by F.M. Rzhavskaya, T.A. Dubrovskaya, A.M. Makarova, and L.V. Pravdina

Transcription

1 FISHERIES AND MARINE SERVICE Translation Series No Study of the qualitative state and the lipid fatty acid composition in relation to their method of isolation from muscle tissues of frozen and salted fishes by F.M. Rzhavskaya, T.A. Dubrovskaya, A.M. Makarova, and L.V. Pravdina Original title: Issledovanie kachestvennogo sostoyaniya i sostava zhirnykh kislot lipidov v Zavistimosti ot metoda ikh vydeleniya iz myshechnoi tkani morozhenykh i solenykh ryb From: Tr. Vses. Nauchno-Issled. Inst. Morsk. Rybn. Khoz. Okeanogr. (123): Translated by the Translation Section (GAD) Department of Fisheries and Environment Department of Fisheries and Environment Fisheries and Marine Service Halifax Laboratory Halifax, N. S pages typescript

2 I J.DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS TRANSLATED FROM - TRADUCTION DE Ruse jan AUTHOR - AUTEUR MULTILINGUAL SERVICES DIVISION DES SERVICES DIVISION CANADA INTO - EN e -19, 11.,a-b.: MULTILINGUES F-éni) F.M. Rzhavskaya, T.A. Dubrovg'keyA, A.M. MAkArovo, And L.V. Prevdina TITLE IN ENGLISH - TITRE ANGLAIS STUDY OF THE QUALITATIVE STATE AND THE LIPID FATTY ACID COMPOSITION IN RELATION TO THEIR METHOD OF ISOLATION FROM MUSCLE TISSUES OF FROZEN AND SALTED FISHES TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÈRES ROMAINS) ISSLEDOVANIE KACHESTVENNOGO SOSTOYANIYA T SOSTAVA ZHIRNYKH KISLOT LIPTDOV X offleimuw OT METODA IKH VYDELENIYA IZ MYSHECHNOT TKANI MOROZHENYKH I REFERENCE IN FOREI GN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLI TERATE tforeign CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION).,AU COMPLET, TRANSCRIRE EN CARACTÈRES ROMAINS. Trudy VNII morskogo rybnogo khozyaistva i okeanografii REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS Proceedings of the A11 Union Scientific Research Institute of Marine Fisheries and Oceanography PUBLISHER - ÉDI TEUR PLACE OF PUBLICATION LIEU DE PUBLICATION USSR YEAR ANNÉE 1977 DATE OF PUBLICATION DATE DE PUBLICATION VOLUME ISSUE NO. NUMÉRO 123 PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 17 REQUESTING DEPARTMENT MINISTÈRE-CLIENT BRANCH OR DIVISION DIRECTION OU DIVISION PERSON REQUESTING DEMANDÈ PAR DFE Fisheries Dr. R.G. Ackman TRANSLATION BUREAU NO NOTRE DOSSIER N 0 TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) LU=M GAD YOUR NUMBER VOTRE DOSSIER NO DATE OF REQUEST DATE DE LA DEMANDE March 7th, 1979 APR SOS (REV. 2/68)

3 ,. Sedretary Secrétariat, of State d'état MULTILINGUAL SERVICES DIVISION - DIVISION DES SERVICES MULTILINGUES TRANSLATION BUREAU BUREAU DES TRADUCTIONS Clients No. No du client Departrnent Ministère Division/Branch Division/Direction City -7 Ville DFE Fisheries Halifax, N.S. Bureau No. No du bureau Language - Langue Translator (Initiais) - Traducteur (Initiales) Russian GAD ez? Trudy VNII morskogo rybnogo khozyaistva i okeanografii -(Proceedings of the All-Union Scientific Research Institute of Marine Fisheries and Oceanography), no. 123, 1977, pp UDC :543 U; EDirfED TRAM F>IATIO'M Vcr cn!y STUDY OF THE QUALITATIVE STATE AND THE LIPID FATTY ACID COMPOSITION IN RELATION TO THEIRMETHOD OF ISOLATION FROM MUSCLE TISSUES OF FROZEN AND SALTED FISHES By F.M. Rzhavskaya, T.A. Dubrovskaya, A.M. Makarova, L.V. Pravdina Proteins - the main components of the muscle tissue of fish and lipids /106* - undergo certain changes in processing of fish by different methods. In developing new technological processes and determining optimal regimes, it becomes necessary to evaluate the degree of change of proteins and lipids to ensure the maximal preservation of the food value of fish. Methods must be used which ensure the extraction of the most altered components of muscles tissues, in particular, the lipids. The absence of a definitive view on the optimal method of extracting lipids, and the difficulty of their exte,:action with a binary solvent (chloroform with methanol) necessitated a study of the effect of the method of isolation of lipids from the muscle tissues of chilled, frozen and salted fish on their composition and properties. When determining the composition of lipids, they are extracted as a rule from fresh or chilled fish; to eval- uate the qualitative state, which is determined by the degree of oxidation, the lipids are most often isolated from frozen and salted fish which has been in storage for varying durations. *The numbers in the right-hand margin indicate the corresponding pages in the original. :- Transi. SEC 6-25 (Rev. 6/78)

