centre de Nantes BP 21105 44311 Nantes cedex03 France (E-mail jpbergeifremerfr)

These results are part of the EU-project ldquoUtilisation and stabilisation of by-productsfrom cod speciesrdquo (QLK1-CT2000-01017) Special thanks go to the staff of IcelandicFisheries Laboratories (IFL) for providing selected by-products We are also gratefulto Novozymes for their gift of enzymes and their technical advices

Journal of Aquatic Food Product Technology Vol 13(2) 2004httpwwwhaworthpresscomwebJAFPT

2004 by The Haworth Press Inc All rights reservedDigital Object Identifier 101300J030v13n02_07 69

Please note that this electronic prepublication galley may contain typographical errors and may be missingartwork such as charts photographs etc Pagination in this version will differ from the published version

lthttpwwwHaworthPresscomgt 2004 by The Haworth Press Inc All rightsreserved]

KEYWORDS By-product cod lipid extraction protease

INTRODUCTION

Characterization of lipid in some by-products (liver roe viscera headmilt backbone and cut-offs) from some cod species has shown that these partscan be utilized as a raw material for production of lipid compounds of interestsuch as fish oils (Rustad and Falch 2002)

Indeed fish oils are an important source of polyunsatured fatty acids(PUFA) and are now widely used in health and nutrition (Shahidi et al 1998Kolanowski et al 1999) For a long time these molecules were shown to be ofgreat importance in the prevention of a number of diseases such as inflamma-tion hypotriglycerimedic effect allergies diabetes or coronary heart disease(Rambjor et al 1996 Simopoulos 1997 Minnis et al 1998 Ackman 1999)However nowadays in industry fat extraction is carried on at high tempera-ture which can lead to some lipid oxidation and little work has been done foroptimizing those extractive conditions The use of enzymes such as proteasescould be an alternative solution to the present process conditions which de-stroy valuable components in the oils and oils with higher quality will createnew markets and obtain higher prices

This preliminary work presents the yield variations of fat extraction afterprotease hydrolysis of cod by-products

MATERIAL AND METHODS

Raw Material

This study focused on cod by-products (Gadus morhua) provided by theIcelandic Fisheries Laboratories (IFL) Those secondary raw materials wereselected on board immediately after the catch in the Icelandic sea in June2001 They were head backbones viscera roes milt skin and cut-offs Theywere kept frozen until landed then they were mixed and stored under vacuumat 80degC before analysis

Enzymes

Enzymes selected were wide spectrum proteases because they may nothave denaturant properties on lipidic compounds Two different kind of en-

70 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY

zymes were used Papaiumlne and Chymotrypsine (Sigma France) which are forldquolaboratory userdquo with a high degree of purity and Protamexreg andFlavourzymereg which are for ldquoindustrial userdquo (Novozymes Denmark)

Hydrolysis

Hydrolysis was realized in non-buffered solutions according to the pH statmethod (Brocklehurst 1995) where pH and temperature were controlledTemperature was stabilized with a continuous flow water bath while pH waskept constant by an auto-titrator with sodium hydroxide (1 N) Experimentalconditions are indicated in Table 1 for each enzyme the optima for pH con-centration and temperature were selected according to their technical sheet(provided by Sigma and Novozymes) As long as a hydrolysis was monitored(see below) the reaction was pursued therefore experiments were carried outat different operating times according to each enzyme

Crush substrates (80 g) were mixed with distilled water (15 volume offresh matter) and when optimal conditions for pH and temperature werereached enzyme and catalyst were added At the end of hydrolysis mixtureswere centrifuged (30 min 20000 g at 4degC) and pellet and supernatant wererecovered Supernatant part was previously freeze-dried before storage at20degC like the pellet part

The rate of hydrolysis (proteolytic activity) was monitored for each experi-ment by measuring the liberation of free amino groups using the DNFB re-agent commonly referred to as Sangerrsquos reagent (Sanger 1949) Periodicallyone milliliter of mixture was drawn off the reactor and heated at 80degC during15 min in order to inactivate enzyme Samples were then centrifuged (5000 g 10 min) and the pellet was eliminated while the supernatant was diluted by200 and sodium tetraborate (2) was added (vv) After mixing 025 ml of asolution of 24-Dinitrofluorobenzene (DNFB 13 in ethanol SIGMAFrance) was added Samples were then heated 10 min at 60degC after cooling 2

