Translation Series No Original title: -20 C Chozochu no gyoniku actomioshin no fuyoka---i

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1 FISHERIES AND MARINE SERVICE Translation Series No Stel CE S for S.1. CMINDI\ MS111111f. ININVOP,1_ RF_SU\RCH COUlett 011AWN ÇNtir1/4 P1 Insolubilization of rainbow trout actomyosin during storage at 20 o C. I. Properties of insolubilized protein formed by reaction of propanal or caproic acitl with actomyosin by Kozo Takama Original title: -20 C Chozochu no gyoniku actomioshin no fuyoka---i Propanaaru oyobi kapronsan niyoru fuyoka tanpakushitsu no seijyo From: Niihion Suisan Gakkaishi, 40'(6) : , 1974 Translated by the Translation Bureau(HH/PS) Multilingual Services Division Department of the Secretary of State of Canada Department of the Environment Fisheries and Marine Service Biological Station St. John's, Nfld. I 1974 TRANSLATION SERVICES CANADA INSTITUTF. rt..;f? S. T. L NATiONAL fl :IL 4; _ere i 8 pages typescript u.

2 UEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE Japanese i NTO - EN English Fo-W 3 3A j 7 AUTHOR - AUTEUR Kozo Takama TITLE IN ENGLISH - TITRE ANGLAIS Insolubilization of Rainbow Trout Actomyosin during Storage at -20 C - I Properties of Insolubilized Protein Formed by Reaction of Propanal or Caproic Acid with Actomyosin TITLE IN FOREIGN LANGUAGE ( TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ETRANGERE ( TRANSCRIRE EN CARACTÉRES ROMAINS) -20 C Chozochu no gyoniku actomioshin no f uyoka---i Propanaaru oyobi kapronsan niyoru fuyoka tanpakushitsu no seijyo. REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE.FOREIGN CHARACTERS. NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÉRES ROMAINS. REFERENCE EN LANGUE ÉTRANGERE ( Nihon suisan gakkai shi REFERENCE IN ENGLISH - REFERE CE EN ANGLAIS Bulletin of the Japanese Society of Scientific Fisheries PUBLISHER- EDITEUR PLACE OF PUBLICATION LIEU DE PUBLICATION YEAR ANNEE 1974 DATE OF PUBLICATION DATE DE PUBLICATION VOLUME 40 ISSUE NO. NUMERO 6 PAGE NUMBERS IN ORIGINAL NUMEROS DES PAGES DANS L'ORIGINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIEES 8 REQUESTING DEPARTMENT MINISTÉRE-CLIENT Environment TRANSLATION BUREAU NO. NOTRE DOSSIER NO BRANCH OR DIVISION DIRECTION OU DIVISION PERSON REQUESTING DEMANDÉ PAR Fisheries Service Office of the Editor TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) HH / FS JAN YOUR NUMBER VOTRE DOSSIER NO DATE OF REQUEST DATE DE LA DEMANDE Nov. 1, 1974 UNEDITED ôrati[fillatic:m For informa'-ion only TRADUCTION N(DN r r'j{see tnfor:r.afion seul^:nent SOS (RE V. 2/66)

3 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS i MULTILINGUAL SERVICES r^- ^^+ _..=1 DIVISION DES SERVICES. G^ a^ CANADA DIVISION MULTILINGUES CLIENT'S NO. DEPARTMENT. DIVISION/BRANCH NO DU CLIENT MINISTERE DIVISION/DIRECTION VI I Environment Fisheries Service office of the Editor BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) NO DU BUREAU LANGUE TRADUCTEUR (INITIALES) Japanese HH/ Fs CITY VILLE Ottawa JAN Bulletin of the Japanese Society of Scientific Fisheries 40 (6) 1974 INSOLUBILIZATION OF RAINBOW TROUT ACTOMYOSIN DURING STORAGE AT -20 C -- I PROPERTIES OF INSOLUBILIZED PROTEIN FORMED BY REACTION OF PROPANAL OR CAPROIC ACID WITH ACTOMYOSIN Kozo TAKAMA*. Properties of insolubilized protein formed by the reaction of lipid oxidation products such as propanal or caproic acid with rainbow trout actomyosin during'storage at -20 C were studied in KCI-phosphate buffer (1=0.5, ph 7.2). These insolubilized proteins retained nearly the same amino acid compositions, but tended to be harder, more elastic and more digestible. In addition, the net-work grade in electron photomicrographs became slender and close. These effects were stronger in the caproic acid addition system than in the propanal addition system.. It was considered that the differences in those effects were due to the various progressive degrees of the intraand/or intermolecular ieactions of the proteins, and that these reaction degrees were attributable to propanal and caproic acid having different interaction mechanisms. It is known that the insolubilization of salt soluble protein of fish meat which occurs during frozen storage is related to the change * Faculty of Fisheries, Hokkaido University, Hakodate UNEDITED TRANàzA_'t'ION Por infnrmcfian oaly TRADUCTION NON REVIS: E Inforr-,iation ssulcmqnt SoS-2DO-10-91

