The effects of soybean protein-derived hydrolysate on lipid metabolism in rats fed a high cholesterol diet

THE

EFFECTS

OF

SOYBEAN

PROTEIN-DERIVED

HYDROLYSATE ON

LIPID METABOLISM IN RATS FED A

H3GH CHOLESTEROL DIET

JIUN-RONG CHEN, SHIAU-FANG CHIOU, MING-ER SHIEH

and SUH-CHING YANG'

Department of Nutrition and Health Sciences

Taipei Medical University

Taipei 110. Taiwan

Received for Publication January 2,2001

Accepted for Publication March 18,2002

ABSTRACT

This study was pe$ormed to investigate the effects of the peptic undigested fraction derivedfrom soybean protein hydrolysate (UDSP) on lipid metabolism in

rats fed a cholesterol-enriched diet (I %). Eighteen male Wistar rats weighing

205-235 g were randomly divided into three groups: the control group (20%

casein), U2 (18% casein + 2% UDSP), and

U5

group (15% casein + 5% UDSP).

Aper 4 weeks, rats were sacrificed, and the lipid profiles of the plasma, liver, and feces were determined. Body weight gain, daily food intake, and liver weight showed no direrences among the groups, but the fieding Mciency ratio in the US

group was significantly lower than that in the other groups (P < 0.05). There were

no changes in plasma cholesterol, LDL-C, HDL-C, and liver cholesterol levels in

each group. However, the

US

group showed a significantly lower VLDL-C

compared to the control and U2 groups. In addition, the plasma and liver TG

content were lower in the U2 and US groups than in the control group (I< 0.05). '

Moreover, the fecal bile acid and total neutral steroid excretions were higher in the

U2 and US groups (P < 0.05) compared to the control group.

'Corresponding author. Fax +886-2-2737-3112; E-mail: [email protected] Journal of Food Biochemistry 26 (2002) 43 1-442. All Rights Resewed.

432 J.-R. CHEN, S.-F. CHIOU, M.-J. SHIEH and S.-C. YANG

INTRODUCTION

Hypercholesterolemia is a major risk factor in cardiovascular disease while maintaining the blood lipids level within normal range can reduce the risk of having cardiovascular disease. Animal proteins such as casein are more hypercholesterolemic than are soybean or other plant proteins (Carroll and Kurowska 1995; Bakhit et al. 1994). Many studies have reported that soybeans have hypocholesterolemic effects, and that soybean protein is one of the effective components (Potter 1998, 1995; Kritchevsky et al. 1987; Jacques et al. 1986). In the Asian dietary pattern, soybean is a traditional food, containing a lot of protein and healthy ingredients, such as dietary fiber, saponin, isoflavone, etc. Recently, soybeans have begun to be used as a health food in Western countries. There are a number of biologically active compounds associated with soybean protein (Merz- Demlow et al. 2000; Crouse et al. 1999); however, the precise mechanism and components of soybean protein have not been fully established. Some studies suggest that, when soybean protein is consumed, cholesterol absorption or bile acid reabsorption, or both, may be impaired (Damascene et al. 2001 ; Nagata et al. 198 1, 1980). As to aspects of digestion, protein is digested to peptides or amino acids in the gastrointestinal tract; thus, the difference in amino acid composition of dietary protein has been considered to be one of the regulatory factors for lipid metabolism in the body. Other workers also reported that the high Arg/Lys ratio of soybean protein lowers serum cholesterol levels (Katan et al. 1982; Sugano et al. 1982). Additionally, West et al. (1983) suggested that the tertiary structure of dietary protein plays an important role in maintaining the plasma cholesterol level.

Soybean protein can be hydrolyzed in vitro by proteases to two parts: a digested fraction containing amino acids and short-chain peptides (low molecular weight fraction) and undigested fraction containing long-chain peptides (high molecular weight fraction). Yashiro et al. (1985) suggested that the various hydrolysates of soybean protein could have different effects on cholesterol metabolism. Moreover, Sugano et al. (1990, 1988) suggested that the undigested high molecule weight fraction of soybean protein digested with endo- and exo-type microbial proteases significantly decreased the plasma cholesterol level. In a review of the research history of the interrelation between dietary protein and cholesterol, Kritchevsky (1995) suggested that early studies were nutritionally naive and poorly detailed, but they still provided similar results to those obtained by today’s studies. He also pointed out that all the early studies and most of the recent ones were conducted using only a single source of protein in the various diets.

