FATTY ACID COMPONENT OF LIPID
OF EUPHAUSIA SUPERBAHIDEO TSUYUKI
ANDSHINGO ITOH
Department of Food Technology, College of Agriculture andVeterinary 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 quantitatedby 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 shown to 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, 6.9% 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 and3.5% 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(16.83-30.49%), palmitoleic (5.92-26.31 %), oleic (19.31-29.17%), eicosapentaenoic (0.52-12.59%) and docosatrienoic (0.50-11.74%) acids.
INTRODUCTION

Euphausia superba is a shrimp-like crustacean of about 5 cm in body length,
and inhabits only in the Antarctic Ocean, though many euphausiid species arefound widely over the world oceans. E. superba composes one of the mostimportant 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 abasic knowledge for utilizing E. superba as to be a possible humans' food.
There have been several reports on the chemical properties and the fatty
acid composition of E. 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 thefractional distillation method, the minute examinations of the fatty acidcomponent have not been reported yet.
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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 thefatty acid components of E. superba by a combination of thin-layer and gasliquid 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 theE. superba harvested was quickly frozen as the raw frozen products andanother part of them was similarly quick frozen as the boiled-frozen products
after flash boiling. Both products were processed as the whole body of E.
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 5.5 cm in the body length.
The method of Bligh and Dyer (1959) was used to extract and purify thelipids 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 E. superba lipids was subsequently separated by thinlayer chromatography using 20x 20 cm 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. phospholipids, 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
ll0°C for 15 min. 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 andphospholipids 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 total lipids, triglycerides, free fatty acids
and phospholipids of E. siiperba lipids esterified by the saponification-transesterification method as described by Metcalfe et al. (1966). 5 ml of a 0.5 N
methanolic KOH solution were added to approximately 30 mg of total lipids,
triglycerides, free fatty acids and phospholipids, and heated over a steam bathfor 5 min. Then, 5 ml of 12.5% boron trifluoride in methanol were added tothese mixtures and boiled for 3 min. Thin-layer chromatography was used todetermine the completeness of the transesterification. Silica gel G plates werespotted with the reacted products and known standards, and developped withpetroleum ether: diethyl ether: acetic acid as previously described. A comparison of the R 1 values of the reacted products, triolein, methyl oleate andSci. Rep. Whales Res. Inst.,
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LIPID OF EUPHAUSIA SUPERBA

oleic acid (Nihon Chromato Works Ltd.) indicated that the conversion of total
lipids, triglycerides, free fatty acids and phospholipids fractions to methyl esters
was complete.
The methy 1 esters were analyzed weth a Shimadzu Gas Chromatograph
Model SA (Shimadzu Seisakusho Co.) equipped with a dual flame ionization
detector. The columns used were 3 m X 3.0 mm I. D. glass coil tubing packed
with Diasolid ZF on 80/100 mesh, and with 3% SE-30 on 80/100 mesh Chromosorb W (Nihon Chromato Works Ltd.). The carrier gas was nitrogen at flowrate 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.
Some of 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 confirm the correctness of identification of the unsaturated acids. Approximately
20 mg of methyl esters, 1 ml of methanol and a pinch of platinum black wereadded 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 thegas-liquid chromatograph with the same condition as above mentioned. The
area of each chromatographic peak representing a fatty acid present was obtained by multiplication of the height of each peak by the width at halfheight. 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 Euphausia superba
are shown in Table 1. The lipid contents are comparatively low i. e., 3.41 %of the raw frozen sample and 5.62% of the boiled-frozen sample on the wet
TABLE I.

PROPERTIES OF TOTAL LIPIDS FROM EUPHAUSIA SUPERBA
LipidsAppearance (20°C)
Oil content (%)
Refractive index (40°C)
Acid value
Iodine value
Saponification value
Unsaponifiables (%)

Sci. Rep. Whales Res. Inst.,
No. 28, 1976.