4 2 in lipids which also occurs during the process. The authors recognized the interrelationship between the degree of sourness of fish meat and the reduction of salt-soluble protein. Using model systems with respect to the reaction of protein in the actomyosin fraction with lower aldehydes and lower fatty acids which are volatile substances produced from the oxidation of lipid and with respect to the effects of coexisting lipids on these reactions, they speculated that aldehyde and fatty acids enhance the insolubilization of actomyosin (AM) through a different mechanism. 1) In the present paper, using propanal and caproic acid which have a higher reactivity to AM, the state of insolubilization of protein during storage at -20 C was examined. EXPERIMENTAL METHOD A rainbow trout AM solution was prepared as described in the previous report, 1) and prepared in KC1-phosphate buffer (I = 0:5, ph 7.2) at a 2 mg/ml concentration. Preparation of insolubilized protein. 3 pmole propanal per 1 mg AM were mixed. After storing three days at -20 C, it was thawed at room temperature, and centrifuged at 10,000xG for. 20 minutes. The precipitated fraction was obtained as insolubilized protein (PIP). In the same manner, insolubilized protein with caproic acid (CIP) was prepared. These insolubilized proteins were dialyzed against water (we used deionized watei.nistillation) until the phosphoric acid qualitative reaction showed negative.

5 3 The water suspensions were prepared as samples for direct electron microscopic examination. The freeze dried materials were used for amino acid analysis and for hydrolitic measurement by trypsin. The hardness and elasticity of insolubilized protein were measured by a rheometer manufactured by Iiodenki Co., model RMT-130 using as such swollen samples obtained by centrifugal separation. Amino acid analysis. The freeze dried insolubilized protein was put in a sealed cylinder with 6 N HC1, hydrolyzed for 24 hours at 110±1 C, and analyzed in the usual way by an amino acid automatic analyzer manufactured by Hitachi Co., model KLA-5. Hydrolysis by trypsin. According to the method of ANDREUS et al. 2) about 40 mg freeze dried insolubilized protein was suspended with 0.8 mg trypsin (obtained from cow pancreas by Eizai Co.) in 5.5 ml phosphate buffer solution (ph 7.7) and reacted for 20 hours at 38 C while shaking. 2 ml, 20% trichloroacetic acid solution was added to the reaction solution; after the reaction was stopped, the solution was filtered. After washing the precipitate with a 5% trichloroacetic acid solution it was made up to a constant 10 ml volume by combining the collected filtrate and the washing solution. Nitrogen of amino form was determined according to the method of Van Slyke. At the same time, it was determined for the dialyzed and dried material of non-frozen AM and expressed as pg of nitrogen of amino form per 1 mg sample. The degrees of hydrolysis by trypsin were expressed as percentages of measuri ements of each sample against the measurement obtained from non-frozen AM sample.

6 4 RESULTS AND DISCUSSION Examination under the électron micrôscopé'ând hardness ând'élasticity of insolubilized protein. The electron photomicrograph of insolubilized protein is shown in Fig. l. 10,000 ^ Fig. 1. Electron photomicrograph of insolubilized protein formed by storage at?() C for 3 days. Control: Insolubilized protein formed by frozen storagt- of actomyosin solution. PIP: Insolubilized protein formed by frozen storage of propanal addition (3 It mole!mg protein) system. CIP: Insolubilized protein formed by frozen storage of caproic acid addition (3 p molle/mg protein) system In the electron photômicrograph of insolubilized protein (control) obtained from freezing and thawing without adding propanal or caprionic acid, inter-woven thick fibers were observed. The network was large and sparse. While in PIP, the network was rather dense and in CIP it was most dense and small. As in Table 1, showing the hardness and the elasticity of these insolubilized proteins, CIP was the hardest and had the highest elasticity, followed by PIP and control in this order. These results explain the network shown in the electron photomicrographs very well. i