Thus,

we thought that it would be important to investigate the hypocholesterolemic reaction to diets containing various protein sources or even protein hydrolysate. In this study, we utilized pepsin, a protease in the stomach, to digest soybean protein, and then distinguished the peptic undigested fraction. Furthermore, we replaced the same

SOYBEAN PROTEIN DERIVED HYDROLYSATE ON LIPID METABOLISM 433

percentage of casein with peptic undigested fraction from soybean protein hydrolysate on>SP), and examined the effects of a mixed casein-UDSP diet on lipid metabolism in rats.

MATERIALS

AND

METHODS

Purification of UDSP from Soybeans

Soybean acid-precipitated protein (AF'P) was prepared fiom GZycine max using the method of Iwabuchi and Yamauchi (1987). Ten grams of

APP

was dissolved in 300 mL of deionized water. The pH of the solution was adjusted to 2.0 with 2

N HC1, and 30 mg of pepsin (from porcine gastric mucosa, EC.3.4.23.1, Merck, Germany) was added. After 24 h of digestion at 37C, the hydrolysates were neutralized and dialyzed against water for 36 h. The undigested fraction derived from soybean protein hydrolysate (UDSP) was lyophilized and stored at 4C until use. The crude protein containing UDSP was determined using the Kjeldahl method.

Animals, Diets, and Experimental Design

Four-week-old male Wistar rats (National Laboratory Animal Breeding and Research Center, National Science Council, Taiwan) were used in this study.

Rats

were individually housed in a room maintained at 23 f 2C with 50%-70% humidity and a 12-h lightldark cycle. Rats were allowed free access to a standard rat chow diet for 1 week before the start of the experiment. Eighteen rats weighmg fiom 205 to 235 g were randomly divided into three groups (6 rats each) according to the UDSP level with isocaloric diet: control (20% casein),

U2

(1 8% casein + 2% UDSP), and U5 (1 5% casein + 5% UDSP). The composition of the experimental diet was modified according to AIN-76 and is shown in Table 1.

Rats

were fed the experimental diets for 4 weeks ad libitum. Experimental diets were changed at the same time everyday. Food intake was measured daily, and body weights were measured once a week. Blood was collected

fiom

tail vein of rats weekly for lipid analysis. At the end of the feeding period, rats were fasted for 6 h (0800-1400), blood was collected, and the liver was perfused with cold normal saline before excision and stored at -7OC until analysis.

Lipid Analysis

Serum total cholesterol and triacylglycerol concentrations were assayed enzymatically with the method described by Richmond (1973) and Mcgowan et al.

(1983), respectively. Liver lipids were extracted by the method of Folch et al. (1957). Cholesterol and triacylglycerol concentrations in the liver were determined

434 Group Ingredient' Casein UDSI" Corn starch Sucrose Mineral mixture Cellulose Vitamin mixture Cholesterol Choline bitartrate Energy ( W g ) Soybean oil

J.-R. CHEN, S.-F. CHIOU, M.-J. SHIEH and S.-C. YANG

Control u2

u5

20.0 18.0 15.0 0.0 2.0 5.0 50.0 50.0 50.0 11.9 11.9 11.9 10.0 10.0 10.0 4.0 4.0 4.0 2.0 2.0 2.0 1.0 1.0 1.0 1

.o

1

.o

1.0 0.1 0.1 0.1 17.75 17.75 17.75 TABLE 1.

COMPOSITION OF THE EXPERIMENTAL DIETS (%)

*Casein (high nitrogen), sucrose (food grade), soybean oil, mineral mixture (AN-76 mineral mixture), cellulose (nonnulritive bulk), and vitamin mixture (AM-76 vitamin mixture) were obtained from ICN Biochemicals, Inc., CA. Com starch was purchased from Samyang Genex Cop. (Seoul, Korea). Cholesterol and choline bitartrate were obtained from Sigma (St. Louis, MO).

'UDSP: peptic undigested fraction derived from soybean protein hydrolysate.

with diagnostic kits (Randox, UK) with cholesterol and glycerol as standards, respectively.

