Trace Nutrients Research 29 : 18−24（2012）

Original Article

Effect of Dietary Oyster-Extract By-Product on Serum and Liver Lipid Contents in Rats
1, 2）

2）

3）

Ryota HOSOMI , Harutaka NODA , Yoshikazu MATSUDA ,4）2）
2）
Toshimasa NISHIYAMA , Munehiro YOSHIDA and Kenji FUKUNAGA
1）＊Division of Human Living Sciences, Tottori College ,2）
Faculty of Chemistry, Materials and Bioengineering, Kansai University＊＊ ,3）Central Research Institute, Japan Clinic Co., Ltd.＊＊＊ ,4）Department of Public Health, Kansai Medical University ＊＊＊＊

Summary
To clarify the possibility of using an oyster-extract by-product as a health maintaining and promoting food material
or supplement, this study investigated the eﬀects of oyster-extract by-product powder (OEBP) on serum and liver lipid
contents in rats. OEBP was prepared from washed and freeze-dried oyster-extract by-product, which contained 58.3 %protein, 19.7 % fat, and 19.7 % carbohydrate. Male, 5-week-old Wistar rats were divided into 2 dietary groups of 7 rats
each, one group receiving a control AIN-93G diet and the other receiving a 5 % OEBP-containing diet. After the rats
had been fed on their respective diets for 31 days, their serum and liver lipids contents and fecal cholesterol, bile acid,and nitrogen excretion were measured. The results demonstrated that OEBP decreased liver cholesterol content, inpart owing to the enhancement of fecal cholesterol and bile acids excretion; however, serum cholesterol content in the
OEBP diet did not decrease. In addition, the OEBP group exhibited lower protein digestibility during

digestion

analyses compared with the control diet. Therefore, in the OEBP group, the excretion of fecal cholesterol and bile acids
was inﬂuenced by the low digestibility of OEBP protein. However, in order to develop OEBP as a lipid-lowering functional food and supplement, we concluded that OEBP required improvements in its formulation and further concentration of the bioactive components because OEBP has not the lowering eﬀect of serum lipid contents.

4）

Introduction

they do not lower serum cholesterol content . Therefore,
there is a possibility that the health functions of seafood

Seafood consumption has preventative effects against

are not solely related to EPA and DHA. The beneﬁcial ef-

coronary heart disease according to epidemiological evi-

fect of seafood protein may have been masked by EPA

1）dence gathered from Greenland Inuit and Japanese ﬁsh2）

and DHA in seafood intake intervention studies. Seafood

ing villages . Many other studies have also described that

protein has beneﬁcial eﬀects such as hypocholesterolemic,

seafood consumption assists in protecting against lifestyle-anti-hypertensive, stimulation of ﬁbrinolysis, and anti-obe-

related diseases, including type 2 diabetes, hypertension,sity properties in animal stdies

3）and hyperlipidemia . The various beneﬁcial eﬀects of sea-

Oysters (5 ‑ 8）.

) contain many nutrients andfood have primarily been attributed to ‑3 polyunsaturat-

are widely used in Japan. Oyster-extract contains main com-

ed fatty acids (PUFAs), such as eicosapentaenoic acidponents including zinc, glycogen, and taurine, and oyster-ex-(EPA) and docosahexaenoic acid (DHA). There has been a

tract powder is marketed as a supplement. Several studies

great deal of research focused on the eﬃcacy of EPA andhave suggested that oyster-extract powder suppresses the

DHA in seafood for human health. However, dietary re-initiator action of carcinogens in mice and promotes the re-gimes also include EPA and DHA, and in addition to thesecovery of proximal tubular epithelial cell function in -amin-

components whole seafood provides many additional nu-

ophenol-induced nephrotoxicity in ratstrients. Dietary ‑3 PUFAs decrease serum triacylglycer-

ter of food by-product disposal had become a concern.

ol (TG) content, although a meta-analysis indicated that

Thereafter, it is also expected that the effective usage of

Address：854, Kurayoshi, Tottori 682‑8555, Japan
Address：3‑3‑35, Yamate-cho, Suita, Osaka, 564‑8680, Japan＊＊＊ Address：10‑15, Fumuzono-cho, Moriguchi, Osaka, 570‑8506, Japan＊＊＊＊ Address：10‑1, Kainichi-cho, Uzumasa, Ukyo-ku, Kyoto, 616‑8555, Japan＊＊＊― 18 ―