4 2 Differences discovered in some cases in the fatty acid composition,d and the degree of oxidation of the lipids of chilled fish extractr\by diff- erent methods [6,7 ] cannot be extended to frozen and salted fish because the products of oxidation and hydrolysis of their lipids can interact with proteins or the products of their hydrolysis with the formation of proteolipid or lipoprotein complexes, whose nature must be essentially different from that of the complexes characteristic of frozen fish. The nature of these complexes can have a definite effect on the characteristics of lipids depending on their method of isolation. We studied lipids extracted by different methods from the muscle tissue of frozen and'salted fish, differing in qualitative state and fatty acid composition. Information on the nature of the change in the composition of lipid, fatty acids of frozen and salted fish are very important for a correct understanding of the extent of their oxidation, since this process can be accompanied by the breakdown of fatty acid radicals, whichhare the basic stuctural element of lipids, subject to the effect of oxygen. The objects of research were frozen sardinella Sardinella sp., Spanish mackerel Scomber japonicus, ocean perch Sebestes marinus, halibut Hippoglossus sp., and salted Pacific herring Clupea harengus pallasi of different fatness (10.4 and 23%). The total amount of lipids was 10.9% in the muscle tissue of halibut and 3.6% in sardinella. The muscle tissue of the ocean perch and the mackerel were similar in lipid content (4.6 and 4.8% respectively). Lipids were isolated from the tissues of ocean perch and mackerel by four methods: infusion with chloroform for 4 and 24 hours [3] and extraction with binary solvent[13,8] - methanol with chloroform and ethanol* with chloroform; lipids were isolated from sardinella by prolonged infusion using methanol; from frozen halibut and salted herring with high lipid content by 5 methods: *Ethanol was used to study the possibility of using it to replace methanol.

5 3 infusion with chloroform of different duration and extraction with binary solvent, whose composition included methanol and ethanol, and also by centrifuging [2]. The indicators which characterize the degree of oxidation, hydrolysis and composition were determined in each lipid sample. The degree of oxidation of lipids was evaluated from the values of the peroxide and aldehyde numbers and the content of oxirane (epoxy) oxygen. The degree of hydrolysis was evaluated from the acid number. The composition of lipids was determined from the ratio of individual fatty acids, and also by some indirect characteristics (the iodine number and the content of unsaponifiable substances). The ratio of fatty acids was determined by the method of gas-liquid chromatography. For the methylation of fatty acids, their separation and identification, as well to determine the degree of oxidation and hydrolysis and the indicators of lipid composition, we used conditions and methods similar to those previously used [4,5,7]. The results obtained show that for slightly oxidized lipids, the method of separation does not substantially effect their qualitative state (Table 1). The content of peroxide, epoxy and carbonyl compounds is practically the same in lipids obtained by centrifuging, infusion with chloroform of different duration, and extraction with binary solvent using ethanol and methanol. However, as can be seen in the case of frozen halibut and ocean perch with slightly oxidized lipids, a tendency is observed towards increase in the aldehyde number, with either extraction of lipids by binary solvent, as with the halibut, or with prolonged infusion with chloroform, as with the ocean perch. In addition, the halibut lipids separated by centrifuging contain very little epoxy compounds (oxirane oxygen) and almost two or three times