Dumay Barthomeuf and Berge 71

TABLE 1 Hydrolysis conditions

Enzyme AS(Unitg)

Ratioenzymesubstrate

(ww)

Temperature(degC)

pH Hydrolysistime (hours)

Catalyst

Papaiumlne 65 200 60 65 2 Cystein 0025

Chymotrypsine 600 001 40 70 3 Calcium chloride2mM

Protamexreg 15 020 60 60 1 -

Flavourzymereg 330 025 50 65 4 -

ml of hydrochloric acid was added in order to stop the reaction A calibrationcurve was established by using glycine (SIGMA France) and by reading theabsorbance at 410 nm Results were expressed in equivalent glycine

Dry Matter Content

Raw materials pellets and supernatants were freeze-dried According tomass differences (before and after lyophilization) water loss and dry mattercontent were determined by the following relation

drymatterfreshmatter g waterloss g

freshmatte()

( ) ( )=

minusr g( )

Lipid Extraction

Lipids were extracted from raw material (by-products) wet pellet and drysupernatant according to the Folch method (Folch et al 1957) Freeze driedsupernatants were previously re-hydrated by adding 4 parts of distilled waterto dry material (41 ww) To 5 g of hydrated material 33 mL of methanol(Carlo Erba Reactifs) was added and mixed for at least 30 min A short mix (1min) with an ultraturax was then done before adding 66 ml of chloroform(Carlo Erba Reactifs) Solution was then stirred for 30 min After eliminatingthe non-soluble part by filtration 02 volumes of calcium chloride (09 indistilled water) were added After decantation in a separatory funnel (12 hoursat 4degC) the organic phase was recovered and the solvents evaporated Lipidfractions were stored in a solution of benzeneethanol (41 vv) at 20degC be-fore use

Phospholipid Assay

Phospholipid content was estimated by a colorimetric method based on theformation of a complex between phospholipids and ammonium ferro-thiocyanate (Stewart 1980) A 2 ml aliquot of chloroform was added to about035 mg of dry lipid extract after mixing 1 ml of a solution of ferric chloride(27 gL) and ammonium ferrothiocyanate (30 gL) was added After vortexingand centrifuging at low speed (750 g 10 min) the lower phase was recov-ered and the absorbance at 488 nm was recorded A calibration curve was pre-viously realized with known amounts of a standard phospholipid solution(phosphatidyl choline) Results were expressed in equivalent phosphatidylcholine


Gas Chromatography Analysis of Fatty Acid Methyl Esters (FAMEs)

Aliquot of lipid was evaporated under nitrogen and transmethylated bycontact with methanolsulphuric acid (982) in excess for one night at 50degCAfter cooling 2 ml of pentane and 1 ml of water were added and vortexed Theupper organic phases containing FAMEs were collected and assayed by GCusing a Perkin Elmer Autosystem equipped with a FID detector Separationwas done using He as carrier gas on a fused silica column (BPX-70 60 m long025 mm id 025 microm film thickness SGE) programmed from 55degC (for 2 min)to 150degC at 20degCmin then to 230degC at 15degCmin Sample was injected with aprogrammable splitsplitless inlet and large volume injection system (PSS) us-ing the following temperature program 55degC (for 2 min) to 350degC at 200degCmin FAMEs were identified by comparison of their equivalent chain lengthwith those of authentic standards (Bergeacute et al 2002) Quantification was doneusing margaric acid (170) as internal standard

RESULTS AND DISCUSSION

Hydrolysis

For each experiment as long as DNP derivatization products increasedwith time the hydrolysis was pursued When a plateau was reached the exo-genic enzyme was considered to be not effective any more and the reactor con-tent was centrifuged in order to separate the two fractions a soluble one (thesupernatant) and a non-soluble one (the pellet) Results obtained for hydroly-sis of the different cod by-products by Protamexreg are presented in Figure 1As the main objective was to study the lipid part no attention was given to the


