J Nutr Sci Vitaminol, 62, 162–169, 2016

Okara, a By-Product of Manufacturing, Modifies Triglyceride Metabolism at the Intestinal and Hepatic Levels

Yasuo Nagata1,2, Shiho Yamasaki1, Norihiro Torisu1, Taishi Suzuki1, Saya Shimamoto1, Shizuka Tamaru1 and Kazunari Tanaka1

1 Department of Nutrition, University of Nagasaki, Siebold, 1–1–1 Manabino, Nagayo-cho, Nishisonogi-gun, Nagasaki 859–2195, Japan 2 Center for Industry, University and Government Cooperation, Nagasaki University, 1–14 Bunkyo-machi, Nagasaki 852–8521, Japan (Received August 21, 2015)

Summary Irrespective of a well-known hypocholesterolemic action, a few studies have shown a hypotriglyceridemic potential of , a by-product of tofu manufacturing. Okara was fed to rats at the level of 2.5 and 5.0% as dietary protein for 4 wk, and serum and hepatic lipid levels were determined. In addition, soy flour, which has a well-known hypolip- idemic action, was used to compare effects on lipid metabolism. Mechanisms of action were further evaluated by measuring hepatic enzyme activity, gene expression of lipid metabo- lism-related proteins and fecal excretion of lipids. Feeding the okara diets resulted in a sig- nificantly lower weight of the liver and adipose tissue in a dose-dependent manner. Serum triglyceride levels were more than 50% lower in rats fed the okara diets compared to those fed the control diet. Enzyme activities of fatty acid synthesis were significantly lowered by the okara diet. Fecal weight was significantly higher in the okara group than in the control group, and fecal excretion of steroids tended to be higher. Therefore, a relatively low amount of okara may exert hypotriglyceridemic action in rats in part through decreased hepatic triglyceride synthesis. The present study also suggests an involvement of intestinal events in altered lipid metabolism in rats fed the okara diets. Key Words okara, hypotriglyceridemic, lipogenesis, fecal weight, dietary fiber

Over centuries, soy has been traditionally used in var- emic (15). Furthermore, okara has which are ious ways in Asia, ranging from tofu to fermented foods known to have hypolipidemic, antioxidant and antican- such as natto and (1). It has been known that soy cer activities (2, 3). For these reasons, okara is thought and soy ingredients including protein, fiber and isofla- to be a useful foodstuff to deliver health benefits. vones have various functions such as anticancer, anti- Using animals, studies have mainly focused on the oxidant, anti-inflammatory and hypolipidemic activi- hypocholesterolemic action of okara. Rats fed a 10% ties (2). Epidemiological and experimental studies have dietary fiber-rich okara diet for 4 wk showed lower total shown the relationship between soy intake and various cholesterol levels compared to those fed the control diet types of diseases including cardiovascular disease (3), (16). Villanueva et al. supplemented diets with okara coronary heart disease (4), cancer (5, 6), diabetes (7) containing 13% or 20% of dietary fiber and also found and bone fracture (8). The FDA has approved a health that okara reduced total plasma lipids and total choles- claim on and coronary heart disease preven- terol in high fat-fed Syrian hamsters (17). When okara tion (9). In addition to the health benefits of soy and soy and okara tempe, a fermented okara in Indonesia, were foods, inclusion of soy foods in daily meals may provide given to rats, plasma cholesterol levels were lower com- minerals such as calcium and magnesium, and reduce pared to those of a casein-based control diet group, indi- saturated fat and cholesterol (10). cating that dietary fiber and protein in okara are respon- Okara is a by-product of tofu manufacturing and a sible for reduced cholesterol levels (15). Matsumoto et rich source of protein and dietary fiber. Dietary fiber in al. also have shown that okara induces hypolipidemic okara accounts for ~50% on a dry matter basis with action in rats fed a dried okara-supplemented diet (18). hemicellulose being a major component (11). Various In that study, mRNA of CYP7A1, a key enzyme convert- beneficial effects of dietary water-soluble fiber have been ing cholesterol to bile acid, was up-regulated by okara reported, including body weight reduction, and hypogly- feeding, suggesting that okara may stimulate cholesterol cemic, hypolipidemic and antioxidant activities (12–14). catabolism. In addition, providing 25 g/d of soy fiber Even after manufacturing for tofu, okara con- to hypercholesterolemic patients reduced plasma total tains a relatively high amount of protein (~20% on a cholesterol, but not triglyceride (19). Furthermore, vari- dry matter basis) which is known to be hypocholesterol- ous constituents in soy (e.g. isoflavones) seem likely to contribute to hypocholesterolemic action (20). Collec- E-mail: [email protected] tively, it is suggested that okara, a good source of pro-

162 Okara and Triglyceride Metabolism 163

Table 1. Nutrient composition in the diets (%).