9, 10）. Recently, the mat-

oyster-extract by-product. To measure the nutrient compo-

was analyzed using a gas-liquid chromatography systemnents of oyster-extract by-product, we found that a high(GC-14B, Shimadzu Co.) with an SE-30 column (Shinwa

content of protein remains in oyster-extract by-product. In

Chemical Industries; Kyoto, Japan), in which 5α-cholestane

this study, to clarify the possible use of oyster-extract by-

was used as an internal standard . Phospholipid content

product as a health maintaining and promoting food materi-

was measured by phosphorus analysis .

12）

13）

al or supplement, the effects of oyster-extract by-product

Table 2 Fatty acid composition of OEBP total lipid (%)

powder (OEBP), obtained from washed and freeze-dried oys-

Fatty acid
14 : 0
15 : 0
16 : 0
16 : 1
17 : 018 : 018 : 118 : 2
20 : 020 : 1
20 : 422 : 2
22 : 6ter-extract by-product, on serum and liver lipid contents
were investigated in rats.

Materials and Methods
1. Preparation of oyster-extract by-product powder
Oyster () extract by-product, obtained

from oyster meat after the procedure of oyster-extract
preparation (extraction methods: hot water and 0.1 N HCl),
was provided by Japan Clinic Co., Ltd. (Kyoto, Japan). Oyster-extract by-product was chopped into small pieces andthen rinsed with water. The resulting meat was freeze-

8.7
1.0
33.4
6.4
2.1
6.2
5.0
11.9
3.0
4.3
2.0
5.1
1.2

‑9
‑6
‑9
‑6
‑3

Othersdried, ground using a mill (GM200, Retsch Co., Ltd. Haan,

9.8

OEBP, oyster-extract by-product powder.

Düsseldorf, Germany) and then sieved to remove any
lump of oyster meat. The resulting product was called
OEBP (particle size: < 1 mm).

Casein and OEBP were hydrolyzed using 6 mol/L HCl
for 24 hours. The amino acid composition in casein and

Table 1 shows the nutrient composition of oyster-ex-

OEBP was determined by reversed phase high-perfor-

tract by-product and OEBP. The crude protein content

mance liquid chromatography (HPLC) with UV detection

was determined by the Kjeldahl method, with an N-to-pro-using derivatization with phenylisothiocyanate (Table 3) .

tein conversion factor of 6. 25. The crude fat content was

The identification and quantitation of each amino acidmeasured by the Soxhlet method. The moisture content

were carried out using commercially available authentic

was estimated as the loss in weight after drying at 105 °Cstandard mixtures.

14）for 24 hours, and the amount of ash was analyzed by di-Table 3 Amino acid composition of casein and OEBP
(g/100g protein)rect ignition at 550 °C for 24 hours. The remaining content
after the elimination of protein, fat, ash, and moisture was

Amino acid
AlanineArginine†
Aspartic acid§
Glutamic acid
GlycineHistidine
Isoleucine
LeucineLysineMethionine
Phenylalanine
ProlineSerineThreonine
Tyrosine
Valineconsidered as carbohydrate.
Table 1 Chemical compositions of oyster-extract by-product and
OEBP (g/100 g)

Crude protein
Crude fat
Ash
Moisture§
Carbohydrate

Oyster-extract by-product
21.5
7.2
0.8
69.7
0.8

†

OEBP
58.3
19.7
1.2
1.1
19.7

†

Consists of oyster meat after the procedure of oyster-extract preparation.
§
Carbohydrate = 100 ‑ (crude protein + crude fat + ash + moisture)
OEBP, oyster-extract by-product powder.

