FATTY ACID COMPONENT OF LIPID
`
`OF EUPHAUSIA SUPERBA
`
`HIDEO TSUYUKI
`
`AND
`
`SHINGO ITOH
`
`Department of Food Technology, College of Agriculture and
`Veterinary Medicine, Nihon University, Tokyo
`
`ABSTRACT
`
`Lipids extracted from the raw frozen and boiled-frozen krill, Euphausia
`superba, harvested in the Antarctic Ocean were identified and quantitated
`by a combination of thin-layer and gas-liquid chromatography.
`The lipid contents of E. superba on the wet weight basis were 3.41%
`of the raw frozen sample and 5.62% of the boiled-frozen sample. There
`were shownto consist of 0.8% of steryl esters, 27.4% of free fatty acids,
`52.3% of triglycerides, 4.7% of sterols, a trace amount of diglycerides,
`2.2% of monoglycerides, 6.99% of phospholipids and 4.1% of pigments in
`the raw frozen sample, and 0.6% of steryl esters, 4.6% of free fatty
`acids, 76.8% of triglycerides, 4.2% of sterols, 9.2% of phospholipids and
`3.5% of pigments in the boiled-frozen sample. The predominant fatty
`acids contained in total
`lipids and the fractions of free fatty acids,
`triglycerides and phospholipids were myristic (5.98-22.16%), palmitic
`(16,83-30.499), palmitoleic (5.92-26.31%), oleic (19.31-29.179), eicosapent-
`aenoic (0,52-12.59%) and docosatrienoic (0.50-11.7494) acids.
`
`INTRODUCTION
`
`Euphausia superba is a shrimp-like crustacean of about 5cm in body length,
`and inhabits only in the Antarctic Ocean, though many euphausiid species are
`found widely over the world oceans. E. superba composes one of the most
`important prey organisms for
`the inhabitants in the oceanic regions of the
`Antarctic ecosystem.
`In addition to utilization by the faunistic inhabitants
`there, E. superba should be regarded as the food resources for mankind in the
`future. The present study stands on such viewpoint, and intends to give a
`basic knowledge for utilizing EH. superba as to be a possible humans’ food.
`There have been several reports on the chemical properties and the fatty
`acid composition of EF. superba lipids (Saiki and Mori, 1953; Saiki et al., 1959;
`Tsuyuki et al., 1964a and b). So far as these works were carried out by the
`fractional distillation method,
`the minute examinations of
`the fatty acid
`component have not been reported yet.
`
`Sci. Rep. Whales Res. Inst.,
`No. 28, 1976, 167-174.
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`168
`
`TSUYUKI AND ITOH
`
`On the other hand, Kayama and Nakagawa (1975), and Kayama and Ikeda
`(1975) reported on the lipids of some micronektonic shrimps.
`The objective of this study is to identify and quantitate the lipids and the
`fatty acid components of E#. superba by a combination of thin-layer and gas-
`liquid chromatography.
`
`MATERIALS AND METHODS
`
`Euphausia superba, used in this study were harvested in the waters off Enderby
`Land, 10°E in the Antarctic Ocean between December, 1974 and February, 1975
`by a expedition of commercial basis Nippon Suisan Co. Ltd. A part of the
`E. superba harvested was quickly frozen as
`the raw frozen products and
`another part of them was similarly quick frozen as the boiled-frozen products
`after flash boiling. Both products were processed as the whole body of £.
`superba. They were in the range from 0.4 to 0.7. g in the body weight and in
`the range from 3.5 to 5.5cm in the body length.
`The method of Bligh and Dyer (1959) was used to extract and purify the
`lipids in these samples. The extracted lipids were then weighed and stored at
`—20°C under nitrogen atmosphere. The chemical properties of the extracted
`lipids were examined by ordinary methods.
`The fractionation of EH. superba lipids was subsequently separated by thin-
`layer chromatography using 20x 20cm glass plates coated with activated Silica
`gel G (Merck Chemical Co.), The solvent systems used were petroleum ether:
`diethyl ether: acetic acid, 85: 15: 1, v/v/v. The pure standards e.g. phospho-
`lipids, cholesterol, steryl palmitate, mono, di,
`triglycerides, oleic acid (Nihon
`Chromato Works Ltd.) were used to identify zones on the thin-layer plates
`which were removed, dried, sprayed with 50% sulfuric acid and charred at
`110°C for 15min. The fractionated zones were scraped into Toyo filter paper
`No. 2 cones (Toyo Roshi Kaisha Ltd.) and eluted with chloroform respectively.