Control Okara (2.5) Okara (5) Soy

Casein 20.0 17.5 15.0 15.0 a-Corn starch 13.2 13.2 13.2 13.2 oil 7.0 7.0 7.0 7.0 Sucrose 10.0 10.0 10.0 10.0 Cellulose 5.0 5.0 5.0 5.0 Mineral mix AIN-93G 3.5 3.5 3.5 3.5 Vitamin mix AIN-93 1.0 1.0 1.0 1.0 t-Butylhydroquinone 0.0014 0.0014 0.0014 0.0014 Choline bitartrate 0.25 0.25 0.25 0.25 L-Cystine 0.30 0.30 0.30 0.30 Sample — 11.3 22.6 13.6 b-Corn starch 39.75 30.95 22.15 31.15 Total (%) 100.0 100.0 100.0 100.0

tein and dietary fiber, has hypocholesterolemic action. protein, carbohydrate, fat, ash, dietary fiber and water However, okara’s hypotriglyceridemic action was found in soy flour was 36.9%, 21.4%, 15.9%, 4.9%, 16.9% only when dietary fiber from okara was high in the diets and 4.0%, respectively, on a dry matter basis. Other (17, 18). Thus, the hypotriglyceridemic effects of okara reagents were of analytical grade and used without fur- remain largely unknown. Given that hypertriglyceride- ther purification. mia is one of the factors affecting the development of Animal experiments. Four-week-old Sprague-Dawley cardiovascular events (21, 22), whether okara has a (SD) rats were purchased from Japan SLC, Inc. (Shizu­ role in the regulation of triglyceride metabolism is thus oka, Japan), individually housed in stainless-steel cages worthy of investigation. In addition, a large quantity of under a controlled atmosphere (temperature, 2261˚C, okara is produced during tofu manufacturing and poses humidity, 5565%, light cycle, 08:00–20:00), and fed a a disposal problem. Okara putrefies quickly because of commercial pellet diet (CE-2, CLEA Japan, Inc., Tokyo) its water activity and high protein content. Thus, inves- for 1 wk. The animal study was carried out under the tigating additional functions of okara would be highly Guidelines for Animal Experiments of University of anticipated for its application as a functional food, and Nagasaki (Nagasaki, Japan), and Law no. 105 and Noti- for the reduction of okara as waste. Thus, our aim was fication no. 6 of the Government of Japan. After 1 wk of to investigate whether okara affects triglyceride metabo- acclimation, the rats were assigned to 4 groups, 6 ani- lism in rats, and, if so, how okara modifies triglyceride mals per group. A control group was given a modified metabolism at the enzyme and molecular levels. Since AIN-93G diet with corn starch as a major source of car- the effects of soy on lipid metabolism have been well bohydrate. As an okara group, okara was added to the documented (23), soy flour that contains a low amount control diet at the level of 2.5 and 5% as dietary protein of dietary fiber and a high amount of protein was used at the expense of corn starch. A soy group was given as a reference in this study. the control diet but with soy flour at the level of 5% as We here show that okara is hypotriglyceridemic. dietary protein. The detailed composition of the diets is Okara reduces the weight of liver and adipose tissue, shown in Table 1. Blood was withdrawn from tail vein in and decreases serum triglyceride levels in part through week 2 after 6 h fasting, and serum was obtained after lowered enzyme activity of fatty acid synthesis in the centrifugation at 1,500 3g for 15 min. Rats were fed liver. This is the first study showing the underlying the diets ad libitum for 4 wk. After 4 wk of feeding, ani- mechanism of okara’s hypotriglyceridemic action. These mals were sacrificed after 6 h fasting. The liver and adi- results may provide a potential application of okara for pose tissue (mesenteric, perirenal and epididymal) were the prevention and treatment of hypertriglyceridemia. dissected and stored at 280˚C prior to further analysis for tissue lipid, enzyme activity or gene expression as MATERIALS AND METHODS described below. Interscapular brown adipose tissue was Chemicals. Dried okara was provided by Kyuichian also removed for determining enzyme activity. The small Company (Oomura, Japan). The level of dietary fiber intestine was collected, and flushed with ice-cold saline, in okara and soy flour was analyzed by Japan Food and the jejunum and ileum sections were isolated. A Research Laboratories using AOAC 985.29. The level of portion of the jejunum was used for RT-PCR analysis. protein, carbohydrate, fat, ash, dietary fiber and water Serum and tissue biochemical analyses. Serum lipid in dried okara was 22.1%, 5.7%, 11.6%, 3.4%, 50.4% levels were assayed enzymatically using commercial kits and 6.6%, respectively, on a dry matter basis. Soy flour (Cholesterol E-Test, Triglyceride E-Test and Phospholipid was prepared from locally-grown soy and purchased C-Test, Wako Pure Chemical Industries, Ltd., Osaka, from Nakajima Mill Co. (Isahaya, Japan). The level of Japan). The liver was weighed, and liver lipids were 164 Nagata Y et al.

Table 2. Effects of 4 wk administration of the okara diets on body weight, food intake and tissue weight in rats.