Table 2 shows the fatty acid compositions of OEBP. The

Casein
2.9
3.6
6.8
20.5
1.7
2.9
5.3
9.1
7.7
2.8
4.8
10.8
5.0
4.0
5.4
6.5

OEBP
3.6
9.2
11.1
14.3
4.4
1.9
2.8
8.4
6.6
3.2
6.4
5.0
5.9
7.5
4.6
5.2

†

Aspartic acid + asparagine.
Glutamic acid + glutamine.
OEBP, oyster-extract by-product powder.
§composition of fatty acids was determined by analysis using a fused silica capillary column, Omegawax 250 (Supelco, Pennsylvania, USA) in a gas-liquid chromatography

2. Animals care and experimental diets

system (GC-14B, Shimadzu Co., Kyoto, Japan) after methyl11）

ation with sodium methoxide . The cholesterol content

The experimental protocol was reviewed and approvedby the Animal Ethics Committee of Kansai Medical Uni-

― 19 ―

17）

versity and followed the “Guide for the Care and Use ofand Dyer . Each total lipid sample was dissolved in an

Experimental Animals” issued by the Prime Ministerʼs Of-

equal volume of dimethylsulfoxide, and the TG content

ﬁce of Japan. Male 5-week-old Wistar rats obtained fromwas determined using an enzymatic assay kit (Triglycer-

Japan SLC, Inc. (Shizuoka, Japan) were kept in an air-con-ide-E-Test Wako, Wako Pure Chemical Industries; Osaka,ditioned room (temperature, 21‑22 °C; humidity, 55‑65 % ;Japan). Cholesterol and phospholipid contents were deter-

lights on, 08 : 00 ‑ 20 : 00), with free access to tap water andmined by gas-liquid chromatography and phosphorus

feed. Fourteen rats were divided into the following 2 di-analysis, respectively, as described previously.

etary groups of 7 rats each: control diet (AIN-93G) andOEBP diet. Table 4 lists the composition of the experimen-

5. Fecal lipid and nitrogen analysis

15）

tal diets, prepared based on the AIN-93G formulation .

Fecal cholesterol content was determined by gas-liquid
chromatography as described above. Fecal bile acids con-Table 4 Composition of the experimental diets (g/kg diet)

CaseinOEBP
Dextrinized corn starchCorn starch
SucroseCellulose
AIN-93G mineral mixture
AIN-93 vitamin mixture
L-Cystine
Choline bitartrate
Soybean oil-Butylhydroquinone

Control
200‑
132397.486
100
50
35
10
3
2.5
70
0.014

tent was determined as 3α-hydroxysteroid based on the

OEBP
170.9
50
132
386.486
100
50
35
10
3
2.5
60.1
0.014

18）

molar extinction coeﬃcient of NADH at 340 nm . Fecal
nitrogen content was determined by the Kjeldahl method.
6. In vitro digestion
Control and OEBP diets were digested by the methodsof Hosomi

5）with some modiﬁcations . Pepsin hydroly-

sis parameters were as follows: diet concentration, 10 %(w/v); enzyme/substrate ratio, 1 : 100 (w/w); adjusted topH 2.0 with HCl at 37 °C (Sigma Chemical Co., Missouri,
USA). After incubation for 180 min, pepsin was inactivatedDiets were prepared based on the AIN-93G formula.
OEBP, oyster-extract by-product powder.

by neutralization with NaOH.
Porcine pancreatin (Sigma Chemical Co.) was added;

Food consumption and body weight were recorded dai-pancreatin hydrolysis parameters were as follows: en-ly. Feces were collected from each group every 24 hours

zyme/substrate ratio, 1 : 30 (w/w); adjusted to pH 7.4 withfor 7 days before being sacrificed. After feeding for 31

NaOH at 37 °C. After hydrolysis for 360 min, digestion wasdays, rats were weighed and sacriﬁced under pentobarbi-

stopped by heating to 95 °C for 15 min. The digested sam-®

tal (Nembutal , Dainippon Sumitomo Pharma Co., Ltd.;ple was centrifuged at 4,500× for 20 min. The sediment

Osaka, Japan) anesthesia for 08 : 00 ‑ 10 : 00. Rats were not

was washed with distilled water two times, centrifuged at

deprived of food prior to being sacriﬁced because food de-

4,500× for 20 min, dried at 105 °C for 24 hours, and thenprivation leads to a significant down regulation of theweighed.