Fecal Steroid Analysis

Feces were collected 2 dayslweek during the experiment, and were freeze-dried for 48 h and stored at -7OC until use. Bile acids and neutral steroids were separated from feces according to the method of Folch et al. (1957) and measured with commercial kits (Randox, UK).

Statistical Analysis

Data are expressed as mean f SD. Differences among groups were analyzed by one-way analysis of variance test using SAS computer program. A P value of less than 0.05 was considered statistically significant.

SOYBEAN PROTEIN DERIVED HYDROLYSATE ON LIPID METABOLISM 435

RESULTS

Body Weight Gain, Food Intake, Feeding Efficiency, and Liver Weight The data are shown in Table 2. The initial body weights of rats were 205-235 g, and the final body weights were 330-370 g after a 4-week experimental period. The final body weights of the experimental groups were sigdicantly lower than the control group. Furthermore, food intakes and liver weights were not significantly different. But, the feeding efficiency ratio in the US group was significantly lower than that of the control and U2 groups (P C 0.05).

Plasma Lipid Profile and Lipoprotein Cholesterol Concentration

Plasma TG levels in the U2 and U5 groups were lower than those of the control group (Table 3). Further, rats in the US group had lower plasma VLDL-C than the control and U2 groups. In addition, there were no differences in plasma cholesterol, LDL-C, and HDL-C concentrations among all groups.

Liver Lipid Profile

The data for liver lipid analyses are also presented in Table 3. The Iiver cholesterol levels did not W e r among the groups. However, liver TG levels in the U5 group were significantly lower than the control group. The whole liver cholesterol and TG contents was lower in the U5 group.

Total Neutral Steroids and Bile Acids in Feces

The data are shown in Table 4. Total neutral steroids excretion in experiment groups

was

significantly

higher

than those of control group. In addition, bile acid excretions were increased in the U2 and US groups compared to that in control groups (P < 0.05).

DISCUSSION

In this study,

UDSP

was used to replace casein in the experimental diet, and lipid metabolism was studied in rats fed a cholesterol-enriched diet. The present results show that the body weight gain and feeding efficiency were significantly lower in the U5

than

in the other groups. We thought that the differences of feeding efficiency resulted from the digestibility of protein in the diet. The crude protein contents of casein and UDSP in this study were 87% and 92%, respectively.

Yashino

et al. (1985) and Sugano et al. (1990,1988) indicated that weight gain and

TABLE 2. CHANGES IN BODY WEIGHT, DAILY FOOD INTAKE, FEED EFFICIENCY, AND LIVER WEIGHT OF RATS FED DIFFERENT DIETS' Diet group Control u2 u5 Initial body weight (g/rat) 227.8 f: 7.4 215.5 k 4.6 216.8 f 2.1 Final body weight (g/rat) 362.0

k

15.1a 341.9 ? 13.3 b 335.6 f 15.7b Weight gain (g/day/rat) 4.8 _+ 0.3 4.5 k 0.2 4.2

f

0.2 Food intake

(g/

day/ rat) 18.0 f 2.0 17.8

f

3.6 18.6 f 2.4 Feeding efficiency 2 (%) 26.6

k

2.0" 25.4

f

1.1 a 22.6 f 0.9b Liver weight (g/rat) 16.9 f 0.7 15.2 f 1.0 14.6 f 0.5 Hepatosomatic index 3 4.7 k 0.2 4.4

i

0.2 4.4 It: 0.2 'Data are expressed as mean f SD (n = 6); values in a row with different superscript letters significantly differ (P < 0.05) as analyzed by one-way analysis of variance 'Feeding efficiency: (daily weight gainldaily food intake) x 100%. 'Heuatosomatic index: (liver weiehtibodv weieht) x 100%. test.

TABLE 3. CHANGES IN PLASMA AND LIVER LIPIDS LEVELS OF RATS FED DIFFERENT DIETS * 4 Diet group W

2

3

Cholesterol 2.52

f

0.25 2.58 2 0.31 2.61

f

0.28

E!