7 Table 1. Hardness and elasticities of insolubilized protein formed by frozen storage. Each 1 g of wet insolubilized protein formed by storage at 20 C for 4 days was placed into a glass tube (7 mm i.d. x 9 mm ht.), and measured by rheolometer. Control* PIP* CIP* Hardness Rheolometcr RMT-1300, 12 Cycles/min, Sens 15V, Clearance 1 min. Sec footnôte of Fig. 1 Elasticity 5 7 Amino acid composition of insolubilized protein. ROUBAL et a1. 3) examined the damage inflicted upon amino acids in many kinds of protein by peroxide of lipids and reported that damage to amino acid differed according to the difference in protein. BUTTKUS 4) reported that the reaction of malonic aldehyde at -20 C was especially high with Met. Lys. and Tyr. of myosin. But as shown in Table 2, which Table 2. Amino acid compositions of actomyosin and insolubilized protein formed by storage at 20 C for 3 days Amino acid Native actomyosin Control*. pmole-n/sarnple-n (m mol) PIP* Asp Thr Ser Glu Pro Gly Ala Val Met , 0 Ileu Leu Tyr Phe Lys His Arg No corrections for hydrolysis losses have been applied. * See footnote of Fig. 1 CIP*

8 6 contains the amino acid composition of insolubilized protein, it is hard to recognize any noticeable difference among the various insolubilized proteins, although therè is a recognizable difference especially in Gly., Ala., Val., Met., and His. when compared with non-frozen AN. PIP and CIP have similar amino acid composition. Even with respect to PIP, the damage inflicted by the reaction of peroxide of lipid or malonic aldehyde upon the specific amino acids as reported was not recognized. Hydrolysis of insolubilized protein by trypsin. It is known that protein denatured by heat becomes easily subject to the enzymatic actions of trypsin etc. because of the appearance of the reacting radical in the molecule by the destruction of the molecule5) and that protein denatured by bridge reaction progress shows resistance2) to those actions. As shown in Table 3, the hydrolytic quality of insolubilized Table 3. Trypsin hydrolysis of insolubilized protein formed by storage at -20 C for 3 days pg of amino-n/mg of sample Hydrolysis Native actomyosin Control* 15.1 PIP* C1P* * See footnote of Fig. 1 Î

9 7 protein formed during frozen storage by trypsin was always lower than that of non-frozen AM. It was especially apparent in the insolubilized protein (control) formed during frozen storage of AM alone, and in the insolubilized protein formed with added propanal (PIP). This fact suggests that insolubilized protein formed mainly from the bridge reaction of inter molecular and/or intramolecular reaction. The relatively higher hydrolytic quality of protein insolubilized by caproic acid addition means that those reaction had not progressed too much. From the above results, it is concluded that the differences in hardness, hydrolytic quality due to trypsin in insolubilized protein generated during frozen storage and that the differences in thickness of fibers or state of network recognized by electron photomicrograph are not the results of changes in composing amino acids but are related to the differences in the degree of progress in intermolecular and/or intramolecular reaction of protein, and it is presumed that those differences are based on the difference in the reaction mechanism of propanal and caproic acid against protein molecules. I would like to express my deep appreciation to Prof. Hisanao Igarashi and Asst. Prof. Koishi Zama of this faculty for their guidance, to Prof. Masamichi Toyomizu of Dept. of Agr., Kyushu University for his advice, and to Dr. Eenichi Kataya, the director of the Central Research Centre of Nippon Kagaku Shiryo Co. for analyzing the amino acids. A part of this study was done with a Science Grant of the Ministry of Education in 1971.

10 8 BIBLIOGRAPHY 1) Kozo Takama, Koichi Zama and Hisanao Igarashi: Bull, of the Jap. So. Sci. Fish. 38, (1972) 2) F. ANDREU% J. BJORKSTEN and F. B. TRENK; A. S. HENICK and R. B. KOCH: J. Am. Oil chemi rs' Soc., 42, (1965). 3) W. T. Rovam. and A. L. Tappel: Arch. Biochem. Biophys., 113, 5-8 (1966). 4) H. Bu-rnms: J. Food. Sc., 32, (1967). 5) Jiro Nikuni and Tadào Hata: Food Chem. Handbook, Asakura Bookstore, 1966, P. 48.