4035

30

25

20

15

10

5

00 10 20 30 40 50 60

Nh

(10

mm

oles

)2

5

SkinMiltRoesHeadVisceraCut-offsBackbone

Time (min)

FIGURE 1 Protamexreg hydrolysis of cod by-products as a function of time ex-pressed in free amino groups liberated

proteinpeptide compounds Indeed with such monitoring the lipid extractionhas only been done on the most hydrolyzed mixture that could be obtain withthe enzyme selected

Dry Matter Repartition

The first level of analysis was the distribution of the dry matter among pel-let and supernatant (Figure 2) Except for three hydrolysis reactions withFlavourzymereg (milt head and cut-offs) the major part of the dry matter waspreferentially located in the supernatant which means that this material hasbeen made soluble under the action of exogenic enzyme The most easilyhydrolysable material seemed to be the viscera Indeed whatever the enzymeused the dry matter was distributed for at least 76 in the supernatant andreached up to 84 (with chymotripsine) The by-products can be divided in 2parts ldquonon-solidrdquo ones (viscera skin roes and milt) and the ldquosolidrdquo ones(backbone cut-offs and head) which contain material more resistant to prote-olysis (cartilage or bone) Therefore the resulting pellet for the second groupof by-products were more important than those obtained for the other one thiswas particularly noticeable for hydrolysis with industrial enzymes

Surprisingly viscera expected the less ldquosolubilizingrdquo enzyme was Fla-vourzymereg Under its action the proportion of dry matter in the supernatantwas always lower than those obtained with the others enzymes tested How-ever Flavourzymereg is a proteasepeptidase complex which contains bothendoprotease and exopeptidase activities Such properties normally lead to ahigher degree of hydrolysis of native proteins by comparison to others en-zymes Thus the weak effectiveness observed was certainly due to parametersnot optimized such as ratio EnzymeSubstrate proportion of water etc


Pellet Supernatant

100

80

60

40

20

0

of

dry

mat

ter

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pr

Fl

Pr

Fl Pr

Fl Pr

Fl

Viscera Cut-offs Milt Backbone Heart Skin Roes

FIGURE 2 Dry matter distribution

Pa Papaiumlne Ch Chymotrypsine Pr Protamexreg Fl Flavourzymereg

Lipid Content

The second level of analysis was the distribution of lipid compoundsamong pellet and supernatant and a comparison with classical extraction with-out hydrolysis results were expressed by proportion of lipid into dry weight(Figure 3)

Without action of exogenic enzymes the quantities of lipids extracted werelower for skin cut-offs head and backbones (lt 6 of dry weight) by compari-son to those obtained from roes and viscera (9 and 15 of dry weight respec-tively) Considering the great variability of the biochemical composition offish and notably cod species (due to factors like size sexual maturity diet age(Dambergs 1963 1964 Ingolfsdottir 1998)) and also the effectiveness of thechemical extraction of lipids (Roose and Smedes 1996) the results obtainedin this study are globally in accordance with those previously published(Dambergs 1963 1964 Jangaard et al 1967 Shahidi et al 1991Ingolfsdottir 1998)

Whatever the enzymes or the by-products used except for the experimentson head with Flavourzymereg more lipids have been extracted when a pre-hy-drolysis has been done (sum of lipids in the supernatant and pellet by compari-son to ldquoclassical extractionrdquo) This ratio (lipids in pellet + lipids insupernatantlipids classically extracted) varied from 095 (head with Fla-vourzymereg) to 716 (skin with Protamexreg) Those expected results reveal thateven with precautions taken such as a pre-incubation phase of fresh tissueswith polar solvent (methanol in this study) and a vigor mix (ultraturax) beforeadding less polar solvent the lipid extraction was not complete The additional


Pellet Supernatant1

09

08

07

06

05

04

03

02

01

0

glip

idg

dry

wei

ght

M Pa Ch Pr Fl M Pa Ch Pr Fl M Pa Ch Pr Fl M Pr Fl M Pr Fl M Pr Fl M Pr Fl

Viscera Cut-offs Milt Backbone Head Skin Roes

FIGURE 3 Lipid distribution

Pa Papaiumlne Ch Chymotrypsine Pr Protamexreg Fl FlavourzymeregM classical exraction by Folch without hydrolysis

lipids obtained by using proteolytic enzymes were probably among the mostdifficult extractable ones by classical methods Of course the yield of extrac-tion of lipids by solvent could be enhanced by a second extraction (Roose andSmedes 1996) however this is very timendashand solvent-consuming