B

Control u2 u5

E

Plasma (mmol/L) U Trig1 yceride 1.25

f

0.218 0.65

f

0.34b 0.73

f

0.32b VLDL-C LDL-C HDL-C Liver Liver weight (g/ rat)

e

0.78

f

0.23" 0.71 f 0.19" 0.58 f 0.15b 0.72 2 0.14 0.69 2 0.11 0.64 f 0.12

2

0.59 k 0.09 0.68

f

0.13 0.73

f

0.14 16.9

f

0.7

s

15.2

f

1.0 14.6

f

0.5 4

G

1747.6

t

205.1 a 1524.8 5 186.6ab 1468.0 5 257.5b 5

&

Cholesterol (pmol/g liver) 103.7

f

13.3 100.4 2 19.3 100.6

f

15.9 Total cholesterol (pmol) Triglyceride (pmollg liver) 3.9

f

0.43 3.7 5 0.4" 3.3

f

0.3b

5

Total triglyceride (pmol) 66.9

f

7.1" 55.7

f

6.8ab 47.5

f

4.6b

@

z

?;

*

Data are expressed as mean f SD (n = 6); values in a row with different superscript letters significantly differ (P < 0.05) as analyzed by one-way analysis of variance~test. P W 4

P W m TABLE 4. CHANGES IN TOTAL NEUTRAL STEROIDS AND TOTAL BILE ACIDS IN FECES OF RATS FED DIFFERENT DIETS' ~~ Diet group Control u2 u5 Total neutral steroids (pmol/day) 228.9

f

12.5a 258.7

t

8.5b 292.9

t

16.1 c Total bile acids (pmol/day) 10.3

t

0.2a 11.0

f

0.2b 11.2 2 0.2b 'Data are expressed as mean f SD (n = 6); values in a row with different superscript letters significantly differ (P < 0.05) as analyzed by one-way analysis of variance test. m

h

SOYBEAN PROTEIN DERIVED HYDROLYSATE ON LIPID METABOLISM 439

food intake showed no changes in rats fed an undigested fraction of soybean protein. However, feeding efficiency or PER was not calculated in those studies.

In addition, it was suggested that soybean protein or the hydrolysate product was not digested in stomach, but digestion occurs in the small intestine, and was absorbed before it reached the colon (Iwami et al. 1986). From this report, we consider that the difference in feeding efficiency is not attributable to the indigestible soybean protein. However, in this study, the final body weights were sigdicantly lower in the experimental groups, this might be explained by the low utilization of UDSP.

The plasma TG was markedly lower in rats fed 2% UDSP. Additionally, plasma TG and VLDL-C levels showed differences in the

U5

group. We suspect that the hypolipidemic reaction might be due to a concentration of UDSP in the experimental diet. In

this

study, fecal excretions of neutral steroids and bile acids were increased by means of UDSP ingestion. In other related studies, a similar phenomenon was also observed, when pepsin was used as a protease (Iwami et al.

1990; Doi et al. 1986). In contrast, neutral and acidic steroids increased in the feces

when rats ingested the undigested fraction derived from soybean protein after microbial protease, tqpsin or Pronase E dialyzation (Tanaka and Nozaki 1983; Huff et al. 1977).

In the present study, the peptic undigested fraction in the soybean protein have an effect on fecal excretions of steroids or bile acids which may consequently influence cholesterol metabolism. The weight of feces in the UDSP group was higher

than

that of the control group (data not shown). The total neutral steroids and bile acids of the feces in the rats fed with UDSP diet were higher than rats fed with control diet.

The plasma or liver TG concentration was significantly lower in rats after ingesting 2% (only for plasma TG) or 5% UDSP.

The

triglyceride-lowering effects of soybean protein or its hydrolysate on liver are ubiquitous not only in animals but also in human studies. Intani et al. (1988) indicated that plasma TG varied as a consequence of dietary patterns. He also pointed out that a deficiency or imbalance of specific amino acids in the diet could &crease the plasma TG level due to lowering of VLDL synthesis in the liver.