6 4 less free fatty acids than when organic solvents are used [for separation]. Moreover, for halibut, as for ocean perch, the use of a binary solvent leads to a higher level of free fatty acids (FFA) (value of acid number), than does brief infusion with chloroform. Of the marine fish, mackerel and sardinella, whose lipids are more, /108 oxidized than those of halibut and ocean perch, show a more obvious effect of the method of separation of lipids on the qualitative state of lipids. This was reflected in the organoleptic features of the muscle tissue. Strong oxidation of the lipids of sardinella is confirmed by the high values of the peroxide and aldehyde numbers, although the content of epoxy compounds is relatively small (Table 1). In addition the FFA level is high in sardinella, just as in mackeral, which shows their rather deep hydrolysis; this also applies to a somewhat lesser extent to mackerel. The lipids of sardinella, when extracted by chloroform with methane, /109 have an equal content of aldehydes which react with benzidine, but contain more FFA and especially peroxide compounds than lipids extracted by prolonged infusion with chloroform. For mackerel, which has lipids, which are less oxidized than those of sardinella, the method of extraction affected the value of the aldehyde number (which was only a tendency in the lipids of ocean perch and halibut), the content of epoxy oxygen and fresh fatty acids. The use of a binary solvent ensured a higher content of these products of oxidation and hydrolysis by comparison with infusion with chloroform, especially brief infusion (4 hours). The lipids of salted herring also have a high level of fresh fatty acids when separated with a binary solvent (Table 2). In addition, unlike frozen sardinella, prolonged infusion with chloroform and extraction by binary solvent using methanol, result in the extraction of lipids with a somewhat lesser

7 1 5 Table 1 Degree of oxidation and hydrolysis and composition of lipids isolated by different methods from the muscle tissue of frozen fish. 1.."...1%., -.,.'',.. rlepe- Cogepr:. Cortep.,,f4..p44o ,.-,?..,f, (.,'.:"- --,.9.,..f? :.*IvieTort mchoe?kaii.ii, --:,f-traedé Jicinibé xiaahe tuien:ti;'';'' : 3,!,,l'....Witëneaxsi, ii4lc.r:i'o,,,, ::, xdar..i.â 4igçno,... ilicno, aeomm- %,,f,i6,14a:?eilloga -,ilosor ': iv.ir Ko- mr. n_zemblx, : KIIC110-: ', p --'.'.,.. ntsc Hir ïéi.iiecte.,-.:. ',-_,,.. Pic;),4aeo'. ::éiiliqe;,.i',,p,4:, 'tarta t,i é îl.iewrprer.xposamie 0 HacTaxaaame c xnopoclepmom B Tetienme xi 0 I24 '0 RaKcrpalawsi 61-map- HBIM paczaopwrenem 13 xnopod)opm+ataaon 0 xnopoci)opm+meta- 0 Ilanryc /5-0,004 2,2 1,5-82,7 0 0,6 4,1 2 14:y 84,7 5 0,6 6,8 2,7 90,9 0,06 0,6 7,6 2,0 0;06 1,3 7,1 2,4 110.?: r -. Mopcxoe oxyab qhactansaalle c xnopodlopmom B Teuelnie /0 4 n 0 7 0,6 12,8 2,2 102,9 /1. 24 u 0 6 2,2, 14,0 2,4 122,9 I plcczpainutsi 6miap- }lbw pactsopatenem /.5)(nopocPopm+DTaxon I tpcnopottopm-fmeta-..-aon oc,i.hactalisamle c xno-. pocpopmo.1 B Teueime 0) 4 u - tepscipalguisi 6liaap- \4i;W4paraoparenem lexiicipo4aopm+meta- llactansalme c xnopoclaopmom a Tenemie ,1 Ct ,3àçTpalourg xnopo- 6 C metano- nor..el- Cxy..16pnn 15,0-2,2.. 2,4 _ 132, ,7 21,9 4,1 142,0. 4 4,4 22,9 4,2 141, ,6 25,6 4,0 141, ,8 25,8 4,2 144,0 Capnaserma g_ ,6: 42,5 3,2 148,0 4 21,6 5 3,2 15 : : Key to table on page 6

