Journal of Oleo Science

Copyright 2010 by Japan Oil Chemists Society

J. Oleo Sci. 59, (9) 457-462 (2010)

Dietary Soybean Phospholipids Increase

the Oxidative Stability of Lipids Extracted from Fish
Fillets
Yoshihiro Murano1 , Tomoko Funabashi1, Hiroyuki Takeuchi2 and Tatsuhiro Matsuo3
1

The Nisshin OilliO Group, Ltd. (1 Shinmeicho, Yokosuka, Kanagawa 239-0832, JAPAN)
Department of Food and Nutrition, Toyama College (444 Gankaiji, Toyama 093-0193, JAPAN)
3
Faculty of Agriculture, Kagawa University (2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, JAPAN)
2

Abstract: We previously reported that the feeding of soybean phospholipids to fish increased the storage
stability of n-3 polyunsaturated fatty acids (PUFA)-rich fish fillets. In this study, we examined the storage
stability of lipids extracted from fish fed a diet containing soybean phospholipids and fish oil. Rainbow trout
were divided into two groups, and were fed an either 2.5% soybean phospholipids (test) or no phospholipids
(control) containing diet for 4 weeks. Lipids were extracted from fish fillets after the feeding period, and
were subjected to an oxidation test. Lipids extracted from the fillets of fish in the test group exhibited lower
values of oxygen absorption than those in the control group, and the degradation of docosahexaenoic acid
(DHA) and eicosapentaenoic acid (EPA) was inhibited. Higher percentages of DHA and EPA were bound to
phosphatidylcholine and phosphatidylethanolamine in the extracted lipids in the test group than in the
control group. These results indicate that the oxidative stability of lipids extracted from fish fed soybean
phospholipids is high, and that the higher percentages of DHA and EPA in PC and PE may have resulted in
the higher stability of the lipids extracted from fish fillet.
Key words: soybean phospholipids, fish fillet, lipid, oxidation, phosphatidylcholine, phosphatidylethanolamine,
docosahexaenoic acid, eicosapentaenoic acid
1 INTRODUCTION
We previously reported that the feeding of soybean
phospholipids to rainbow trout increased the storage stability of fish fillets1. Soybean phospholipid feeding did not
influence the level or fatty acid composition of total lipids,
or the content of antioxidants, tocopherol and phospholipid in fish fillets. Rainbow trout fed a soybean phospholipid
diet had a higher percentage of docosahexaenoic acid
DHAin phosphatidylcholinePCand phosphatidylethanolaminePEin fillets than those fed a soybean phospholipid-free diet2. The oxidative stability of DHA-bound PC
and PE has been reported to be high3, 4. We speculate that
one of the factors associated with the increased storage
stability of fish fillets was the high percentage of DHA were
bound to PC and PE. However, in addition to lipids, watersoluble vitamins, amino acids, and proteins may also influence the storage stability of fish fillets 5, 6. If the high
amounts of DHA-bound PC and PE increased the storage
stability of fish fillets, the oxidative stability of lipids ex-

tracted from them is also expected to be high. Fish oils are

known to contain abundant DHA and eicosapentaenoic
acid EPAand have various physiological activities 7, 8;
however, their low oxidative stability poses limitations to
their commercial use.
In this study, rainbow trout were fed a diet supplemented with 2.5soybean phospholipids and 10fish oil or
2.5soybean oil and 10fish oil for 4 weeks. Subsequently, lipids were extracted from the fillets of fish in each experimental group. Extracted lipids were subjected to an
oxidation test to examine whether the oxidation stability of
lipids extracted from soybean phospholipid-fed fish is increased. In addition, the amounts of DHA-bound PC and
PE in the extracted lipids were compared.

Correspondence to: Yoshihiro Murano, The Nisshin OilliO Group, Ltd., 1 Shinmeicho, Yokosuka, Kanagawa 239-0832, JAPAN
E-mail : y-murano@nisshin-oillio.com
Accepted March 4, 2010 (received for review January 28, 2010)

Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online

http://www.jstage.jst.go.jp/browse/jos/
457

Y. Murano, T. Funabashi, H. Takeuchi and T. Matsuo

2 EXPERIMENTAL
2.1 Materials
The soybean phospholipids and soybean oil used in this
study were provided by Nisshin OilliO Group Ltd., Tokyo,
Japan. The fish oil was obtained Nippon Suisan Kaisha,
Ltd., Tokyo, Japan.
2.2 Procedure
2.2.1 Fish and feeding experiment
A purified diet, the composition of which is shown in
Table 1, was used in the experiment. Vitamins and minerals were added, as described by Kaushik et al.9and Ogino
et al.10. Two types of experimental diet containingtest
dietand not containing soybean phospholipidscontrol
dietwere prepared as follows. Fish oil10 g/100 gwas
added to each diet. In addition, soybean oil
5 g/100 gwas
added to the control diet while soybean phospholipids
2.5
g/100 gand 2.5 g of soybean oil were added to the test
diet. The fatty acid composition of the total lipids in each
type of experimental diet is shown in Table 2.
Test fish were maintained and fed as previously described 1. Juvenile rainbow trout Onchorhynchus
mykisspurchased from a local supplier were used in the
feeding experiment. At the start of the feeding experiment,
a total of 140 fisheach weighing 2.1 g on average
were divided into 2 groups70 fish per group. At the end of the
feeding period, fish fillets were collected as previously described, and used as samples.
2.2.2 Biochemical analysis
Lipids were extracted from samples according to the
method of Folch et al.11. The phospholipid content, fatty
acid composition, and tocopherol content were determined
as previously described1. Triacylglycerols were fractionated by thin-layer chromatography on Silicagel 70 plates
Wako Pure Chemical Industries, Ltd., Osaka, Japanusing

Table 1 Experimental Diet Composition.

Treatment group
Control

Test
(g/100 g)

Casein

50

Dextrin

20

Corn starch

Mineral mix

Vitamin mix

Lipids
Fish oil

10

10

Soybean oil

2.5

Soybean phospholipids

2.5

Table 2 Fatty Acid Composition of the Experimental

Fats.
Treatment group
Control

Test
(g/100 g)

12:0 *

0.1

0.1

14:0

4.0

4.0

15:0

0.3

0.4

16:0

12.7

14.2

16:1

4.9

4.9

17:0

0.2

0.3

18:0

3.1

3.1

18:1

21.4

19.0

18:2 (n-6)

18.3

19.2

18:3 (n-3)

2.7

2.8

20:0

0.3

0.2

20:1

9.0

8.9

20:2 (n-6)

0.2

0.2

22:0

0.6

0.6

22:1

8.0

8.0

20:5 (n-3)

7.1

7.1

24:0

0.4

0.4

24:1

0.5

0.5

22:5 (n-3)

1.0

1.0

22:6 (n-3)

5.2

5.2

* Number of carbon atoms : number of double bonds.

a solvent consisting of chloroform and methanol
9:1, and
weighed12. Total phospholipids, PC, and PE were analyzed
according to the method of the Japan Oil ChemistsSociety13. Fatty acids at positions sn-1 and sn-2 in PC and PE
were analyzed by the phospholipase A2 method14. These
fatty acid analyses were performed by gas-liquid chromatography6898 series; Agilent Technologies, Santa Clara,
CA, USAwith a capillary column
OMEGAWAX320; Supel1
.
co, Bellefonte, PA, USA
2.2.3 Oxidation test
Each sample consisted of lipids extracted from 14 fish.
Three control and three test samples were prepared for
use in oxidation test. Lipids were dispensed in 200 mg aliquots into 10 ml glass vials allowing sealing with butyl-rubber septa and aluminum seals
Agilent Technologies, Santa
Clara, CA, USA
. After sealing, the vials were stored in the
dark at 60 for 20 days. All samples were prepared in duplicate, and the mean of two measurements was used for
analysis.
The oxygen concentration in the vial headspace and the
amount of fatty acids in the lipids16:0, 18:1, 20:5, and

458

J. Oleo Sci. 59, (9) 457-462 (2010)

Dietary Phospholipids Increase Oxidative Stability of Fish Lipids

22:6
were measured as indicators of the storage stability of
extracted lipids. The oxygen concentration in the headspace of each vial was analyzed with an oxygen meter
RO-102; Iijima Electronics, Corp. Aichi, Japan
. Fatty acids in each vial were quantified by gas chromatography.
2.2.4 Statistical analysis
Data are expressed as the meanSEM. Significant differences between the diet groups were determined by Students t-test. Differences with a P-value 0.05 were considered significant. A statistical software packageToukei
version 2, Esumi, Tokyo, Japanwas used for all statistical
analyses.

lipids extracted from fish fillets between the two groups

Table 4
.
In the oxidation test, the oxidative stability of extracted
lipids was compared in terms of the amount of oxygen in
the headspace that decreases with lipid oxidation in a
sealed vial and the amounts of DHA and EPA that remained
without undergoing oxidative degradation during lipid oxi-

Table 5 Fatty Acid Composition of Lipids from

Fillets.
Treatment group
Control

Test
(g/100 g)

3 RESULTS
Table 3 shows the growth of rainbow trout fed a control
or test diet for 4 weeks. There was no significant difference
in body weight or feed conversion efficiency between the
test and control groups. The supplementation of soybean
phospholipids did not influence growth.
The total lipid content of fish fillets after the feeding period in the control and test groups was 5.90.0 and 6.0
0.1g/100 g, respectively, showing no significant difference. There was no significant difference in the content of
triacylglycerol, phospholipids, PC, PE or tocopherols in the