As for the dry matter in most of the experiments Flavourzymereg seemed tobe less effective for extracting lipids by comparison to other enzymes How-ever even if the enzymatic conditions were not optimized the use of Fla-vourzymereg led to a better extraction of lipids (except for head) The best yieldwas obtained by using this enzyme on viscera (086 g of lipidsg of dry matter)Regarding the efficiency of enzymes for extracting lipids no global tenden-cies can be drawn the results varied according to the by-products Indeed ifProtamexreg was the most effective for the skin experiment and Flavourzymereg

was the worst results were inversed for the viscera experiments Thus it ap-pears that no enzyme can be preferentially selected whatever the fresh materialto study and preliminary assays have to be done

If the dry matter was preferentially made soluble under the action of prote-olysis this was not the case for the lipids which still mainly distributed into thepellet (Figure 3) The proportion of lipids into the supernatant never exceeded44 with the exception of the roes hydrolysis by Flavourzymereg (63)Moreover for half of the experiments no more than 10 of the total lipids ex-tracted were recovered in the pellet (backbones hydrolysis by Flavourzymereg

and Protamexreg all the cut-offs hydrolysis head hydrolysis by Flavourzymereg

and viscera hydroysis by Flavourzymereg papaiumlne and chymotrypsine) Underthese conditions if the main goal is to collect lipids such supernatants couldbe discarded without important losses As for total lipids the repartitionamong pellet and supernatant of lipid compounds could not be predicted ac-cording to the enzyme or the raw material selected On the other hand miltlipids had the highest tendencies to be partially located into the supernatantwhatever the enzyme used However the global tendency observed was a con-centration of lipids into the non-soluble part which will be of great benefit fortheir organic extraction in that much less organic solvent can be used in the ex-periments

Phospholipid Content

The third level of analysis was the distribution of phospholipids among pel-let and supernatant and a comparison with classical extraction without hydro-lysis results were expressed by proportion of phospholipids into dry weight(Figure 4)

With classical extraction the phospholipids were ranging from 04(cut-offs) to less than 9 (milt) of the dry weight With the exception of twoby-products (roes and milt) all others were found quite low in phospholipids


(not more than 132 mgg of dry weight) Those results were not surprising asit is well known that the phospholipids are generally located in cellular mem-branes and some tissues like gonads and muscle are richer than others(Jangaard et al 1967 Standsby et al 1990)

With a proteolysis before the quantities of extracted phospholipids in-creased whatever the enzyme or the by-products used except for Protamexreg

on viscera (7 phospholipids lost) and for Flavourzymereg on head (40phospholipids lost) Thus with proteolytic enzymes the content ofphospholipids compared to classical extraction has been multiplied by 11(milt hydrolyzed by Protamexreg) up to 230 (cut-offs hydrolyzed by papaiumlne)The highest increase has been obtained for the cut-offs whatever the enzymeassayed and yield was multiplied by at least 57 (cut-offs withFlavourzymereg) As for the global lipids the comments on extraction effi-ciency (see above) are also valid here

Also like total lipid phospholipids were mainly located into the pellet(Figure 4) Different groups can be created regarding the distribution ofphospholipids one group where none were found into the supernatant (back-bones and cut-offs hydrolyzed by Protamexreg) a second group with only fewphospholipids into the supernatant (less or equal to 15 chymotrysinehydrolysate of cut-offs and viscera Flavourzymereg hydrolysis of backbonecut-offs head milt and skin papaiumlne hydrolysis of cut-offs and viscera andhead hydrolysis by Protamexreg) one group where phospholipids in thesupernatant was ranging from 22 to 47 (milt hydrolysis by Protamexreg


Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t

M PaChPr Fl M PaChPr Fl M PaChPr Fl M Pr Fl M Pr Fl M Pr Fl M Pr Fl


FIGURE 4 Phospholipid distribution


chymotrypsine and papaiumlne hydrolysis of viscera by Protamexreg andFlavourzymereg Protamexreg hydrolysis of roes) and at least one group wherephospholipids were found in majority in the supernatant (57 when skin washydrolyzed by Protamexreg and 59 when roes were hydrolyzed byFlavourzymereg) Those observations indicated that some parts of phos-pholipids could be recovered into the aqueous soluble fraction without any useof organic solvent

In order to know if enzymes can preferentially help the extraction of thosecomplex lipids with regards to others lipids the ratio phospholipidstotallipids was calculated (Table 2) A classification in 3 different clusters can thenbe made in the first one whatever the enzyme used the pre-hydrolysis stephas lead to an increase of the ratio (backbones and cut-offs) at the opposite inthe second one were located the by-products where less phospholipids ex-tracted after hydrolysis were found than without the use of enzyme (head miltand roes) and in the last group the remaining by-products have been broughttogether which according to the enzyme the yield was increased or lowered(skin and viscera)

Fatty Acid Content

The final level of analysis was the composition of fatty acids of lipid ex-tracts with a focus on the two more interesting ones the Eicosapentaenoic acid(EPA 2053) and the Docosahexaenoic acid (DHA 2263) They werequantified by gas chromatography (see MampM section) an example of achromatogram is presented in Figure 5

As shown in Figure 6 without proteolysis the quantities of EPA were atleast 50 less for skin cut-offs roes head and backbones (less than 4 mgg ofdry matter) than those found in viscera and milt (more than 8 mgg dry matter)This was also found true for the DHA content with less than 5 mgg dry matterand about 10 mgg of dry matter respectively (Figure 7) Thus it appearedthat viscera and milt were the most interesting by-products for extracting clas-sically valuable polyunsaturated fatty acids such as EPA and DHA they werein fact twice richer than the other secondary raw material studied

By using enzymes before extraction the quantities of EPA increased for allthe experiments except the one with head and Flavourzymereg The ratio EPAextracted with pre-hydrolysis (pellet + supernatant)EPA ldquoclassically ex-tractedrdquo varied from 055 (head with Flavourzymereg) to 938 (skin withProtamexreg) These results were not surprising as they reflected those obtainfor total lipids Another expected result was the distribution of EPA mainlyinto the pellet as for total lipids and phospholipids Thus only two experi-ments recovered higher quantities of EPA in the supernatant compared to pel-let (roes with Flavourzymereg 65 and skin with Protamexreg 53)


TABLE 2 Comparison of total lipids phospholipids EPA and DHA extracted with or without a proteolysis of theby-products

Enzyme By-products Total lipids Phospholipids EPA DHA Ratio

(mgg dry weight) PLlipids EPAlipids DHAlipids EPADHA

Classical extraction Backbones 6420 976 385 498 015 006 008 077

Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082

Hydrolysis Ch Cut-offs 13747 4918 724 917 036 005 007 079

Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80

Moreover for all the experiments carried out on cut-offs viscera and back-bones less than 10 of EPA were found in the soluble part

For the DHA the results were quite different In addition to the experimentusing head and Flavourzymereg (ratio 047) some others also led to a diminu-tion of the extracted DHA backbones with Protamexreg (074) and viscera withchymotrypsine (089) On the other hand in some cases the yield was greatlyincreased up to 5 (skin and roes with Protamexreg) and even 6 (cut-offs withpapaiumlne) As for the EPA the major part of extracted DHA was located intothe pellet Indeed only two hydrolysis reactions had more than 10 of DHA


4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)

FIGURE 5 Fatty acids chromatogram obtained for miltrsquos pellet hydrolysed byProtamexreg

Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t



FIGURE 6 Eicosapentaenoiumlc acid distribution


in the soluble part skin with Protamexreg (13) and roes with Flavourzymereg

(26) Furthermore all the hydrolysis realized on cut-offs viscera and back-bones were unable to liberate DHA into the soluble part