8 Key: 1 - method of isolation; 2 - peroxide number, %, iodine; 3 - content of oxirane oxygen, %, 4 - aldehyde number, mg% of cinnamic aldehyde;5 - acid number, mg KOH/gm; 6 - content of unsaponifiable substances, %; 7 - iodine number, % iodine; 8 - centrifuging; 9 - infusion with chloroform; 10-4 hours; hours; 12 - extraction with binary solvent; 13 - chloroform plus ethanol; 14 - chloroform plus methanol; 15 - halibut; 16 - ocean perch; 17 - mackerel; 18 - sardinella. difference in the amount of fresh fatty acids. However, this difference increases with increase in the degree of hydrolysis. At the same time, the method of separation of _ lipids typically affects salted herring with strongly oxidized lipids. Moreover, centrifuging of lipids from fat and lean herring produces lipids with a low level of fresh fatty acids, maximal content of primary products of oxidation - (peroxide compounds) and minimal content of secondary oxidation products (aldehydes). /110 Lipids, extracted with a binary solvent using ethanol have the least amount of peroxide compounds and a low amount of aldehydes (lean herring). Peroxide compounds and aldehydes are at a similar level in lipids extracted by prolonged infusion with chloroform and by extraction with binary solvent using methanol (see Table 2). The method of extraction of lipids has little effect on the amountof epoxy compounds, although a tendency to increase in epoxy compounds is observed in lipids extracted by prolonged infusion with chloroform and by binary solvent, especially for fat herring. Thus the use of a binary solvent with methanol makes it possible /111 to separate the most deeply oxidized lipids, which also contain ther greatest amount of fresh fatty acids. It is sometimes also possible to extract oxidized lipids by prolonged infusion with chloroform, but with this method the lipids always contain a lesser amount of fresh fatty acids; this intensifies with intensification of the hydrolytic dissociation of lipids. Extraction by binary solvent, using the less polar ethanol instead of methanol, produces lipids with a lesser amount

9 Degree of oxidation, hydrolysis and composition of lipids methods from the muscle tissue of salted herring. _ Km,c- LtlyZ`plicPyrxposaline, fietalw.anile c xnopo- i, 4,! tt '2, 24 0; pax!,z.ug 61mapilbim \p4c.n36pwrenem. xnopotopm -FaTaHciii ' opocilopm+metation flepexucnoe koela 2$11enTpladoenpoBalalie 0,60 tillacralmame c xnopo-,(popmom Tenexime xi 5 11,3xerpalux.usi 6nHapmem pactsopprrenem pi xnopocliopm-ketahon,0,08 xn9po4>ppremetahon 8 I if - Con,ep- >igiiew oxenpalmsorb KHC1101- a /0 3. mr KoPg71" KOH/r (51'so; anbizel HeempHa2 cent 0,03 :13,7 CorteP- ')i,œfiâel.embut. 0;04 29,6 22,5 1,6 0,04 35,9 26,4 2,0 i:), 18,4, 0,06 32,7 31,6 )KapHasi cesubm, 9 0,05 8,1 8,7 1,3, ,6 14, ,6. 14,2 c? Table 2 7 isolated by different 2,0 1.17,4 2,2 128,0,,, 105;6,1 0,07. 38,3 12,6 2;0 5 0,08 36,9 13,6! 1, ,8 '100,8 1,7 125,8 Key: 1-14 same as in Table 1; 15 - lean herring; 16 - fat herring. of primary and secondary products of oxidation, even by comparison with brief infusion with chloroform. The method of isolation of lipids is reflected both in the indicators of lipid composition (see content of unsaponifiable (as in frozen halibut and Table 1 and 2) and in the ratio of fatty acids. The substances is sometimes low in centrifuged lipids, salted herring) while the values of the iodine numbers, that is, the degree of nonsaturation, are high in the case of lipids extracted

10 with chlorofonnusing methanol. This is correlated with the ratio of individual components of fatty acids. A low degree of unsaturation is typical of either centrifuged lipids (as in halibut) or those extracted by chloroform with ethanol (as in fat herring). The method of extraction of lipids affects the ratio of fatty acids even with a relatively low degree of oxidation and completely satisfactory organoleptic features of the fish. This has been observed in the high-quality frozen halibut and ocean perch (Table 3). Lipids isolated from frozen halibut by centrifuging and by infusion with chloroform, especially brief infusion, have a relatively high level of monounsaturated acids (mainly 18:1 and 20:1) and a minimal amount of polyunsaturated acids (mainly acid 22:6). Lipids extracted with binary solvent have a minimal content of monounsaturated acids;when ethanol is used, this is due to acids 18:1, 20:1 and 22:1, and when methanol is used, it is due to acids 16:1 and 18:1. The use of ethanol, in addition, favors an increase in the level of saturated acids, mainly 14:0. The more polar alcohol (methanol) in the binary solvent ensures the highest total of polyunsaturated acids, mainly due to the high amount of acid 22:6. The lipids of the ocean perch, when separated by brief infusion with chloroform (see Table 3), have a minimal amount of polyunsaturated acids because of the large deficiency of acids 20:5 and 22:6, despite a certain predominance of arachidonic acid; these lipids contain the greatest amount of saturated acids. Lipids extracted by 24 hr. infusion with chloroform and chloroform with methanol are practically equivalent in terms of the total of saturated acids and the ratio of their individual components, but there are slightly more monounsaturated acids in the second case; the dominant polyunsaturated acids are represented in almost equal amounts. The lipids of the muscle tissue of frozen sardinella, when>extracted /116 with chloroform and methanol, contain somewhat less monounsaturated acids (16:1) than when extracted by prolonged infusion with chloroform. In addition,