genes involved in fatty acid synthesis and cholesterol me16）

tabolism . Blood was collected from the abdominal aorta

7. Protein digestibility

without the use of anti-coagulant, and serum was obtained

For the monitoring of protein digestibility at intervals

by centrifugation at 1,500× for 15 min and then stored atduring digestion, aliquots of the digestion reaction were

‑ 70 °
C until analysis. The liver and abdominal white adi-removed at 0, 2, 5, 10, 15, 20, 30, 45, 60, 120, and 180 min

pose tissue (WAT) from the epididymis, mesentery, peri-

during the pepsin and pancreatin

nephria, and retroperitoneum were removed rapidly, thenaliquots were submerged in a boiling water bath for 15

weighed, rinsed with saline, and frozen in liquid nitrogen,min to inactivate pepsin and pancreatin, and then cooled

followed by storage at ‑ 70 °C until analysis.

and stored at ‑ 35 °C until analysis.

digestions. The

Protein digestibility was calculated by the determina3. Serum biochemical testtion of free amino groups by reaction with 2, 4, 6-trinitro-

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (SUN), total lipid, phos-19）

benzene sulfonic acid . L-Leucine was used as a standard
in the protein digestibility assays.

pholipids, cholesterol, and TG were measured using an au8. Statistical analysis

tomatic analyzer (AU5431, Olympus Co. Tokyo, Japan).

Data represent means and standard deviations (SD).
4. Liver lipids analysis

The statistical significance of differences was evaluatedLiver lipids were extracted using the method of Blighusing Studentʼs -test and means were considered signiﬁ-― 20 ―

cantly diﬀerent at < 0.05. The correlation was determined

glycine, and threonine but lower levels of glutamic acidby simple analysis. Analyses were performed using Stat-(glutamic acid + glutamine), isoleucine, and proline.
Table 5 shows the growth parameters and organ

View-J version 5.0 software (Abacus Concepts, California,

weight. There were no differences in body weight gain,USA)food intake, liver weight, and total abdominal WAT

Results and Discussion

weight between the control and OEBP groups.
Table 6 shows the biochemical parameters of the se-

OEBP, prepared by rinsing and freeze-drying oyster-ex-

rum, liver, and feces. Diﬀerences in serum AST, ALT, andtract by-product, contained 58.3 % protein, 19.7 % fat, andSUN were not observed as a result of OEBP administra-

19.7 % carbohydrate. In this study, OEBP was added totion. The serum total lipid, phospholipid, cholesterol, andthe control diet (AIN-93G) at the expense of cornstarch,

TG contents did not diﬀer between the control and OEBP

casein, and soybean oil in the preparation of the OEBP

diets, but the OEBP group showed signiﬁcantly lower liv-

diet, hence the contents of carbohydrate, protein, and fat

er cholesterol content than the control group. The liver to-

between the control and OEBP diets were nearly thetal lipid, phospholipid, and TG contents did not diﬀer be-same. The phospholipids and cholesterol contents in OEBP

tween the control and OEBP group.

were 85.2 % and 1.2 %, respectively. The amino acid com-

One of the reasons for the decrease in liver cholesterol

position in OEBP, compared with casein, showed higher

content was thought to be related to the absorption oflevels of arginine, aspartic acid (aspartic acid + asparagine),cholesterol and bile acids from the small intestine . Previ-

20）

Table 5 Growth parameters and organ weights for rats fed experimental diets for 4 weeks
Control

OEBP

Growth parameters
Body weight gain (g/day)Food intake (kcal/day)

7.16 ± 0.20
18.0 ± 2.0

6.76 ± 0.29
17.5 ± 1.8

Organ weight (g/100 g BW)
Liver weight
Epididymal WAT weightMesentery WAT weight
Perirenal and retroperitoneal WAT weight†
Total WAT weight

3.61 ± 0.26
1.44 ± 0.10
2.13 ± 0.20
1.34 ± 0.42
4.98 ± 0.90

4.01 ± 0.53
1.54 ± 0.21
2.28 ± 0.34
1.39 ± 0.43
5.18 ± 0.73

Data represent means ± SD (n = 7).
†
Total WAT reshows the sum of WAT weights from the epididymis, mesentery, perinephria, and retroperitoneumBW, body weight; OEBP, oyster-extract by-product powder; WAT, white adipose tissue.