Those elementary analyses however must be completed by the study ofother ratios such as EPAtotal lipids and DHAtotal lipids in order to know ifpreferential extraction of fatty acids (ie specific lipids) could be obtained bysuch enzymatic hydrolysis (Table 2) The proportion of EPA among totallipids varied from 26 (viscera hydrolyzed by Flavourzymereg) to 73 (skinhydrolyzed by Flavourzymereg) By comparison to classical extraction a globalenrichment of total lipids in EPA was noticeable for about half the experi-ments all those conduced on cut-offs roes and skin and the hydrolysis of vis-cera by Protamexreg For the other ones whatever the enzyme used aproteolysis of the raw material led to a thinning of extracted fat in EPA Re-garding the DHA (Table 2) its proportion among total lipids varied from 24(viscera hydrolyzed by Flavourzymereg) to 84 (cut-offs hydrolyzed bypapaiumlne) As for the EPA exactly the same experiments led to a global enrich-ment or at the opposite a thinning of fat in DHA The ratio EPADHA variedfrom 064 (cut-offs hydrolyzed by Protamexreg) to 265 (roes hydrolyzed byFlavourzymereg) However as expected this ratio was only slightly modifiedby the hydrolysis step as the EPA and DHA varied in the same way Thus itappeared that for EPA and DHA notably in addition to a global increase oftheir extraction (few cases excepted) a modification of the fatty acid composi-tion of fat could be obtained by doing a pre-hydrolysis step


Pellet Supernatant30

25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght



FIGURE 7 Decosahexaenoiumlc acid distribution


CONCLUSION

Despite using non-optimizing hydrolysis conditions the use of proteolyticenzymes for partially disrupting lean fish tissues had most of the time en-hanced the fat extraction Thus proteolysis acted favorably on the phos-pholipids extraction by enhancing them However this increase wassometimes higher for the total lipids than for those complex lipids which infact led to a reduction of the proportion of the phospholipids among the totallipid compounds extracted this way It was also the case for the two most inter-esting fatty acids namely EPA and DHA which despite of their better extrac-tion with the pre-hydrolysis could be found in less quantities in the totalextracted fat

The greatest benefit of such a proteolytic step in addition to the global ex-tracting fat enhancement is the concentration of most of the lipids into thenon-soluble part (pellets) which will lead to less solvent consuming Howeveradditional works have to be done notably the optimization of the hydrolysisthe use of others enzymes (alone or in combination) in order to improve theyield and perhaps making complex lipids soluble in order to collect themwithout any use of organic solvents

REFERENCES

Ackman RG 1999 Docosahexaenoic acid in the infant and its mother Lipids 31125-128

Bergeacute JP Debiton E Dumay J Durand P and Barthomeuf C 2002 In vitroanti-inflammatory and anti-proliferative activity of sulfolipids from the red algaPorphyridium cruentum J Agric Food Chem 50 6227-6232

Brocklehurst K 1995 Electrochemical assays the pH-stat In Enzyme assays a prac-tical approach The practical approach series Eisenthal R and Danson MJ (Eds)New York Oirl Press pp 191-216

Dambergs N 1963 Extractives of fish muscle 3 Amounts sectional distribution andvariation of fat water solubles proteins and moisture in cod (Gadus morhua L) fil-lets J Fish Res Board of Canada 20 909-918

Dambergs N 1964 Extractives of fish muscle 4 Seasonal variation of fat water-solu-bles proteins and water in cod (Gadus morhua L) fillets J Fish Res Board of Can-ada 21 703-710

Folch J Lees M and Sloan Stanley GH 1957 A simple method for the isolationand purification of total lipids from animal tissues J Biol Chem 226 497-509

Ingolfsdottir S Stefansson G and Kristbergsson K 1998 Seasonal varioation inphysiochemical and textural properties of North Atlantic cod (Gadus morhua)mince J Aquatic Food Prod Technol 7(3) 39-61

Jangaard PM Ackman RG and Sipos JC 1967 Seasonal changes in fatty acidcomposition of cod liver flesh roe and milt lipids J Fish Res Board of Canada 24613-627


Kolanowski W Swiderski F and Berger S 1999 Possibilities of fish oil applicationfor food products enrichment with w-3 PUFA Int J Food Sci Nutr 50 39-49

Minnis RC Haq IU Jackson PR Yeo WW and Ramsay LE 1998 Oily fishand fish oil supplements in the prevention of coronary heart disease J Human NutrDietetics 11 13-19