11 ' Table 3 The composition of fatty acids (in %) of lipids isolated by various methods form the muscle tissue of frozen fish. Kecncrre 12:0 13:0 14:0. 14: :0 16:0 16:1W7 16:2111'4 16:4 17: :0 18:1019* * 18:2e 18:3(1)6 1 18:4(1131 itentpx.- WH130- rue 12,7 13,4 16,6 16,1 mac-raw:tau» c exctpaxelle 611- potbopmom a Tee-I uapii1tm pactao- HHe de' pmenem 4 g 4 13,5 13,8 15,5 15,0 0,6. 24 g 1 xnopo- IxnopooPm+ pm+ 9 =IL peetanspl'-"1 = 11,2 13,2 14,6 15,0. 15,0 14,0 16,0 '14,4 0,7 11,5 12,7 13,3 *. 14,0 MODCK nacrauaanne c xné-exctpexusui 6apotbopmom a Tome- 1 impaled pactaoaxe -- prrelem 7. 4q 14, ,2.15,7 11M0 1.4,9 1,3 1,3 ' 24 gl xnopol.) LcinciPo-,f _ cleptvea eopro+.7 P- ermon_ vratten. 13,1-18,9 14,4 13,6 0,9 13,7. 15,1 14, ,8 1,0 1, ,0. 18,5 14,5 gel 14,7. - mogia_an nacraxaa axe c ;cafe?, cleprem -fh D.nae. 24g 16,8 0,6 18,5 16,0 0,8 1,2 6,8 1,4 0,7 a -exctpoxunnxno-. polep- M014 C reetanonom i 17,7 I 0,7 18,2 14,6 0,7 1,1 6,2 1,3 1,3 1,0 Key to table on page 11. L 19:0 19: :1019 * 20: ' 20:3 20:3q3 ' 20:411f6 20: :5d3 22:1( :5d6 22:5(C/3 22: 6(03 / z.. 1 -Lacbauennble / 3 mohollenacm, meanme n itm CbIllieHHb10 17,6 1,6 1,7 1,4 15,5 WM 0,9 26,8 66,1 7,1 17,5 0,7 1,3 1,1 16,8 1,5 27,9 65,4 6,7 17,3 0,6 1,5 2,8 1,4 16,0 Mal 4,4 24,7 63,1 1.2,2 0,6 15,4 1,5 0, ,4 MM1 4,2 3 69,6 1 16,8 0,6 1,6 2,0 2,7 1.5,3 6,0 25,0 60,0 15,0 0,8 0:2. 0,8 0,7 :o 3 ' 6,8 5,9 7,1 7,7 ' 1,0 1,4 1,1 0,6 0,8' 0,6 0,6 0,8 0,7 0,8 0,6 0,8 ' 0,6 -- 2,8 ' 0,9 0,6. 2,1. 1,4 1,5 1,8 1,5 1,3 1,8 2,6 2,8 5,9 6,4 5,5 11,8 9,1-8,6 7,9 8,8 7,7 8,4. 6,2 1,2 1,2 0,7 Z. 0,6 0,8 0,9 2,6 : 3,2 10,7 8,8 1 8,4. 9,9 ; 3(3,6 33,1 3 33,3 7,8 38,5 46,8 42,7 46,3 44,9.uos. 30, 5 16,6 24, 2 23, ,6 31,0