Table 6 The biochemical parameters of serum, liver and feces in rats fed experimental diets for 4 weeks
Control

OEBP

Serum
AST (IU/L)
ALT (IU/L)
SUN (mg/dL)
Total lipid (mg/dL)
Phospholipids (mg/dL)
Cholesterol (mg/dL)
TG (mg/dL)

79.9 ± 6.8
43.1 ± 3.6
23.1 ± 3.0
205 ± 21119 ± 1266.1 ± 7.8
22.0 ± 5.3

81.3 ± 5.5
47.4 ± 6.1
17.9 ± 5.5
220 ± 19129 ± 1171.3 ± 5.8
20.4 ± 5.3

Liver (mg/g Liver)
Total lipid
Phospholipids
Cholesterol
TG

7.03 ± 0.90
2.62 ± 0.06
0.45 ± 0.15
3.96 ± 0.80

6.89 ± 0.53
2.66 ± 0.18
0.24 ± 0.05 *3.77 ± 0.97

Feces
Dry weight (g/day)
Cholesterol (mg/day)Bile acids (µmol/day)Nitrogen (mg/day)

3.92 ± 0.35
5.58 ± 1.03
8.18 ± 0.64
32.9 ± 5.3

4.33 ± 0.38
12.04 ± 1.04 *13.62 ± 2.48 *51.5 ± 6.7 *Data represent means ± SD (n = 7). Value statistically diﬀerent at * <0.05 using a Studentʼs -test compared to control group.
AST, aspartate aminotransferase; ALT, alanine aminotransferase; OEBP, oyster-extract by-product powder; SUN, urea
nitrogen; TG, triacylglycerol.

― 21 ―

ous studies suggested that the extent of the decrease inserum and liver cholesterol depends on the extent of fecalcholesterol and bile acids excretion

5, 21）. Therefore, we ana-lyzed the eﬀect of dietary OEBP on cholesterol and bile
acids absorption by determining the total amount of cholesterol and bile acids in feces. Table 6 shows the fecal dry
weight, cholesterol, bile acids, and nitrogen excretion inrats fed on control and OEBP diets. The fecal dry weightin the OEBP group tended to be higher than that in thecontrol group. The fecal cholesterol and bile acids contents
were significant higher in the OEBP group than in thecontrol group. The enhancement of fecal cholesterol excretion by OEBP administration was partly inﬂuenced by thecholesterol content of the OEBP diet. Based on the fecal
steroids excretion, the decreased liver cholesterol content

Fig. 2 Course of protein digestibility of control (◆) and OEBP (■)
diets using pepsin (added at time 0 min) and pancreatin(added at time 180 min).
Each point represents means ± SD (n = 3). Values are statistically different compared with the control diet at* < 0.05 using Studentʼs -test.

in rats fed the OEBP diet was in part as a result of theenhancement of fecal cholesterol and bile acids excretion.

gestibility of control and OEBP diets using andi-In this study, the OEBP diet increased fecal nitrogengestion model. The OEBP diet had a signiﬁcantly higher

excretion compared with the control diet. Previous studies

contained insoluble sediment compared with the control

showed that dietary soybean, buckwheat, and ﬁsh protein

diet (Control and OEBP: 16.0 ± 0.33 and 19.4 ± 0.45, respec-

simultaneously increased fecal steroids and nitrogen con-tively). Fig. 2 shows the time course of protein digestibility

tents; moreover, positive correlation has been observedof the control and OEBP diets measured using

between fecal steroids and nitrogen contents

5, 22, 23）di-. In thisgestion. The protein digestibility of the control and OEBP

study, the fecal cholesterol content correlated positively

diets with pepsin was very high in the ﬁrst 10 min, afterwith the fecal nitrogen content, and a signiﬁcant correla-that hydrolysis proceeded more slowly, and at 180 min,tion between fecal bile acids and nitrogen was also ob-

the protein digestibility of the OEBP diet was signiﬁcantly

served (Fig. 1-A and 1-B). Therefore, the eﬀect of OEBP

lower than that of the control diet. During pancreatin di-on the liver cholesterol content in partly due to reﬂect thegestion, the protein digestibility of the control and OEBP

enhancement of fecal cholesterol and bile acids excretion

diets were dramatically increased, and the protein digest-owing to the increased fecal nitrogen content.

