`
`1‘
`
`El
`32 E35.
`fa“
`
`HOKKAIDO UNIVERSITY
`
`Title
`
`Compositional Changes in Molecular Species of Fish Muscle
`Phosphatidylcholine during Frozen Storage
`
`/\utlC1or(s)
`
`TAKAHASHI, Koretaro ; ZAMA, KO ichi
`
`Citation
`
`jtitE3Ek¥%%xJ<fi€¥%r%l$lfii§%iili = BULLETIN or THE
`FACULTY OF FISHERIES HOKKAIDO UNIVl,+)RSI'l‘Y,
`37(1): 80-84
`
`Issue Date
`
`1986-02
`
`http://hdl.handle.net/2115/23908
`Doc URL
` ! _
`__
`_.__-,_. W.-.
`,
`
`_..v~-,._
`
`._.._, _,, ,_, .,_____,,_j.
`
`_,,,
`
`.,
`
`Right
`
`Type
`
`bulletin
`
`Additional
`Information
`
`9%
`
`Instructions for use
`
`Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
`
`°°°°°1
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1022
`
`000001
`
`
`
`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 do11bt 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 temperature”) 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 japomeus) caught olf the
`coast of Hachinohe, Japan, in May 1984 and Alaska pollack (T/zeraga chalcogramma)
`from Uchiura Bay, Hokkaido, Japan in Dec. 1981.
`The mean body Weight of live mackerel examined Was 446 g and that of Alaska
`pollack was 610 g.
`
`Methods
`
`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
`
`_“;‘_—Ilaboi-atory of flhemistry I, F‘aculty of Fisheries, Hokkaido University ( ,1h~}’f§iffi:')\"»‘}': gg 1,53-1‘-f:\L‘q§f{
`fit Matt ‘fifiklél
`
`so ——
`
`000002
`
`000002
`
`
`
`TAKAHASIII & ZAMAZ Changes in phospholipid during storage
`
`method (Ozumor Densitometer model 82, Tokyo) after charring the thin layer
`chromatographic plate at 150—160°C. The spray reagent used for thin layer
`chromatography was 3% copper acetate in 8% phosphoric acid." The developing
`solvents used for thin layer chromatography were n-hexane/ethyl ether/acetic acid
`(80 2 20 : 0.5, v/v) for nonpolar lipids and cliloroform/Inethanol/acetic acid,/water
`(25 : 15 : 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 an.d the result is shown in
`Fig. 2 as high performance liquid chromatograms (IIPLC). Outstanding differences
`in the chromatographic patterns were observed between mackerel and Alaska
`
`Dark muscle
`
`Mackerel
`
`white muscle
`
`Scales are In Hold g/1009 muscle
`
`
`
`Triglyceride Free fatty acid
`
`
`ii. Phosohatidvlethanolamine + Phosohatldvlserlne
`
` ' Sterol
`
` '
`
` PhOSDh0tldVlCh0l l ne
`
`[W others
`
`Fig. l. Lipid compositional change in fish flesh during frozen storage at —~20°(l.
`m 31 -...
`
`000003
`
`000003
`
`
`
`Bull. Fac. Fish. Hokkaido Univ. 37(1), 1986.
`
`
`
`IV
`Alaska Dollack
`<0 month)
`
`Mackerel DM
`(4,
`(0 month)
`_,;/ LAe\_M__M_
`
`1
`
`i
`
`
`
`ln/A
`
`i My
`
`I J
`
`comm
`
`Mackerel WM
`(0 month)
`
`‘
`:
`
`‘
`/l
`)
`J "u/xx’
`
`)
`\,f’lJ\,r\w_J\i
`Mackerel NM
`(6 months)
`‘
`()
`) _/"/jkiw
`i
`L J)
`_,. W” "
`‘
`.
`Mackerel NM
`_
`<9 WW3)
`I
`_
`A
`3
`I
`I
`or tn ‘t**=m..,/N
`
`Ill
`
`
`
`l
`2
`I3
`ll
`L.i...»’i My
`20
`
`'I_l‘
`
`.
`
`
`
`«
`"
`
`1
`
`20
`
`W
`
`Mackerel D"
`'
`(6 months)
`\~"\.,V\_,_.J!.l2\_.
`L0
`so
`so
`ufmin
`/"~~/J\"'“““
`ellll Alaska pollack
`Mflckerel D”
`Fl)
`(6 months)
`(9 months)
`I
`K
`/J
`A
`M
`M \..,W_¢_. ,_ M/\_
`/\
`no
`so
`no
`E1. W l\/()(\’
`M Alaska Dollack
`1......;?..E9:¥gi1%m_( ,1
`“
`
`Fig. 2. Changes in HI’LC chromatographic patterns of fish muscle PC molecular species during
`fronzen storge at 200.
