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`HOKKAIDO UNIVERSITY
`
`Title
`
`Compositional Changes in Molecular Species of Fish Muscle
`Phosphatidylcholine during Frozen Storage
`
`TAKAHASHI, Koretaro ; ZAMA, KO ichi
`Author(s)
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`37(1): 8034
`
`Issue Date
`
`1986-02
`
`http://hdl.handle.net/Zl15/23908
`Doc URL
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`Additional
`Information
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`fl
`
`Instructions for use
`
`Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
`
`000001
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
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`ENZYMOTEC - 1022
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`000001
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`

`

`Bull. Fae. Fish. Hokkaido Univ.
`37(1), 80-84. 1986.
`
`Compositional Changes in Molecular Species of Fish Muscle
`
`Phosphatidylcholine during Frozen Storage
`
`Koretaro TAKAHASHI and Koichi ZAMA“
`
`Abstract
`
`Compositional changes in phosphatidylcholine molecular species of mackerel and Alaska
`pollack muscle during frozen storage were studied.
`During storage, the molecular species of (20:5) (20: 5), (20 : 5) (22 : 6) and (22: 6) (22:
`6) drastically decreased in Alaska pollack, in contrast to mackerel which exhibited a relative
`increase in the amount of the same molecular species.
`
`Organoleptic and chemical changes observed in taste, flavors and other prop—
`erties of fish during frozen storage are of great commercial importance. Changes in
`lipids often make frozen fish less acceptable. Though there is no doubt that the
`development of oxidative rancidity is one of the main problems with fatty fish that
`are rich in neutral lipids,“ changes in phospholipids also effect the quality of fish
`especially when it has been stored, under low temperaturel'” conditions. The
`susceptibility of attack from hydrolytic enzymes or oxygen is considered to be
`effected not only by the fatty acid composition, but also by the molecular species of
`the phospholipids?’ Therefore, the compositional changes of molecular species of a
`representative phospholipid, phosphatidylcholine (PC), were examined.
`
`Materials
`
`Experimental
`
`Fish used for this research were mackerel (Scomber japonicus) caught off the
`coast of Hachinohe, Japan, in May 1984 and Alaska pollack (Theraga chalcogramma)
`from Uchiura Bay, Hokkaido, Japan in Dec. 1981.
`The mean body weight of five mackerel examined was 446 g and that of Alaska
`pollack was 610 g.
`
`MeLdes
`
`The dorsal muscle of mackerel was collected and separated into dark muscle
`(DM) and White muscle (WM). Dorsal muscle of Alaska pollack was also collected
`but it was not separated into DM and WM since the amount of DM was negligible.
`Muscles from both fish were chopped into small pieces with a kitchen knife and were
`packed in polyethylene bags. These bags were stored in a freezer at ~20°C for 6
`months and 9 months. Total lipid was extracted from the sample according to the
`method of Bligh-Dyer. Lipid composition was determined by the densitometric
`
`
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`TAKAHASIII & ZAMAZ Changes in phospholipid during storage
`
`method (Ozumor Densitometer model 82, Tokyo) after charring the thin layer
`chromatographic plate at
`l50—160°C. The spray reagent used for thin layer
`chromatography was 3% copper acetate in 80/0 phosphoric acid.“ The developing
`solvents used for thin layer chromatography Were n-hexane/ethyl ether/acetic acid
`(80 : 20 : 0.5, V/v) for nonpolar lipids and chloroform/methanel/acetic acid/water
`(25 : l5 : 4 : 2, v/V) for polar lipids. Purification and identification of PC molecular
`species were done in the same manner as previously reported.”
`All the molecular species analyzed were calculated as mg/ 100 g muscle for the
`principal component analysis (PCA).“
`
`Results and Discussion
`
`Lipid compositional changes in frozen fish stored at »-—20°C is shown in Fig. l.