ibility of the OEBP diet was signiﬁcantly lower compared

To clarify the enhancement of fecal nitrogen content by

with that of the control diet from 210 min to 360 min. The

the OEBP diet, we determined the production rate of in-high production rate of insoluble sediment weight and thesoluble sediment weight and the time course of protein di-

low protein digestibility of OEBP using thisdiges-

Fig. 1 Correlation between fecal cholesterol and nitrogen contents (A), fecal bile acids and nitrogen contents (B) in the control (◆) andOEBP (■) group. The values were signiﬁcantly diﬀerent ( < 0.05), as determined by simple correlations.
― 22 ―

Referencestion model and the increased fecal nitrogen excretion in
OEBP group were probably caused by the low digestibility of OEBP. From these results, OEBP contains resistant1）Kagawa Y, Nishizawa M, Suzuki M, Miyatake T,

proteins that are undigested remnants of dietary protein,

Hamamoto T, Goto K, Motonaga E, Izumikawa H, Hirata

24）

which have the same function of ﬁber in the intestine . A

H, Ebihara A (1982) Eicosapolyenoic acids of serum

previous study suggested that a high-molecular-weightlipids of Japanese islanders with low incidence of car-fraction of dietary soybean, which has 25 ‑ 30 % indigestible

diovascular diseases. J Nutr Sci Vitaminol (Tokyo)

remnants, decreased serum and liver cholesterol contents

28 : 441‑453.

owing to its enhancement of fecal cholesterol and bile acids

2）Bang HO, Dyerberg J, Sinclair HM (1980) The compo-

24）

excretion . Because digestion-resistant proteins in OEBP

sition of the Eskimo food in north western Greenland.

inhibit cholesterol and bile acids absorption in the intestine,

Am J Clin Nutr 33 : 2657‑2661.

this results in a decrease in the liver cholesterol content.

3）Gopinath B, Buyken AE, Flood VM, Empson M,

However, in this study dietary OEBP did not aﬀect the se-

Rochtchina E, Mitchell P (2011) Consumption of poly-rum cholesterol content despite the increase in fecal cho-unsaturated fatty acids, ﬁsh, and nuts and risk of in-lesterol and bile acids excretion. We estimated that the in-flammatory disease mortality. Am J Clin Nutr 93 :crease in the serum cholesterol content associated with the1073‑1079.

OEBP diet contained approximately 118 mg/kg cholesterol4）Balk EM, Lichtenstein AH, Chung M, Kupelnick B,

and liver cholesterol homeostasis. Further research is re-

Chew P, Lau J (2006) Eﬀects of omega-3 fatty acids onquired to clarify this issue.

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Our previous studies suggested that dietary phospholipids (18 g/kg) decreased the serum cholesterol and liver5）Hosomi R, Fukunaga K, Arai H, Kanda S, Toshimasa N,

contents through the enhancement of fecal cholesterol ex-

Yoshida M (2011) Fish protein decreases serum cho-cretion compared with a diet containing soybean oil alone

lesterol in rats by inhibition of cholesterol and bile

25）

acid absorption. J Food Sci 76 : H116‑H121.

(AIN-93G) . The OEBP diet contained 8.4 g/kg phospholipids, thus there is a possibility that phospholipids in the6）Boukortt FO, Girard A, Prost JL, Ait-Yahia D,

OEBP diet affected cholesterol metabolism. Further re-Bouchenak M, Belleville J (2004) Fish protein im-

search is necessary to clarify the eﬀects of phospholipids

proves the total antioxidant status of streptozotocin-

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In conclusion, this study showed that OEBP decreased
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R, Uchida M (2004) Fish protein stimulated the ﬁbri-

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ever, in terms of the aim of developing lipid-lowering functional foods or supplements, the current formulation of9）Hosomi R, Matsuda Y, Ishimaru A, Takemura S,OEBP has not the lowering eﬀect of serum lipid contents.

Fukunaga K, Yoshida M (2007) Eﬀect of dietary oys-

We concluded that OEBP requires further improvement

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