Table 3 Growth and Feed Performance of Rainbow

Trout after a 4-Week Feeding Period.
Treatment group
Control

Test

Initial body weight (g)

2.1 0.0

2.1 0.0

Final body weight (g)

6.2 0.2

6.2 0.2

118.0

118.5

Feed conversion efficiency (%)

14:0*

4.1 0.1

4.0 0.1

16:0

19.7 0.3

20.5 0.2

16:1

7.7 0.2

7.6 0.1

18:0

4.2 0.1

4.2 0.0

18:1

27.1 0.4

26.8 0.2

18:2 (n-6)

11.9 0.7

10.4 0.5

18:3 (n-3)

1.4 0.0

1.3 0.0

20:1

6.4 0.3

6.9 0.4

20:2 (n-6)

0.6 0.0

0.6 0.1

22:0

0.5 0.0

0.6 0.0

22:1

3.9 0.3

4.3 0.2

20:5 (n-3)

2.7 0.1

2.8 0.1

22:5 (n-3)

0.8 0.0

0.8 0.0

22:6 (n-3)

7.5 0.2

7.9 0.0

*Number of carbon atoms : number of double bonds.

Values are means SEM, n=5. No significant differences
were observed.

Values are means SEM, n=70. No significant differences

were observed.

Table 4 Triacylglycerol, Phospholipid and Tocopherol

Contents of Lipids from Fillets at the End of the
Feeding Period.
Treatment group
Control

Test

Triacylglycerol (g/100 g lipid)

69.6 2.1

68.2 4.7

Phospholipids (g/100 g lipid)

16.9 0.5

16.7 0.9

Phosphatidylcholine (g/100 g lipid)

12.5 0.3

12.7 0.5

Phosphatidylethanolamine
(g/100 g lipid)

4.2 0.2

4.0 0.5

0.43 0.04

0.43 0.03

Total tocopherol (mg/g lipid)

Values are means SEM, n=5. No significant differences were

observed

Fig. 1 Effects of Dietary Soybean Phospholipids on the

Stability of Extracted Lipids from Fish Fillets
during the Oxidation Period.
The oxidative stability was evaluated by oxygen
consumption of the head space in a vial by
lipid oxidation. Values are means SEM (n=3).
Asterisks indicate significant differences (P<0.05).
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J. Oleo Sci. 59, (9) 457-462 (2010)

Y. Murano, T. Funabashi, H. Takeuchi and T. Matsuo

Fig. 2 Effects of Dietary Soybean Phospholipids on the Stability of Extracted Lipids from Fish Fillets during the
Oxidation Period.
The oxidative stability was evaluated by the residual fatty acid content based on lipid oxidation. Values are means
SEM (n=3). Asterisks indicate significant differences (P<0.05).
dation. No significant difference was noted in the fatty acid
composition of extracted lipids used in the oxidation test
Table 5. Measurement of the oxygen concentration in
the headspace during the oxidation test showed that the
amount of oxygen absorption on day 10 or later was significantly lowers in the test than in the control group
Fig. 1
.
Analysis of the amount of fatty acids remaining during the
oxidation test revealed no significant difference in the
amount of palmitic or oleic acid between the test and control groups. In contrast, the amounts of residual DHA and
EPA decreased with the duration of storage in both groups,
but were significantly higher in the test than in the control
group, indicating the inhibition of fatty acid oxidation in
the test group
Fig. 2.
Table 6 shows the fatty acid composition of triacylglycerol in lipids from fish fillets. The amount of 22:1 in triacylglycerol was significantly higher in the test group than the
control group, however, the amounts of DHA and EPA
were not significantly different between the two groups.
Analysis of the amount of fatty acids in PC and PE showed
that DHA and EPA were bound predominantly to the sn-2
position of PC in both groups; larger amounts of DHA and
EPA were bound to the sn-1 and sn-2 positions of PC in the
test than in the control group
Fig. 3 A, B
; a significantly
higher percentage of DHA and EPA were bound to the sn-1
position of PE in the test than in the control group
Fig. 4
A; and significantly higher percentages of DHA was bound

Table 6 Fatty Acid Composition of Triacylglycerols

in Lipid from Fillets.
Treatment group
Control

Test
(g/100g)

14:0*

4.0 0.0

4.1 0.1

16:0

20.3 0.2

20.4 0.5

16:1

7.9 0.3

7.9 0.2

18:0

4.6 0.1

4.6 0.2

18:1

29.9 0.5

28.9 0.6

18:2(n-6)

11.5 0.2

10.5 0.4

18:3(n-3)

1.1 0.1

1.2 0.1

20:1

6.8 0.1

7.2 0.1

20:2(n-6)

0.6 0.0

0.6 0.0

22:1

4.9 0.1a

5.3 0.1b

20:5(n-3)

1.5 0.1

1.8 0.1

24:1

0.5 0.0

0.5 0.0

22:5(n-3)

0.6 0.0

0.7 0.0

22:6(n-3)

3.7 0.3

4.3 0.3

*Number of carbon atoms : number of double bonds.