Studies citing the protective effects of soybean protein for cardiovascular disease have been in the literature since the 1940s. This effect seems to be mediated in large part by effects on plasma cholesterol and TG (Anderson et al. 1995). However, it is unfeasible to intake daily protein source from only one type of food. Furthermore, Huff and Carroll (1980) indicated that hypercholesterolemia could be improved by replacing 25% casein from total protein with soybean protein. The hypolipidemic components of soybean protein have not been identified. Sugano et

al. (1984) reported that only the soybean protein hydrolysate with the molecular

weight of more

than

5000 Da had the hypolipidemic effect. Additionally, Iwami

et al. (1990) indicated that the hydrophobic character of the protein was related to

440 .I.-R. CHEN, S.-F. CHIOU, M.-J. SHIEH and S.-C. YANG

easy to hydrolyze the hydrophobic part of protein, the high molecular weight protein particle remained intact. The hydrophobic domain of the high molecular weight protein particle could combine with cholesterol and bile acid further to decrease the absorption of cholesterol and bile acids.

In summary, diets supplemented with 5% UDSP had a hypocholesterolemic effect in this study. In addition, an apparent TG-lowering action was observed. Further experiments are needed to investigate the hypolipidemic reaction when the amount of UDSP replacer in the experimental diet is increased. Furthermore, because the effects of soybean protein hydrolysate on human cholesterol metabolism are variable and more moderate than that in experimental animals, human studies will be necessary. In conclusion, it is impossible to have only one type of protein source in our dietary patterns. Therefore, the search for an adaptive ratio of animal and plant proteins is important for improving health.

REFERENCES

ANDERSON, J.W., JOHNSTONE, B.M. and COOK-NEWELL, M.E. 1995. Meta-analysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 333, 276-282.

and POTTER, S.M. 1994. Intake of 25 g of soybean protein with or without soybean fiber alters plasma lipids in men with elevated cholesterol concentrations. J. Nutri. 124,213-222.

CARROLL, K.K. and KUROWSKA, E.M. 1995. Soy consumption and cholesterol reduction: review of animal and human studies. J. Nutri. 125, 594-597. CROUSE, J.R., MORGAN, T.M., TERRY, J.G., ELLIS, J., VITOLINS, M. and

BURKE, G.L. 1999. A randomized trail comparing the effect of casein with that of soy protein containing varying amounts of isoflavones on plasma concentrations of lipids and lipoproteins. Arch. Intern. Med. 159,2070-2076. DAMASCENO, N.R., GIDLAND, M.A., GOTO, H., DIAS, C.T., OKAWABATA,

F.S. and ABDALLA, D.S. 2001. Casein and soy protein isolate in experimental atherosclerosis: influence on hyperlipidemia and lipoprotein oxidation. Ann. Nutr. Metab. 45,38-46.

DOI, H., IWAMI, K., IBUKI, F. and KANAMORI, Mi 1986. Effect of feeding peptic digest of soy protein isolate on rat serum cholesterol. J. Nutr. Sci. Vitaminol. 32,373-379.

FOLCH, J., LEES, J.M. and SOLANE-STANLEY, G.H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226,497-509.

HUFF, M.W. and CARROLL, K.K. 1980. Effects of dietary proteins and amino acid mixtures on plasma cholesterol levels in rabbits.

J. Nub.

11 0, 1676- 1685. BAKHIT, R.M., KLEIN, B.P., ESSEX-SOFUIE, D., HAM, J.O., ERDMAN, J.W.

SOYBEAN PROTEIN DERIVED HYDROLYSATE ON LIPID METABOLISM 441

HUFF, M.W., HAMILTON, R.M.G. and CARROLL, K.K. 1977. Plasma cholesterol levels in rabbits fed low fat, cholesterol-fiee semipurified diets: effects of dietary proteins, protein hydrolysates and amino acid mixture. Atherosclerosis 28, 187-195.

IRITANI, N., SUGA, A., FUKUDA, H., KATSURADA, A. and TANAKA, T.

1988. Effects of dietary casein and soybean protein on triglyceride turnover in

rat liver. J. Nutr. Sci. Vitaminol. 34, 309-315.

IWABUCHI, S. and YAMAUCHI, F. 1987. Determination of glycinin and

9-

conglycinin in soybean proteins by immunological methods. J. Agric. Food Chem. 35,200-205.

I W M , K., KITAGAWA, M. and IBUKI, F. 1990. Effect of dietary proteins andlor their digestive products on intestinal taurocholate absorption. J. Nu@. Sci. Vitaminol. 36, 141-146.