`The eluting solvents were evaporated off with a stream of nitrogen and weighed.
`The three major fractions were recovered triglycerides, free fatty acids and
`phospholipids for analyzed the fatty acid components. The other minor fractions
`were noted on the plates but were not in sufficient quantity to be recovered.
`The fatty acid methyl esters from totallipids, triglycerides, free fatty acids
`and phospholipids of E. superba lipids esterified by the saponification-trans-.
`esterification method as described by Metcalfe et al. (1966). 5ml of a 0.5N
`methanolic KOH solution were added to approximately 30 mg oftotal lipids,
`triglycerides, free fatty acids and phospholipids, and heated over a steam bath
`for 5min. Then, 5ml of 12.5% boron trifluoride in methanol were added to
`these mixtures and boiled for 3 min. Thin-layer chromatography was used to
`determine the completeness of the transesterification. Silica gel G plates were
`spotted with the reacted products and known standards, and developped with
`petroleum ether: diethyl ether: acetic acid as previously described. A com-
`parison of the R, values of the reacted products, triolein, methyl oleate and
`Sei. Rep. Whales Res. Inst.,
`No, 28, 1976.
`
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`LIPID OF EUPHAUSIA SUPERBA
`
`169
`
`oleic acid (Nihon Chromato Works Ltd.) indicated that the conversion of total
`lipids, triglycerides, free fatty acids and phospholipids fractions to methylesters
`was complete.
`The methyl esters were analyzed weth a Shimadzu Gas Chromatograph
`Model 5A (Shimadzu Seisakusho Co.) equipped with a dual flame ionization
`detector. The columns used were 3mX3.0mm I.D. glass coil tubing packed
`with Diasolid ZF on 80/100 mesh, and with 3% SE-30 on 80/100 mesh Chromo-
`sorb W (Nihon Chromato Works Litd.). The carrier gas was nitrogen at flow
`rate of 40 ml per min. The column were operated isothermally at 185°C for
`Diasolid ZF and 225°C for SE-30. The injector block and detector were at
`-215°C for Diasolid ZF and 245°C for SE-30, respectively.
`Someof the gas-liquid chromatographic peaks were identified by comparison
`with standard peaks obtained from pure methyl ester mixture (Nihon Chromato
`Works Ltd.). Also, equivalent chain-length values were determined according
`to the method of Miwa (1963) and were compared with those reported by
`Hofstetter et al. (1965) for identifying peaks for which no pure methyl!esters
`were available. A portion of each sample esterified was hydrogenated to con-
`firm the correctness of identification of the unsaturated acids. Approximately
`20 mg of methyl esters, 1 ml of methanol and a pinch of platinum black were
`added to a screw cap vial. Hydrogen was bubbled in for 5 min, the vial sealed
`and then reacted for 15 min with frequent shaking. After reaction, the methyl
`esters were transferred to n-hexane, dried, then taken up and injected into the
`gas-liquid chromatograph with the same condition as above mentioned. The
`area of each chromatographic peak representing a fatty acid present was ob-
`tained by multiplication of the height of each peak by the width at half-
`height. The areas of each peak was then compared with the total combined
`area at all of the peaks to obtained the percentage of each specific fatty acid.
`
`RESULTS AND DISCUSSION
`
`The chemical properties of the total lipids extracted from Huphausia superba.
`are shown in Table 1. The lipid contents are comparatively low 7.e., 3.41%
`of the raw frozen sample and 5.62% of the boiled-frozen sample on the wet
`
`TABLE 1. PROPERTIES OF TOTAL LIPIDS FROM EUPHAUSIA SUPERBA
`
` Lipids
`
`Boiled-frozen sample
`Raw frozen sample
`Reddish brown liquid
`Reddish brown liquid
`5. 62
`3.41
`1, 4820
`1.4788
`18.2
`74.1
`127.9
`142.3
`197.6
`181.3
`5.6 5.8
`
`
`Appearance (20°C)
`Oil content (%)
`Refractive index (40°C)
`Acid value
`Todine value
`Saponification value
`Unsaponifiables (%)
`
`Sci, Rep. Whales Res. Inst.,
`No, 28, 1976.
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`170
`
`TSUYUKI AND ITOH
`
`weight basis. The total lipid of the raw frozen sample was higher in iodine
`and acid values, and lowerin saponification value than those of the boiled-frozen
`sample. These are seemed to be autoxidized during frozen storage or thawing.