Raw frozen sample
Reddish brown liquid

Boiled-frozen sample
Reddish brown liquid

3.41

5.62

I. 4788
74. I

142.3
181. 3
5.6

I. 4820

18.2
127.9
197.6
5.8

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TSUYUKI AND ITOH

weight basis. The total lipid of the raw frozen sample was higher in iodine
and acid values, and lower in 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 chromatography is markedly different between the samples of the raw frozen and boiledfrozen. By visual evaluation of these chromatograph as shown in Fig. 1, thezones 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 thecompositions of triglycerides and free fatty acids between two samples. The
lipid compositions of these fractions are reported in Table 2. In the extractsof the boiled-frozen sample, triglycerides are the preponderant fraction comprising 76.8% of the total lipid fractions, whereas in the raw frozen sample,
triglycerides fraction is much lower. On the other hand, the composition offree fatty acids fraction is much higher in the raw frozen sample than in the
boiled-frozen sample. These appearently explained to the difference of acidvalue in Table I. Also, the raw frozen sample had '.?.2% of monoglycerides- Front

SE

TG

Ill

FFAlll
p
DG

s
p

MG
PL

--• • •
.....

·(;

'···

0

~:::-

.
0

s

p

..PL

A

e Strong

(;

~:·>

:·:.~

~:~

-::::

--• • •TG ' '
FFAO
0 '
0
p

• • - Origin
0{~:-• • •B

O MediumC":·....··

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.
Sci, Rep. Whales Res. Inst.,
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LIPID OF EUPHAUSIA SUPERBA

171

and a trace of diglycerides fractions while the boiled-frozen sample was notdetected them.
The fatty acid methyl esters of the total lipids and the main three fractions
contained fatty acids i. e., triglycerides, free fatty acids and phospholipids wereanalyzed by gas-liquid chromatography, but no attempt was made to further
study the component of other fractions. The presence of the fatty acids contained 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 compositionof 14: 0, 16: 0, 16: 1 and 18: 1 acids accounted for more than 85% of thetotal 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 thetotal 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 contained 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: I 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 thoseof 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 SUPERBABY THIN-LAYER CHROMATOGRAPHY.
LipidsComposition
Trigl ycerides
Digl ycerides

Raw frozen sample (%)

Boiled-frozen sample (%)

52.3

76. 8

trace

Monogl ycerides

2.2

Free fatty acids
Phospholipids
Sterols

27.4
6.9
4. 7

4.6
9.2
4.2

Stery 1 esters
Pigments

0.8

0.6

4. 1

3.5

Unknowns

I. 6

I. I

Sci. Rep. Whales Res. Inst ..
No. 28, 1976.

TSUYUKI AND !TOH

172

TABLE 3.

FATTY ACID COMPOSITION OF EUPHAUSIA SUPERBA LIPIDS.
(percentage of total Ii pids)
Lipids in raw frozen sample

Fatty
acids

12: 0
12: I
13 :0
lso-14: 0

14: 0
14: I
lso-15:0

15: 0
15: I
lso-16: 0
16: 0

16: I
16: 2
lso-17: 0

17: 0
17: I
lso-!8: 0
I8: 0
18: I
18: 2
18: 3
19: 0

20: 0
20: I
20: 2
20: 3
20: 4
20: 5
21:0
22: I

22: 2
22: 3
22: 5
22: 6

24: I

Lipids in boiled-frozen sample

Total
lipidsTriglycerides

Free
fatty
acids

Phospholipids

Total
lipidsTriglycerides

Free
fatty
acids

Phospholipids

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
I. 02
0.28
I. 45
0.94
12.59
0.21
0.34
0.56
11.29
0.48
0.67
0.37

0.27
0.05
0.08
0.03
22. 16
0. 15
0.05
0.84
0. 13

0.16
0.02
0.03
0.01
9.07
0. 13
0.03
0.52
0.07
0.04
26.01
7.96
0. 71
0.01
0.57
0.07
0.04
0.96
29. 17
3.47
0.80
0. 20
0. 13
0.52
0. 12
0.95
I. 15
7.04
0. 18
0.92
0.31
6.89
0.13
0. 13
0.27

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
I. 78
28.49
2.53
0.54
0.07
0.20
0.52
0.47
0.83
0.47
6.59
0. 13
0.43
0.25
2.66
0.33

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
0.55
IO. 96
0.07
0.41
0.30
11. 74
0.23
0.41
0.36

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
I. 02
25.87
2.03
0.49
0. 13
0.55
I. 47
0. 74
0.60
0.87
I. 40
0.10
0.11
0.07
I. 20