`The amount of free fatty acid (FFA) increased both in mackerel and Alaska pollack
`during the 6 months storage while all other lipid components decreased. After 9
`months storage, all
`the lipid components,
`including FFA, decreased though the
`relative amount of FFA increased in mackerel DM. The drastic decrease in total
`
`lipids is considered to be mostly caused by the decrease in triglyceride in the case of
`mackerel and PC was considered to be responsible in the case of Alaska pollack.
`From an idealistic viewpoint, changes in molecular species of all the lipid
`classes should be studied, but unfortunately, the methodology and theory in deter—
`mining the molecular species composition of marine sources has been established
`only for PC and phosphatidylethanolamine.3) Compositional change of the PC
`molecular species during frozen storage was investigated and the result is shown in
`Fig. 2 as high performance liquid chromatograms (lll’LC). Outstanding differences
`in the chromatographic patterns were observed between mackerel and Alaska
`
`Dark muscle
`
`Mackerel
`
`White muscle
`
`
`
`Scales are In Hold 9/1009 muscle
`
`Alaska Dollack
`
`
`
`Triglyceride % Free fatty acid
`
` Sterol
`
`E Phosphatidylcnol 1 ne
`
`
`
` ' i=1 Phosphatidylethcnolamine + Phasnhatldylserlne
`
`[W others
`
`
`
`5 months
`
`
`9 months
`
`Fig. l. Lipid compositional change in fish flesh during frozen storage at ~20°(l.
`m 81 W.
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`Bull. Fae. Fish. Hokkaido Univ. 37(1), 1986.
`
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`Fig. 2. Changes in III’LC chromatographic patterns of fish muscle I’C molecular species during
`fronzen storge at 20°C.
`1 : (20: 5) (20:
`I : Groups composed of highly unsaturated fatty acids, that is,
`5), 2: (20:5) (22:6), 3: (22:6) (22:6) and 4: (20:11) (22:6).
`III: Groups composed of highly unsaturated fatty acids in combination with
`generally found fatty acids, that is, 5: (18 : 1) (20 : 5),
`6:
`(18 : 1) (22 : 6),
`7: (20:5),(1610), 8: (16:0) (20:5), 9: (22:6) (10:0) and
`10: (16:0)
`(22:6).
`II&IV: Others.
`
`11: (18:1) (16:0) and
`
`12: (10:0) (18:1).
`
`pollack. The molecular species that belongs to group .I. i.e. (20 : 5) (20:5), (20: 5)
`(22:6) and (22:6) (22:6), drastically dercascd in Alaska pollack during storage
`contrasting with mackerel which exhibited a relative increase in the amount of
`molecular species that belongs to group I. Molecular species of group IV relatively
`increased in both fish though the composition of molecular species in group IV is
`quite different for both fish. (16 : O) (18 : 1) was the representative molecular species
`of group IV in Alaska pollack while (16:0) (22 : 5), (16:0) (20: 4), (17: 1) (22:6),
`(18:0) (20 : 5) and (1.8 : 0) (22 : 6) were the representatives of group IV in mackerel.
`Results of PCA of the compositional change in PC molecular species are
`illustrated in figures 3 and 4. Contribution of this PCA was 85% up to the second
`principal component. (16 : 0) (22 : 6) was observed to be the molecular species closest
`to the first principal component as shown in these figures. Eigenvectors of (16:0)
`(18: l) and (22 : 6) (22 : 6) appeared to have a large angle against the axis of the first
`principal component in both figures when compared with other molecular species.
`This suggests that the decrease in the amounts of (16 : 0) (18 :
`'1) and (22 : 6) (22 : 6)
`are small compared to other molecular species.
`It has been pointed out that 22 : 6
`is the fatty acid most susceptible to oxidation”) But the results obtained in this
`study show that the efi'ect of (22 : 6) (22 : 6) on the decrease in the amount of PC is
`smaller than that of (16 : 0) (22 : 6), especially in mackerel, even though these
`molecular species are composed of the same component, 1.0. 22 : 6.
`()hshima et a1.