`The lipid composition of each extract as determined by thin-layer chromato-
`graphy is markedly different between the samples of the raw frozen and boiled-
`frozen. By visual evaluation of these chromatograph as shown in Fig. 1, the
`zones of steryl esters,
`triglycerides,
`free fatty acids, monoglycerides, sterols,
`diglycerides, phospholipids and pigments are appearently present
`in the raw
`frozen sample while the boiled-frozen sample is not found the presence of
`diglycerides and monoglycerides, There are also appearent differences in the
`compositions of triglycerides and free fatty acids between two samples. The
`lipid compositions of these fractions are reported in Table 2.
`In the extracts
`of the boiled-frozen sample, triglycerides are the preponderant fraction com-
`prising 76.8% of the total
`lipid fractions, whereas in the raw frozen sample,
`triglycerides fraction is much lower, On the other hand, the composition of
`free fatty acids fraction is much higher in the raw frozen sample than in the
`boiled-frozen sample. These appearently explained to the difference of acid
`value in Table 1. Also, the raw frozen sample had 2.2% of monoglycerides
`
`Front
`
`@ Strong © Medium ; Weak
`Fig. 1, Thin-layer chromatograms of
`total
`lipids extracted from E. superba, A:
`the raw frozen sample. B:
`the boiled-frozen sample,
`SE: steryl esters. TG:
`triglycerides, FFA: free fatty acids. P: pigments. DG: diglycerides. S: sterols,
`MG: monoglycerides. PL: phospholipids.
`
`Sei, Rep. Whales Res, Inst.,
`No, 28, 1976.
`
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`LIPID OF EUPHAUSIA SUPERBA
`
`.
`
`171
`
`and a trace of diglycerides fractions while the boiled-frozen sample was not
`detected them.
`The fatty acid methyl esters of the total lipids and the main three fractions
`contained fatty acids 7. ¢., triglycerides, free fatty acids and phospholipids were
`analyzed by gas-liquid chromatography, but no attempt was made to further
`study the component of other fractions. The presence of the fatty acids con-
`tained in E. superba lipid is a very wide variety as reported in Table 3, plus
`trace quantities of unidentified fatty acid methyl esters. With a few exceptions,
`the fatty acids of the total lipids and the three main fractions in both samples
`had nearly similar chromatographic patterns and distributions with each other.
`' The major fatty acid compositions of both total lipids were 14: 0, 16: 0, 16:
`1, 18:1, 20:5 and 22:3 acids and contained more than 87% of the total fatty
`acids. Both triglyceride fractions contained 14: 0, 16: 0, 16: 1 and 18:1 acids
`as the predominate fatty acids whereas were not detected the fatty acids of 22:
`5, 22: 6 and 24:
`1 which contained in the total lipids. The total composition
`of 14: 0, 16: 0, 16:
`1 and 18:
`1 acids accounted for more than 85% of the
`total fatty acids. The free fatty acid fractions of both samples were mainly
`consisted of 14: 0, 16: 0, 16: 1, 18: 1, 20: 5 and 22: 3 acids similar to the
`total lipids. The total composition of the six main fatty acids were more than
`77% of the total fatty acids. The fatty acids of 22: 5 and 22: 6 which con-
`tained in the total lipids were not found in this fraction of the boiled-frozen
`sample.
`In both phospholipid fractions, the fatty acids of 14: 0, 16: 0, 16: 1,
`18:1 and 20: 5 as the predominant fatty acids comprised approximately 76%
`of the total fatty acids.
`The compositions of the total saturated acids in the raw frozen sample
`were 39.96% of the total lipids, 56.17% of the triglyceride fraction, 37.96% of
`the free fatty acid fraction and 27.14% of the phospholipid fraction, and those
`of unsaturated acids were 58.95%, 42.83% 60.84% and 71.56% respectively. On
`the other hand,
`those of the total saturated acids in the boiled-frozen sample
`
`TABLE 2, LIPID COMPOSITION OF EUPHAUSIA SUPERBA
`BY THIN-LAYER CHROMATOGRAPHY.
`
`
`Composition
`
`Lipids
`
`Boiled-frozen sample (%)
`Raw frozen sample (%)
`76.8
`52.3
`Triglycerides
`——
`trace
`Diglycerides
`—
`2.2
`Monoglycerides
`4.6
`27.4
`Free fatty acids
`9,2
`6.9
`Phospholipids
`4.2
`4.7
`Sterols
`0.6
`0.8
`Steryl esters
`3.5
`4.1
`Pigments
`11
`,
`1.6
`Unknowns
`ipg
`
`Sci. Rep. Whales Res. Inst..