IWAMI, K. SAKAKIBARA, K. and IBUKI, K. 1986. Involvement of post- digestion ‘hydrophobic’ peptides in plasma cholesterol-lowering effect of dietary plant protein. Agric. Biol. Chem. 50, 1217-1222.

JACQUES, H., DESHAIES, Y. and SAVOIE, L. 1986. Relationship between dietary proteins, their in vitro digestion products, and serum cholesterol in rats. Atherosclerosis 61, 89-98.

KATAN, M. B., VROOA4J3N, L. and H E W S , R. J. J. 1982. Reduction of casein- induced hypercholesterolemic and atherosclerosis in rabbits and rats

by

dietary glycine, arginine and alanine. Atherosclerosis 43,38 1-391.

KRITCHEVSKY, D. 1995. Dietary protein, cholesterol and atherosclerosis: A review of the early history.

J.

Nutr. 125,589-593.

KRITCHEVSKY, D., TEPPER, S.A. and KLURFELD, D.K. 1987. Dietary protein and atherosclerosis. J. Am. Oil Chem. SOC. 604, 1 167- 1 17 1.

McGOWAN, M.W.,

ARTISS,

J.D., STRANBDERGH, D.R. and ZAK, B. 1983. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin. Chem. 29,538-542.

PHIPPS, W.R. and KURZER, M.S. 2000. Soy isoflavones improve plasma lipids in normocholesterolemic, premenopausal women. Am. J. Clin. Nutr. 71, NAGATA, Y., JMALZUMI, K. and SUGANO, M. 1980. Effects of soybean protein

and casein on serum cholesterol levels in rats. Br. J. Nutr. 44, 1 13- 12 1. NAGATA, Y., TANAKA, K. and SUGANO, M. 1981. Further studies on the

hypocholesterolemic effect of soya-bean protein in rats. Br. J. Nu&. 45,233- 241.

POTTER, S.M. 1995. Overview of proposed mechanisms for the hypocholesterolemic effect of soy. J. Nutri. 125,6063-61 1s.

POTTER, S.M. 1998. Soy protein and cardiovascular disease: the impact of bioactive components in soy. Nutri. Rev. 56,231-235.

MERZ-DEMLOW, B.E., DUNCAN, A.M., WANGEN, K.E., XU,

X.,

CARR, T.P.,

442 J.-R. CHEN, S.-F. CHIOU, M.-J. SHIEH and S.C. YANG

RICHMOND, W. 1973. Preparation and properties of a cholesterol oxidase from

Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin. Chem. 19, 1350-1356.

SUGANO, M., GOTO, S., YAMADA, Y., YOSHIDA, K., HASHIMOTO, Y., MATSUO, T. and KIMOTO, M. 1990. Cholesterol-lowering activity of various undigested fractions of soybean protein in rats. J. Nutr. 120,977-985. SUGANO, M., ISHIWAKI, N., NAGATA, Y. and IMAIZUMI, K. 1982. Effects of arginine amd lysine addition to casein and soya-bean protein on serum

lipids, apolipoproteins, insulin, and glucagon in rats. Br. J. Nutr. 48,211-221. SUGANO, M., ISHIWAKI, N. and NAKASHIMA, K. 1984. Dietary protein-dependent modification of serum cholesterol level in rats. Ann. Nutr. Metab. 28, 192-199.

SUGANO, M., YAMADA, Y., YOSHIDA, K., HASHIMOTO, Y., MATSUO, T. and KIMOTO, M. 1988. The hypocholesterolemic action of the undigested fraction of soybean protein in rats. Atherosclerosis 72, 115-122.

TANAKA, C. and NOZAKI, Y. 1983. Effect of partial hydrolysates of casein and soybean protein on serum lipoproteins and fecal neutral steroids. J. Nutr. Sci. Vitaminol. 29,439-446.

WEST, C.E., BEYNEN, A.C., TERPSTRA, A.H.M., SCHOLZ, K. E., CARROLL, K.K. and WOODWARD, C.J.M. 1983. The nature of dietary protein and serum cholesterol. Atherosclerosis 46,253-256.

YASHIRO, A., ODA, S. and SUGANO, M. 1985. Hypocholesterol effect of soybean protein in rats and mice after peptic digestion. J. Nutr. 115, 1325-