`1 : (20 : 5) (20:
`I : Groups composed of highly unsaturated fatty acids, that is,
`5), 2: (20:5) (22 : 6), 3: (22 :6) (2216) and 4: (20 : ll) (22 : 6).
`III : Groups composed of highly unsaturated fatty acids in combination with
`generally found fatty acids, that is, 5: (18 : 1) (2015),
`6:
`(18 : 1) (22 : 6),
`7: (20:5).(16:0), 8: (16:0) (2025), 9: (22: 6) (16:0) and
`I0: (1620)
`(22 : 6).
`II & IV: Others.
`
`11: (18: 1) (16 : 0) and
`
`12: (1620) (1821).
`
`pollack. The molecular species that belongs to group .1. Le. (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 diflerent for both fish. (16 : 0) (18 2 1) was the representative molecular species
`of group IV in Alaska pollack while (16 :0) (22 :5), (1610) (20:11), (17: l) (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°/0 up to the second
`principal component. (16 2 0) (22 : 6) was observed to be the molecular species closest
`to the first principal component as shown in these figures. Eigenvectors of (1620)
`(18: l) and (22 : 6) (22 2 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 2 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 effect 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, i.c. 22 : 6. Ohshima et al.
`had carefully renu)ved the surface porthon of the frozen.stored sarnrfle before the
`lipid extraction in order to concentrate their discussion on enzymatic hydrolysis of
`
`000004
`
`000004
`
`
`
`TAKAHASHI & ZAMA: Changes in phospholipid during storage
`
`II
`
`II
`
`c".__¢n_.. %~
`
`
`
`('1.o:5)(2o:5)
`/ -(l6:0)(20:5)
`///
`
`_’,,-.»-420:5) (22:52)
`~——-- '‘(16:[)) (22:6)
`_ ,.n, _.
`
`(l6:0)(18:1)
`
`Fig. 3. Plots of principal loadings of the frozen samples and eigcnvectors of the PC molecular
`species displayed on the first a.nd second principal component plane on PCA.
`—————-- Mackerel DM, — - - — - — Mackerel WM,
`Alaska pollack.
`I: First principal component, II: Second principal component.
`.DM (9 months),
`1: Mackerel DM (0 month), 2: Mackerel DM (6 months), 3: Mackerel
`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
`l
`
`III
`
`\(22:s)(22;e)
`
`
`
`(l6:O)(20:5)
`.:
`(
`:')
`*“_‘9(—)«~5~)~-ills?!) (22:6)
`—=_'__~;7'fé;1>(-20:5)
`-------_‘ -~~<1s:o)(22:6)
`\
`I X ““r~(2o:5)(22:e)
`\\
`1
`
`('l6:0)(1B:1)
`
`Fig. 4. Plots of principal loadings of the frozen samples and eigenveetnrs of the PC molecular
`species displayed on the first and third principal component plane on PCA.
`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
`phosph0lipid.7"’
`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.
`.In
`_g3_
`
`000005
`
`000005
`
`
`
`Bull. rm. 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 eifect on PC molecular species degradation by discerning
`both reactions.
`
`References
`
`(‘In
`
`Zama, K. and Igarashi, II. (1972). Changes in the flesh lipids of fish during frozen
`Takama,
`storage. Part II. Flesh lipids of several species of fish. Bull. Fae. Fish. Ilukkaido Umc,
`22, 290--300.
`(In Japanese with English abstract).
`Toyomizu, M. (1974). Sakana no Hinshitsu. 1237-137 pp. Koseisha Koseikaku, Tokyo.
`J apancse).
`Takahashi, K. (1985). Suisan I)0'bu.Lsu no Kinniku Shtshitsu. 24~~37 pp. Koseisha Koseikakti,
`Tokyo.
`(In Japanese).
`Fewster, M.E., Burns, B.J. and Mead, J.F. (1969). Quantitative densitometric TLC of lipids
`using copper acetate reagent.
`J. Chmmatogn, 43, 120-426.
`Takahashi, K., Hirano, T., Takama, K. and Zama, K. (1982).
`lecithin. Bull. Japan. Soc. Sci. Fish., 48, 1803-181/1.
`Watari, M. and Kishi, M. (1982). Personal computer library 3, 9 1-9 12 pp. Kogaku Tosho,
`Tokyo.
`(In Japancse).
`Ohshima, T., Wada, S. and Koizumi, C. (1983).
`Enzymatic hydrolysis of phospholipids in cod
`Bull. Japan. Soc. Sci". Fish., 49, 1397—-1404.
`(111 Japanese with
`flesh during cold storage.
`English abstract).
`Ohshima, ’l‘., 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).
`
`Molecular species of fish muscle
`
`__ 34 _.
`
`000006
`
`000006