12 Composition of fatty acids (in %) of lipids isolated from muscle tissue by various methods. Table 4 I/ Knonorà 12:0 13:0 14: : :0 16,0 16:1(977 * 16:2(974 17:0 18:0 18:1(9, 1.8:2(06 18: 3( :4(13 )1, 19: :1(09. 20:2076 ' 20: : 3(113 20:4(06 g0: : 5 (9T3 22:10/11 22:6(97'3 9 Haeblutennbté / 0 M01.10HeHacbluen- 'we lionunexacbttrunmo uentplicpyrxpopanne 1 3, ,5 20,7 1,4 1,2 3,2 0,8 0,9 7,7 4,3 5,2 35,5 46,1 18,4 2 Hamm-fag cameo. HaCTEMB8110e C xno- xctp la3xu7g 61:7-7Le; 3 ACTBO 'pre em xnopo- a.._!2... tvforv 4 it 24 q xnopà- (bopm+ 9T8H0/17,t , ,2 21,7 1,9 1,7 3,0. 0,7 1,0 0,9 4,9 3,4 4,5 39,1 44,5 16,4 14,0 24,6 16,4 20,8 0, ,5 0,9 1,1 2,7 3,9 6,3 39,5 46, \ 12,7 20,9 16,2 20;4 28 1,0 0,6 4,1. 0, ,0 3,4 5,6 34,9 44,9 2 tilopm+ MeTano.. 11,9 22,4. 15,5 1,9,4 1,9. 1,0 - * 2,0 j. 0,6 1,6 4,9 3,9 10,9, 35,6 ' 41,3 j 23,1. )Knonan ce fib Il8CTaltB/111HOCxno- MCCTpLIKURM 6uHapn04)onmom a Tegent'è HIAMPaCirsomyeneg UeliTpH trriipo- 4 it It 24 q xnopo- I xnopo ellopm+ timpm+ a, meramon 19,2 18,9 18,0. 18, ,8 ' 16,3. 16,0 19,7... 0,8 0,8 0,8 1.4,2 13,7 14, ,2 1,7 '. 0,6.. 5,4. 7,4 6,2 0, ,7 0,8 0,7 1,3 2,7 1,3 0,6 5,6 3,7 4,9 4,9 8,8 6,1 6,0 5,6 5,1 38,13 37,8 35,9 41,1 46,2 47,2 2 I 16,0 I 16,9 18,8 17,5 18,8 0,8 1.3,9 1, ,1 1,3 4,2 7,7 2,7 37,6 49,1 13,3 17,6 1(3,8 1.6,0 12,6 1,8 7, ,6 1,1 5,6 7.2 â, 8 35,4 43,7 20,9 Key to table on page 11.

13 1, 11 Table 3 Key: 1 -, acid; 2 7 halibut; 3 - ocean perch; 4- sardinelia; 5- centrifuging; infusion with chloroform for a) 4 hours; h) 24 hours; 7 - extraction with binary solvent; 8 - chloroform plus ethanol; 9 - chloroform plus methanol; 10 - infusion withcchloroform for 24 hours; 11 - extraction by chloroform with methanol; 12 - saturated acids; 13 - monounsaturated; 14 - polyunsaturated. -*Other isomers are also possible. Table; Key: 1 - acid; 2 - lean herring; 3 - fat herring; 4 - centrifuging; 5 infusion with chloroform for a) 4 hr. b) 24 hr; 6 - extraction with binary solvent; 7 - chloroform plus ethanol; 8 - chloroform plus methanol; 9 - saturated; 10 - monounsaturated; 11 - polyunsaturated. *Other isomers are also possible. polyunsaturated acids (mainly 22:5 w 3 and 22:6) predominate in them. The method of extraction of lipids from salted fish also influences the composition of fatty acids (Table 4). The most saturated lipids are extracted from lean herring by infusion with chloroform: they contain the maximal amount of saturated acids (16:0) and the least amount of polyunsaturated acids. Centrifuged lipids and those extracted by chloroform with ethanol somewhat surpass those obtained by infusion with chloroform in degree of nansaturation. This is shown by the relatively low total of saturated acids and the high total of polyunsaturated acids. However, the most unsaturated lipids are extractedbby a binary solent consisting of chloroform and methanol. They are almost equivalent in the level of saturated acids to the lipids obtained by centrifuging and by extraction by chloroform with ethanol, but they have a lower total of monounsaturated acids and the highest total of polyunsaturated acids,