`had carefully removed the surface portion of the frozen stored sample before the
`lipid extraction in order to concentrate their discussion an enzymatic hydrolysis of
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`TAKAHASHI 8L ZAMA: Changes in phospholipid during storage
`
`II
`
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`4“,,»(205) (22:6)
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`(l6:0)(18:1)
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`Fig. 3. Plots of principal loadings of the frozen samples and eigenvcctors of the PC molecular
`species displayed on the first and second principal component plane on I’CA.
`——————— Mackerel DM, — - - - - - Mackerel WM,
`Alaska pollack.
`I: First principal component, [1: Second principal component.
`1: Mackerel DM (0 month), 2: Mackerel DM (6 months), 3: Mackerel DM (9 months),
`4 : Mackerel WM (0 month), 5: Mackerel WM (6 months), 6 : Mackerel WM (9 months),
`7 : Alaska pollack (0 month), 8: Alaska pollack (6 months), 9: Alaska pollack (9
`months).
`
`III
`I
`
`III
`
`(16:0)(18:1)
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`(16:0)(20:5)
`20:5 (20:5)
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`
`Fig. 4. Plots of principal loadings of the frozen samples and eigcnvectnrs ol' the PC molecular
`species displayed on the first and third principal component plane on I’CA.
`I: First principal component, III: Third principal component.
`Symbols and numbers are the same as in Fig. 3.
`
`In their discussion it was demonstrated that, both in skipjack and
`phospholipidjfil
`cod muscle, the relative percentage of (22 : 6) (22 : 6) increased. This coincides with
`the HPLC chromatograms of mackerel shown in Fig. 2 which illustrate the relative
`increase in the amount of (22 : 6) (22 : 6) and differ from that of Alaska pollack.
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`Bull. Fae. Fish. Hokkaido Univ. 37(1), 1986.
`
`our study, the surface portion of the frozen stored sample was not removed before
`Therefore, some oxidative efi'ect might have contributed to a
`lipid extraction.
`Compositional changes in PC
`decrease in some kinds of PC molecular species.
`molecular species is considered to be the result of a complex reaction, namely
`hydrolysis in combination with oxidation.
`The next step in this study will be to clarify the extent of the oxidative effect
`as well as the hydrolytic efl'ect on PC molecular species degradation by discerning
`both reactions.
`
`References
`
`(In
`
`Takama, K, Zama, K. and Igarashi, II. (1972). Changes in the flesh lipids of fish during frozen
`storage. Part II. Flesh lipids of several species of fish. Bull. Fae. Fish. Hokkaido Unto,
`22, 290300.
`(In Japanese with English abstract).
`'I‘oyomizu, M. (1974). Sultana no Hinshitsu. 12371137 pp. Koseisha Koseikaku, Tokyo.
`Japanese).
`Takahashi, K. (1985). Suisan Do'butsu no Kinm'ka Shtshitsu. 24-37 pp. Koseisha Koseikaku,
`’I‘okyo.
`(In Japanese).
`chster, M.E,, Burns, B.J. and Mead, J.F. (1969). Quantitative densitometric TLC of lipids
`using copper acetate reagent.
`J. Chromatogr., 43, 120-«126.
`Takahashi, K, Hirano, T., Takama, K. and Zama, K. (1982). Molecular species of fish muscle
`lecithin. Bull. Japan. Soc. Sci. Fish, 48, 1803—1814.
`Watari, M. and Kishi, M. (1982). Personal computer library 3, 9 19 12 pp. Kogaku Tosho,
`Tokyo.
`(In Japanese).
`Ohshima, T., Wada, S. and Koizumi, C. (1983). Enzymatic hydrolysis of phospholipids in cod
`flesh during cold storage. Bull. Japan. Soc. Sci. Fish, 49, 1391-1404.
`(In Japanese with
`English abstract).
`Ohshima, ’1‘., Wada, S. and Koizumi, C. (1984). Enzymatic hydrolysis of phospholipids in cod
`flesh during storage in ice.
`Ibid., 50, 107—114.
`(In Japanese with English abstract).
`
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