`No. 28, 1976.
`
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`172
`
`TSUYUKI AND ITOH
`
`TABLE 3. FATTY ACID COMPOSITION OF EUPHAUSIA SUPERBA LIPIDS.
`(percentage of total lipids)
`
`
` Fatt Lipids in raw frozen sample Lipids in boiled-frozen sample
`
`a
`acide
`Total
`Trigly-
`Free
`Phospho-
`Total
`Trigly-
`Free
`Phospho-
`lipids
`cerides
`fatty
`lipids
`lipids
`cerides
`fatty
`lipids
`acids
`acids
`0. 16
`0.47
`0.02
`0.03
`0,03
`0.09
`0.01
`0,02
`9.07
`8. 66
`0. 13
`0.19
`0.03
`0. 04
`0, 52
`0. 33
`0,07
`0.09
`0. 04
`0, 06
`26.01
`24, 82
`7,96
`6. 49
`0.71
`0.41
`0.01
`0.02
`0. 57
`0.73
`0. 07
`0,11
`0. 04
`0.02
`0. 96
`1.60
`29.17
`21.82
`3.47
`3.03
`0. 80
`4.10
`0. 20
`0.39
`0.13
`0, 24
`0. 52
`0.77
`0, 12
`1.46.
`0.95
`4.61
`
`0. 21
`0.02
`0.05
`0.01
`8.71
`0. 14
`0. 07
`0. 37
`0.06
`0, 04
`28,94
`5, 92
`0. 14
`0.02
`0.69
`0.09
`0. 04
`0. 40
`19,84
`2.79
`0.12
`0.15
`0, 10
`1,02
`0. 28
`1.45
`
`0.27
`0.05
`0.08
`0.03
`22. 16
`0.15
`0.05
`0. 84
`0. 13
`0. 10
`30. 49
`8, 86
`0,53
`0. 02
`0,55
`0.17
`0. 05
`1.18
`28, 25
`1, 87
`0. 12
`0, 20
`0, 09
`0.76
`0. 13
`0.35
`
`0, 54
`0.03
`0.08
`0.04
`5. 98
`0.10
`0.01
`0. 46
`0.15
`0. 12
`16. 83
`26, 31
`0.58
`0.01
`0. 88
`0.06
`0.01
`1.78
`28, 49
`2.53
`0. 54
`0.07
`0. 20
`0.52
`0.47
`0.83
`
`0, 22
`0.05
`0.07
`0,02
`11.54
`0.09
`0.08
`0. 59
`0. 12
`0,05
`29. 20
`6.27
`0, 25
`0, 02
`0. 64
`0. 09
`0.07
`0. 78
`19, 31
`2.43
`0. 06
`0.16
`0. 04
`0.65
`0. 24
`0. 82
`
`0.32
`0.01
`0. 11
`0,02
`21.35
`0.15
`0.04
`0.73
`0.10
`0.06
`24.04
`13,37
`0. 83
`0.03
`0.68
`0.06
`0.05
`1,02
`25, 87
`2.03
`0. 49
`0.13
`0.55
`1.47
`0.74
`0.60
`
`0. 85
`0.12
`0. 19
`0.19
`9. 62
`0. 24
`0.06
`1.08
`0. 28
`0. 12
`25. 22
`9, 53
`0.45
`0. 02
`0.90
`0.10
`0.09
`4,52
`26. 24
`2,42
`0.41
`0.11
`0.15
`0, 53
`1,37
`0, 29
`
`12:0
`12:1
`13:0
`Iso-14:0
`14:0
`14:1
`Iso-15:0
`15:0
`15:1
`Iso-16:0
`16:0
`16:1
`16:2
`Iso-17:0
`17:0
`17:1
`Iso-18:0
`18:0
`18:1
`18:2
`18:3
`19:0
`20:0
`20:1
`20:2
`20:3
`
`20:4
`20:5
`21:0
`22:1
`22:2
`22:3
`22:5
`22:6
`
`24:1
`
`0. 47
`1e15
`0, 26
`0, 94
`0.63
`1.64
`0. 87
`0.55
`6.59
`7,04
`0. 52
`12,59
`6.12
`8. 84
`1,40
`10. 96
`0.13
`0. 18
`0. 06
`0.21
`0.12
`0, 22
`0. 10
`0.07
`0.43
`0.92 ~
`0. 08
`0. 34
`0.37
`0.51
`0.11
`0. 41
`0. 25
`0.317
`0. 10
`0. 56
`0. 53
`0. 06
`0.07
`0.30
`2.66
`6. 89
`0. 50
`11,29
`4,12
`6. 38
`1,20
`11.74
`0.33
`0.48 — 0.13
`0,12
`oe
`—-
`0.23
`0. 67 —.