14 12 which is due to the acid 22:6. Lipids, extracted by a similar method [binary solvent] from fat herring, are also the richest in polyunsaturated acids, due to the predominance of the same acid (22:6). However, unlike lean herring, the most saturated lipids are extracted, not by infusion with chloroform, but by extraction with chloroform and ethanol. The minimal nonsaturation of these lipids is due to the low amount of acid 22:6. Lipids extracted by chloroform with methanol have a similar total of polyunsaturated acids to lipids obtained by centrifuging, but as has already been mentioned, the most highly unsaturated acid predominates, there are less saturated acids (14:0 and 16:0) and somewhat more monounsaturated acids (20:1 and 22:1) in the former. Lipids obtained by centrifuging are similar in the high level of saturated acids to those separated by brief infusion with chloroform and those extracted by chloroform with ethanol, buttthe 'lipids obtained by centrifuging contain a substantially lesser amount ofmonounsatûrated acids (mainly 20:1 and 22:1) and more polyunsaturated acids (20:5 and 22:6). Lipids separated by prolonged infusion with chloroform are the closest in total saturated acids to the lipids extracted with chloroform with methanol, but because of the different ratio of acids 16:1 and 18:1 and the shortage of acid 22:6, the lipids obtained by infusion contain a predominance of monounsaturated acids and a lesser amount of polyunsaturated acids. These differences in the ratio of lipid fatty acids depending on the method of their isolation ehow that a binary solvent consisting of chloroform and methanol makes it possible to extract not only the most highly oxidized but also the most unsaturated lipids. The differences, in,the ratio of fatty acids of lipids depending on the method of separation are determined by the degree of their hydrolysis and oxidation, which is apparently

15 13 conditioned by the nature of proteolipid complexes. With a relatively low degree of oxidation and a certain degree of hydrolysis, corresponding to the lipids of some of the frozen fish studied (halibut and ocean perch - see Table 1), prolonged infusion with chloroform is of similar effectiveness to extraction with chloroform and methanol. Further increase in the degree of oxidation and hydrolysis, coinciding, for example, with the lipids of frozen mackerel and sardinella, and also with the degree of oxidation of the lipids of salted fat herring or with the hydrolytic dissociation of the lipids of salted lean herring (see Tables 1 and 2), requires extraction of lipids by chloroform and methanol. The method of isolation affects the composition of fatty acids appears not only in oxidized, but also in slightly oxidized lipids, obtained from very high quality fish (see Table 3). This apparently shows the formationpof proteolipid complexes in frozen fish at the earliest stages of oxidation and hydrolysis of lipids. This is not true of chilled fish, as the method of extraction of its lipids has no substantialeffect on the ratio of fatty acids. [6]. The high content of acid 22:6 in lipids extracted with chloroform and methanol confirms its h4h reactivity. The substantial effect of the method of separating lipids on the degree of their oxidation, hydrolysis and the ratio of fatty acids suggests that when studying chenges in fish lipids during prolonged storage or in any technological processes, they shouldbe extracted with chloroform and methanol. Conclusions 1. The qualitative state and the ratio of fatty acids of lipids, extracted from some species of frozen fish and salted herring by centrifuging, brief and prolonged infusion with chloroform, and also by extraction with a binary solvent, consisting of chloroform and methanol or ethanol, has been studied. 2. The use of chloroform with methanol makes it possible to extract

16 14 the most highly oxidized and the most unsaturated lipids with a maximal level of free fatty acids; this difference intensifies with intensification of the degree of oxidation and hydrolytic dissociation of lipids. 3. The replacement of methanol by ethanol in the binary solvent results in obtaining lipids with a lesser amount of primary and secondary products of oxidation and with a different composition of fatty acids, which is characterized, as a rule, by a shortage of the most highly unsaturated acid - docosahexaenoic acid. 4. The differences in the qualitative state and the composition of fatty acids of lipids depending on the method of their extraction show that extraction with chloroform and methanol should be used when studying the changes in fish lipids. This method most fully reveals the degree of change in lipids, and consequently provides the best basis for evaluating different methods and conditions of processing fish.

17 15 References _ 1. Kel'man, L.F., Lyaskovskaya, Yu. N. An accelerated method for the extraction and quantitative determination of the lipids of musle tissue. - "Myasnaya industriya SSSR", 1965, No. 1, pp Lyubavina, L.A. The extraction of fat from the meat of salted herring by the method of centrifuging. - "Rybnoe khozyaistvo", 1964, No. 6, p Procedure for the separation of lipids from fish tissues. Moscow, ONTI VNIRO, 1973, p. 9. Authors: F.M. Rzhavskaya, T.A. Dubrovskaya, A.M. Makarova, L.V. Pravdina. 4. Rzhavskaya, F.M., Dubrovskaya, T.A., Makarova, A.M. Polar phases in gas-liquid choromatography of lipid fatty acids of marine organisms. ONTI VNIRO, 1972a,p Rzhavskaya, F.M., Dubrovskaya, T.A. Isolation of fat from the sterilized liver of cod for the determination of the composition of fatty acids. - "Trudy VNIRO", 1972, vol. 88, pp Rzhavskaya, F.M., Dubrovskaya, T.A., Pravdina, L.V. The effect of the method of isolating fat from the muscle tissue of chilled fish on its composition and properties. - "Trudy VNIRO", 1974, vol. 95, pp Rzhavskaya, F.M., Makarova, A.M. The effect of unsaponifiable substances of the lipids of the liver of cod and baleen whales on the separation of the mixture of methyl esters of fatty acids by the method of gas-liquid chromatography. - "Trudy VNIRO", 1974, vol. 95, pp Remaining reference items listed in English on attached copy of original.