Rambjor GS Walen AI Windsor SL and Harris WS 1996 Eicosapentaenoicacid is primarily responsible for hypotriglyceridemic effect of fish oil in humansLipids 31 45-49

Roose P and Smedes F 1996 Evaluation of the results of the QUASIMEME lipidintercomparison the Bligh amp Dyer total lipid extraction method Mar Pol Bull 32674-680

Rustad T and Falch E 2002 Making the most of fish catches Food Science andTechnology 16(2) 36-39

Sanger F 1949 The terminal peptides of Insulin Biochem J 45 563-573Shahidi F Naczk M Pegg RB and Synowiecki J 1991 Chemical composition

and nutritional value of processing discards of cod (Gadus morhua) Food Chem42 145-151

Simopoulos AP 1997 Essential fatty acids in health and chronic disease Food RevInt 13623-631

Stansby ME Schlenk H and Gruger EH 1990 Fatty acid composition of fish InFish oil nutrition Stansby ME (ed) pp 6-37

Stewart JCM 1980 Colorimetric determination of phospholipids with ammoniumferrothiocyanate Anal Biochem 104 10-140


lthttpwwwHaworthPresscomgt 2004 by The Haworth Press Inc All rightsreserved]

KEYWORDS By-product cod lipid extraction protease

INTRODUCTION

Characterization of lipid in some by-products (liver roe viscera headmilt backbone and cut-offs) from some cod species has shown that these partscan be utilized as a raw material for production of lipid compounds of interestsuch as fish oils (Rustad and Falch 2002)

Indeed fish oils are an important source of polyunsatured fatty acids(PUFA) and are now widely used in health and nutrition (Shahidi et al 1998Kolanowski et al 1999) For a long time these molecules were shown to be ofgreat importance in the prevention of a number of diseases such as inflamma-tion hypotriglycerimedic effect allergies diabetes or coronary heart disease(Rambjor et al 1996 Simopoulos 1997 Minnis et al 1998 Ackman 1999)However nowadays in industry fat extraction is carried on at high tempera-ture which can lead to some lipid oxidation and little work has been done foroptimizing those extractive conditions The use of enzymes such as proteasescould be an alternative solution to the present process conditions which de-stroy valuable components in the oils and oils with higher quality will createnew markets and obtain higher prices

This preliminary work presents the yield variations of fat extraction afterprotease hydrolysis of cod by-products

MATERIAL AND METHODS

Raw Material

This study focused on cod by-products (Gadus morhua) provided by theIcelandic Fisheries Laboratories (IFL) Those secondary raw materials wereselected on board immediately after the catch in the Icelandic sea in June2001 They were head backbones viscera roes milt skin and cut-offs Theywere kept frozen until landed then they were mixed and stored under vacuumat 80degC before analysis

Enzymes

Enzymes selected were wide spectrum proteases because they may nothave denaturant properties on lipidic compounds Two different kind of en-



Hydrolysis






Enzyme AS(Unitg)


(ww)

Temperature(degC)


Catalyst



Protamexreg 15 020 60 60 1 -



Dry Matter Content



freshmatte()

( ) ( )=

minusr g( )

Lipid Extraction


Phospholipid Assay






Hydrolysis



4035

30

25

20

15

10

5

00 10 20 30 40 50 60

Nh

(10

mm

oles

)2

5


Time (min)







Pellet Supernatant

100

80

60

40

20

0

of

dry

mat

ter

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pr

Fl

Pr

Fl Pr

Fl Pr

Fl




Lipid Content





Pellet Supernatant1

09

08

07

06

05

04

03

02

01

0

glip

idg

dry

wei

ght




















Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t







Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES






















Hydrolysis






Enzyme AS(Unitg)


(ww)

Temperature(degC)


Catalyst



Protamexreg 15 020 60 60 1 -



Dry Matter Content



freshmatte()

( ) ( )=

minusr g( )

Lipid Extraction


Phospholipid Assay






Hydrolysis



4035

30

25

20

15

10

5

00 10 20 30 40 50 60

Nh

(10

mm

oles

)2

5


Time (min)