`0.13 —.
`0.41 — — ——
`0, 22 0. 78 0. 36 —— 0.59
`
`
`
`
`
`
`
`
`
`0.37
`
`—_——
`
`0, 27
`
`were 43.60%, 49.20%, 38.71% and 43.289 respectively, and 55.34%, 49.40%,
`61.19% and 55.69% in the total unsaturated acids of them, respectively. Among
`the saturated acids, 14: 0 and 16:0 acids were the most constituents in the
`
`Sci. Rep. Whales Res. Inst.,
`No, 28, 1976.
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`LIPID OF EUPHAUSIA SUPERBA
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`
`total lipids and all fractions of both sample lipids, Although the fatty acids of
`16:1, 18:1, 20:5 and 22: 3 among the unsaturated acids were the main con-
`stituents in the total lipids, free fatty acid and phospholipid fractions of both
`samples,
`these were the least of amount in both triglyceride fractions. The
`most pronounced difference between the trigylceride fractions and otherlipids
`was in the higher level of 14:0 acid and the lowerlevel of the fatty acids more
`than 20 carbon atoms. Moreover, the phospholipid fraction of the raw frozen
`sample contained very higher level (26.31%) of 16:
`1 acid as compared to one
`of other lipids whereas 14: 0 acid comprised slightly lower level of 5.98%.
`The fatty acids of 18: 3 (4.10%) and 20: 3 (4.61%) in the free fatty acid fraction
`_ and 18:0 acid (4.52%) in the phospholipid fraction of the boiled-frozen sample
`were slightly higher levels compared to others. Finally, there were little dif-
`ferences in the fatty acid compositions and distribution patterns of both samples
`except of the phospholipid fraction in the raw frozen sample.
`Saiki e¢ al. (1959) reported that the fatty acid composition of E. superba
`lipid were 16.0% of 14, 33.0% of 16, 36.1% of 18 and 13.6% of 20 carbon
`fatty acids. Also, Tsuyuki et al. (1964a) reported that these were 6.2% of 14,
`25.9% of 16, 35.2% of 18, 24.5% of 20 and 8.2% of 22 carbon fatty acids. We
`have found that these were 8,86-11.65% of 14, 35.04-35.77% of 16, 22.65-23.19%
`of 18, 13.26-16.38% of 20 and 13.09-13.34% of 22 carbon fatty acids in both
`total lipids.
`Although the fatty acid components analyzed by gas-liquid chromatography
`between the raw frozen and boiled-frozen euphausiacea lipids was very low
`difference, the total lipid thin-layer chromatogramsindicated that the free fatty
`acid fractions had appearently greater difference with each other. The results
`obtained with the free fatty acid fractions fractionated by thin-layer chromato-
`graphy in this study indicated that the boiling procedure supported considerably
`in the maintenance of the EF. superba lipid quality during handling and
`frozen storage.
`
`ACKNOWLEDGMENTS
`
`Euphausiacea examined in this study was collected through the courtesy of
`Nippon Suisan Kabushiki Kaisha Ltd.
`In particularly, we are much indebted
`to Mr. Masahiro Makuta who is a member of the Central Research Laboratory
`of Nippon Suisan. We also thank to Prof. Dr. Sadami Kadota,
`the Faculity
`of Fisheries, College of Agriculture & Veterinary Medicine, Nihon University,
`for identifying the material, Huphausia superba.
`
`SUMMARY
`
`1. The chemical properties of lipid contained in Euphausia superba,
`were studied.
`9. The lipid fractions of the euphausiacea were quantitated by thin-layer
`chromatography.
`
`Sci. Rep. Whales Res. Inst.,
`No. 28, 1976.
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`174
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`TSUYUKI AND ITOH
`
`3. The main lipid compositions were triglycerides, phospholipids and free
`fatty acids fractions.
`4, The component fatty acids of the total lipids and each fraction fraction-
`ated by thin-layer chromatography were analyzed by gas-liquid chromatography.
`9. The predominant fatty acids were myristic, palmitic, palmitoleic, oleic,
`eicosapentaenoic and docosatrienoic acids.
`
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`No. 28, 1976.
`
`RIMFROST EXHIBIT 1172 Page 0008
`RIMFROST EXHIBIT 1172 Page 0008
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