18 16 Cnucox mcnonbàoseumoit,,kerib.m a H.11.(1)., il g cxos cïc a si 10.H. YtIçopemei- mé-port" amp,',1#4,h,ii.:1;:oinrixecz:sehhoro onpeaeneiiii.rihrui,uca. TKaHH. sichas egriydri;kkicccp", 1965., No 1, c Ilio 6 HH a 11.A. Bbme.rikine iurpa 113 Msiça. cciip1-ïo.11,cenbah Me- Toi:(pm HeHTpHillyrxposaHHn. "Pm6Hoe ao ;'; 1964, M 6, c MeToiluxa.empenenus.rninHAos Hs TicaHea ph16. M., OFITHB1-1111P0, c. Aar.: c13.m. P)KaacKaz, T.A. )3yepoEscizaz, A.M. Maxap'o.-.Ba, IlpaaeHHa:..4..P)Ketacicasi c1).m.,fly6poscxasi T.A., Maxapoaa A.M. flo-, JIH/JHBI3 t11a3at a ra36-)knh1kocrhot1 xpomatorpadaxa X.HpHbIX HHCJIOT 1111 IIHROBJM0pCKHX.opramnamos. OHTH BHMPO, 1972a. 39 c.. 5exasc.xasi cla.m.,.13.3r613013cka5i T.A. BarizeneHHe achpa 113 '2I CTepeiH30133HH011. nenehh TpecKH jms onpertenehhh coctasa mphmx : IcHcp:riç 'Tpyiet BHHPO", 1972, T. 88, C ?)içasc.Kam RY6Poscizan TA., ripasp,hha z,;wroa Bbmenemin Nuipa ia manuetiac'el TicaHn ox,riaxaehhhix pb16 Ha eriic coctaa H csoekersa. 7 'TpyHEd BHMPO", 1974, T. 95, C. 1i Dxcaacicas1 Maxaposa A.M. Bruisume Heombinsiemaix Betàè CTB zninnizoa nenem4 Tpecx.H H ycatbix KHTOB Ha.paseteiieHHe crvie r: s. : 'CH metanoabrx acimpos KHpnbix KHC.TIOT merquom ra.ao,-)nifixocushoft marorpadmi. 'TpyAbi BM/WO", 1974, T. 95, c pyer VI.J. A rapid method for total 11- ed :extraction and _purification.. Canad. J. Bioch. 1959, V.,37N 8, p STUDIES ON THE QUALITATIVE STATE AND FATTY ACID COMPOSITION OP LIPIDS OBTAINED BY DIFFERENT =HODS FROM THE MUSCLE TISSUE OF FROZEN AHD SALTED PISH P.M.Rshavskaya, T.A:Dubroyskaya, A.M.Makarova - -. and L.V.Fravdina SrUMMARY Fatty acid compasffion, -extent of--oxida-tion-and-hydro - lysis of lipids obtained fi76-m. muscre tissue Of-frozen but, sea bass, mackerel, sardinella and sa3.ted lean oriel fatty herrir_g by five different methods ( centrifugation, short-time and. prolonged infusion in chloroform and ex- 118

19 17., traegoni:with chloroform and methand$1,'or.- ethaei;,,ïj, have beea Studied. hloroiorm e''use of chloroform with methanol lias been shown to l, extr'act most strongly oxidized and most unsatnrated lipids with a maximum content of free fatty acide. Differences in.:the qualitative state and degree of unsaturation of lipids depending on the extraction process become more pronounced as oxidation and hydrolytic disintegration get more intensive. Substitution of ethanol for methanol led to the 'extraction of lipids with a lower content of oxidation products and a different composition of fatty acids characterized, as a rule, by a small amount of docosahexaenoic acid.