0.47
0.03
0.09
0.02
8.66
0. 19
0.04
0.33
0.09
0.06
24.82
6.49
0.41
0.02
0. 73
0.11
0.02
I. 60
21. 82
3.03

0.85
0.12
0.19
0.19
9.62
0. 24
0.06
I. 08

0. IO

30.49
8.86
0.53
0.02
0.55
0. 17
0.05
I. 18
28.25
I. 87
0. 12
0.20
0.09
0. 76
0.13
0.35
0.26
0.52
0.06
0.08
0.10
0.50

0.22

4. IO

0.39
0.24
0. 77

I. 46
4.61
I. 64
8.84
0.22
0.51
0.06
6.38

0.59

0.28
0. 12

25.22
9.53
0.45
0.02
0.90
0. IO

0.09
4.52
26.24
2.42
0.41
0. 11

0. 15
0.53
I. 37
0.29
0.63
6. 12
0. 12
0.37
0.53
4. 12
0. 12
0. 78

were 43.60%, 49.20%, 38.71 % and 43.28% respectively, and 55.34%, 49.40%,
61.19% and 55.69% in the total unsaturated acids of them, respectively. Amongthe saturated acids, 14 : 0 and 16 : 0 acids were the most constituents in theSci. Rep. Whales Res. Inst.,
No. 28, 1976.

LIPID OF EUPHAUSIA SUPERBA

173

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 constituents 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 other lipids
was in the higher level of 14 : 0 acid and the lower level of the fatty acids morethan 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 oneof 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 differences in the fatty acid compositions and distribution patterns of both samples
except of the phospholipid fraction in the raw frozen sample.
Saiki et 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. (l 964a) 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 chromatograms indicated 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 chromatography in this study indicated that the boiling procedure supported considerably
in the maintenance of the E. siiperba lipid quality during handling andfrozen storage.
ACKNOWLEDG MENTS

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 Laboratoryof Nippon Suisan. We also thank to Prof. Dr. Sadami Kadota, the Faculityof Fisheries, College of Agriculture & Veterinary Medicine, Nihon University,
for identifying the material, Eiiphausia superba.
SUMMARY

1. The chemical properties of lipid contained m Eiiphausia superba,
were studied.
2. The lipid fractions of the euphausiacea were quantitated by thin-layer
chromatography.
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TSUYUKI AND !TOH

3. The main lipid compositions were triglycerides, phospholipids and freefatty acids fractions.
4. The component fatty acids of the total lipids and each fraction fractionated by thin-layer chromatography were analyzed by gas-liquid chromatography.
5. The predominant fatty acids were myristic, palmitic, palmitoleic, oleic,eicosapentaenoic and docosatrienoic acids.

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Can.]. Biochem. Physiol., 37: 911-917.
HOFSTETTER, H. H., N. SEN and R. T. HOLMAN, 1965. Characterization of unsaturated fatty
acids by gas-liquid chromatography. ]. Am. Oil Chemists. Soc., 42: 537-540.
KAY AMA, M. and Y. IKEDA, 1975. Studies on the lipids of micronektonic fishes caught in
Sagami and Suruga Bays, with special reference to their wax esters. ]. Jap. Sci. Fisheries,
24: 435-440.
KAYAMA, M. and H. NAKAGAWA, 1975. Studies on the lipids of some micronektonic shrimps
caught in Sagami and Suruga Bays, with special reference to their wax esters. Ibid, 24:
441-445.
MIWA, T. K., 1963. Identification of peaks in gas-liquid chromatography. ]. Am. Oil Chemists'
Soc. 40: 309-313.
SAIKI, M. and T. MORI, 1953. Studies on the whale oil-I. Properties of oils contained invarious parts of a whale body (Part-I). On the sei-whale and fin whale. ]. Jap. Soc. Sci.
Fisheries, 19: 611-613.
SAIKI, M., S. C. FANG and T. MORI, 1959. Studies on the lipid of plankton-II. Fatty acidcomposition of lipids from Euphausiacea collected in the Antarctic and Northern Pacific
Oceans. Ibid, 24: 837-839.
TSUYUKI, H., U. NARUSE, A. MOCHIZUKI and S. !TOH, 1964a. Studies on the Euphausiacea,
Euphausia superba I. Acetone-soluble lipid. ]. Jap. Oil Chemists' Soc., 13: 203-206.
TSUYUKI, H., U. NARUSE, A. MOCHIZUKI and S. lTOH, 1964b. Studies on the Euphausiacea,
Euphausia superba II. Properties of phospholipids. Ibid, 13: 477-480.

Sci. Rep. Whales Res. Inst.,
No. 28, 1976.