Pellet Supernatant

100

80

60

40

20

0

of

dry

mat

ter

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pr

Fl

Pr

Fl Pr

Fl Pr

Fl




Lipid Content





Pellet Supernatant1

09

08

07

06

05

04

03

02

01

0

glip

idg

dry

wei

ght




















Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t







Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES






















Dry Matter Content



freshmatte()

( ) ( )=

minusr g( )

Lipid Extraction


Phospholipid Assay






Hydrolysis



4035

30

25

20

15

10

5

00 10 20 30 40 50 60

Nh

(10

mm

oles

)2

5


Time (min)







Pellet Supernatant

100

80

60

40

20

0

of

dry

mat

ter

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pr

Fl

Pr

Fl Pr

Fl Pr

Fl




Lipid Content





Pellet Supernatant1

09

08

07

06

05

04

03

02

01

0

glip

idg

dry

wei

ght




















Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t







Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES
























Hydrolysis



4035

30

25

20

15

10

5

00 10 20 30 40 50 60

Nh

(10

mm

oles

)2

5


Time (min)







Pellet Supernatant

100

80

60

40

20

0

of

dry

mat

ter

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pr

Fl

Pr

Fl Pr

Fl Pr

Fl




Lipid Content





Pellet Supernatant1

09

08

07

06

05

04

03

02

01

0

glip

idg

dry

wei

ght




















Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t







Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES


























Pellet Supernatant

100

80

60

40

20

0

of

dry

mat

ter

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pa

Ch Pr Fl

Pr

Fl

Pr

Fl Pr

Fl Pr

Fl




Lipid Content





Pellet Supernatant1

09

08

07

06

05

04

03

02

01

0

glip

idg

dry

wei

ght




















Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t







Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES





















Lipid Content





Pellet Supernatant1

09

08

07

06

05

04

03

02

01

0

glip

idg

dry

wei

ght




















Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t







Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES




































Pellet Supernatant

200

150

100

50

0

mg

phos

phol

ipid

gdr

yw

eigh

t







Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES























Fatty Acid Content









Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES

























Cut-offs 4910 369 205 262 008 004 005 078

Head 5314 1312 305 339 025 006 006 090

Milt 15386 8677 892 1002 056 006 007 089

Roes 8840 5357 276 208 061 003 002 132

Skin 4199 1138 203 207 027 005 005 098

Viscera 14575 612 813 989 004 006 007 082


Ch Milt 42575 20395 1745 1059 048 004 002 165

Ch Viscera 28080 2945 1031 877 010 004 003 118

Fl Backbones 9557 4154 511 520 043 005 005 098

Fl Cut-offs 7001 2089 319 427 030 005 006 075

Fl Head 5071 780 167 160 015 003 003 104

Fl Milt 30464 10243 1088 970 034 004 003 112

Fl Roes 23741 8220 1539 581 035 006 002 265

Fl Skin 5118 2538 372 333 050 007 006 112

Fl Viscera 86334 1022 2286 2072 001 003 002 110

Pa Cut-offs 19197 8505 1199 1611 044 006 008 074

Pa Milt 32131 15449 1728 1283 048 005 004 135

Pa Viscera 44765 3284 2065 2527 007 005 006 08279

TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES





















TABLE 2 (continued)



Pr Backbones 10023 4059 408 369 041 004 004 111

Pr Cut-offs 8845 2441 474 742 028 005 008 064

Pr Head 16596 2543 688 814 015 004 005 084

Pr Milt 36146 9787 1417 1136 027 004 003 125

Pr Roes 28382 9888 1959 1062 035 007 004 184

Pr Skin 30076 7182 1904 1021 024 006 003 187

Pr Viscera 29600 571 1711 2114 002 006 007 081

80




4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES
























4500000

4000000

3500000

3000000

2500000

2000000

0 10 20 30 40 50 60

DHAEPAint Std

Are

a(micro

Vm

in)

Time (min)


Pellet Supernatant

25

20

15

10

5

0

mg

EPA

gD

ryw

eigh

t










25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES


























25

20

15

10

5

0

mg

DH

Ag

Dry

wei

ght





CONCLUSION



REFERENCES





















CONCLUSION



REFERENCES





















How Enzymes May Be Helpful for Upgrading Fish By-Products

Documents

Transcript of How Enzymes May Be Helpful for Upgrading Fish By-Products