Values are means SEM, n=5. Letters indicate statistically
significant differences (P<0.05).

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J. Oleo Sci. 59, (9) 457-462 (2010)

Dietary Phospholipids Increase Oxidative Stability of Fish Lipids

Fig. 3 Fatty Acid Composition of Phosphatidylcholine

at sn-1 (A) and sn-2 (B) Positions of Lipids from
Fillets.
Values are means SEM (n=5). Asterisks indicate
significant differences (P<0.05).

Fig. 4 Fatty Acid Composition of Phosphatidylethanolamine at sn-1 (A) and sn-2 (B) Positions of Lipids
from Fillets.
Values are means SEM (n=5). Asterisks indicate
significant differences (P<0.05).
to the sn-2 position of PE in the test than in the control
group
Fig. 4 B.

4 DISCUSSION
The oxidation test on lipids extracted from fish fillets
showed that the amount of oxygen absorption was significantly lower, and the amounts of residual DHA and EPA
were significantly higher, in the test than in the control
group. These results indicate that lipids contained in the
fillets of soybean phospholipid-fed fish exhibit a higher oxidative stability than those of soybean oil-fed fish. The analysis of lipids extracted from fish fillets revealed no significant difference in the content of phospholipids or
tocopherols, but higher percentages of DHA and EPA were
bound to PC and PE in extracted lipids in the test than in
the control group.
We previously reported that the feeding of soybean
phospholipids to rainbow trout increased the storage stability of fish fillets1, 2. The present study showed that the
feeding of fish with soybean phospholipids increased the
oxidative stability of lipids extracted from them, thus con-

tributing to the high storage stability of fish fillets.

The addition of phospholipids increases the oxidative
stability of n-3 PUFA-containing lipids, and this effect varies with the composition of the phospholipids and content
of coexisting tocopherols. Kashima et al.15reported that
the addition of PC or PE, particularly the latter, to fish oil
increased its oxidative stability. Cho et al.16investigated
the storage stability of squid-derived lipids, n-3 PUFA-rich,
and reported that the oxidative stability of lipids derived
from internal organs with a high content of PE was higher
than that of lipids derived from muscles. Takeuchi et al.17
reported that tocopherols and phospholipids, particularly
PE, synergistically increased the storage stability of n-3
PUFA. The lipids used in the oxidation test of the present
study also contained PE and tocopherols. However, their
content and composition did not vary between the treatment groups; therefore, the difference in the oxidative stability cannot be explained by the phospholipid content,
phospholipid composition, or tocopherol content.
Since DHA and EPA in triacylglycerols contain many
double bonds, they show low oxidative stability and tend to
form hydroxyperoxides in oils, thereby triggering lipid autoxidation. In contrast, in phospholipids, DHA- or EPAbound PC and PE are known to exhibit higher oxidative
stability18, 19. In addition, DHA- or EPA-bound PC and PE
have high-level antioxidant activity when added to lipids.
King et al.20reported that phospholipids bound to a high
percentage of DHA increased the oxidative stability of fish
oil. Sugino et al.21found that PC and PE bound to large
amounts of n-3 PUFA increased the oxidative stability of
DHA-containing oils compared with PC and PE bound to
small amounts of n-3 PUFA. In the present study, the percentages of DHA and EPA in PC and PE were higher in the
test than in the control group. Thus, the presence of highly
antioxidant PC and PE in the test group may have resulted
in the greater inhibition of lipid oxidation in this group
than in the control group.
In addition, another anti-oxidative mechanism of phospholipids has been proposed. Judde et al.22suggested that
PC, PE and PS increased vegetable oil stability because
their phospholipids had metal-chelating properties. In this
study, metal ions in fish fillets may be chelated by administrated soybean phospholipids.
In this study, we confirmed the high oxidative stability of
lipids extracted from the fillets of fish fed soybean phospholipids. The higher percentages of DHA and EPA in PC
and PE may have resulted in the higher stability of the lipids extracted from fish fillet. The fish oils with higher oxidative stability may be obtained from fish fed soybean
phospholipids.

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J. Oleo Sci. 59, (9) 457-462 (2010)

Y. Murano, T. Funabashi, H. Takeuchi and T. Matsuo

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