Control Okara (2.5) Okara (5) Soy

Final body weight (g) 409619 37969 40367 400612 Food intake (g/d) 23.161.0 22.060.4 22.060.3 21.560.5 Liver weight (g/100 g body weight) 3.7560.09b 3.4960.07ab 3.3660.08a 3.7060.14b White adipose tissue weight (g) Perirenal 10.261.8 7.5760.87 5.8460.67 7.8560.72 Epididymal 6.2460.72 5.2160.24 5.0560.31 5.5060.43 Mesenteric 5.3260.51b 3.9660.30ab 3.4560.28a 4.6460.46ab Total 21.862.9b 16.761.4ab 14.461.2a 18.061.6ab White adipose tissue weight (g/100 g body weight) Perirenal 2.4560.32b 1.8960.19ab 1.4560.16a 1.9560.15ab Epididymal 1.5260.14 1.3160.05 1.2560.06 1.3760.08 Mesenteric 1.2960.07c 0.9960.06ab 0.8660.07a 1.1560.09bc Total 5.2660.49b 4.1960.28ab 3.5660.28a 4.4760.31ab Brown adipose tissue weight (g) 0.53060.056b 0.45860.029ab 0.36660.039a 0.46460.028ab Brown adipose tissue weight (g/100 g body weight) 0.12960.01 0.11660.01 0.09160.01 0.11760.01

Data are mean6SE (n56). abc Different superscript letters show significant difference at p,0.05.

Table 3. Effects of 2 and 4 wk administration of the okara diets on serum and hepatic parameters.

Control Okara (2.5) Okara (5) Soy

2 wk serum lipids (mg/dL) Triglyceride 230632b 121611a 112617a 168619ab Cholesterol 98.062.8 85.965.9 77.767.5 96.065.6 Phospholipid 192610b 164611ab 135613a 16468ab 4 wk serum lipids (mg/dL) Triglyceride 200621b 96.6618.5a 94.5616.4a 147632ab Cholesterol 10167 82.065.0 86.168.6 88.265.3 Phospholipid 17565b 133610a 11967a 12968a Serum insulin (ng/mL) 3.8260.12b 3.0860.05a 3.2060.21a 3.1460.10a Hepatic lipids (mg/g liver) Triglyceride 43.962.7 24.363.5 35.668.0 38.465.5 Cholesterol 16.563.2 9.360.9 14.062.1 11.960.9 Phospholipid 17.660.3 17.861.6 18.760.3 17.360.7

Data are mean6SE (n56). ab Different superscript letters show significant difference at p,0.05.

extracted by the method of Folch et al. (24). Hepatic mitochondrial carnitine palmitoyltransferase (CPT) levels of cholesterol, phospholipid and triglyceride were (29) were also determined. Interscapular brown adipose measured as described above. Insulin level was mea- tissue was also homogenized as described above, and sured by an ELISA kit (Morinaga Institute of Biological CPT activity was measured. Protein was assayed by the Science, Yokohama, Japan). method of Lowry et al. (30). Hepatic enzyme activity. To determine how okara Fecal lipid analyses. Feces were collected for 2 d alters triglyceride metabolism, activity of lipid metab- before sacrifice, lyophilized, and ground. A small aliquot olism-related enzymes in the liver and brown adipose of feces was extracted by the method of Ikeda et al. (31) tissue was measured. A small aliquot of the excised and measured for lipid excretion with a gas chromato- liver was homogenized in 6 volumes of 0.25 m sucrose graph. Neutral and acidic steroids were measured by solution containing 10 mm Tris-HCl and 1 mm EDTA 5a-cholestane and 23-nordeoxycholic acid as an inter- (pH 7.4) to obtain isolated hepatic subcellular frac- nal standard, respectively. tions. The activity of cytosol fatty acid synthase (FAS) Analysis of gene expression by RT-PCR. To further (25), glucose-6-phosphate dehydrogenase (G6PDH) determine how okara affects lipid metabolism at the (26) and malic enzyme (ME) (27) was measured. Micro- molecular level, gene expression of enzymes and pro- somal phosphatidate phosphohydrolase (PAP) (28) and teins involved in the synthesis of fatty acid and cho- Okara and Triglyceride Metabolism 165

Fig. 2. Effects of the diets on fecal weight and lipid excretion. Data are mean6SE (n56). a, b, c show a sig- nificant difference at p,0.05.

means6SE. Statistical analyses were performed by one- way ANOVA, followed by the Tukey-Kramer test. Values were considered to be significantly different when the p-value was less than 0.05. RESULTS Fig. 1. Effects of the diets on activities of hepatic enzymes involved in lipogenesis. Data are mean6SE Body weight, tissue weight, and serum and hepatic lipid levels (n56). a, b show a significant difference at p,0.05. After a 4-wk feeding of the experimental diets, no significant difference was found in final body weight or food intake, as shown in Table 2. Liver weight was dose- dependently lower in okara-fed rats with a significant lesterol was analyzed since hepatic very low density difference being observed between the control and 5.0% lipoprotein (VLDL) assembly was regulated by hepato- okara diets. Weight of total adipose tissue and brown cellular triglyceride and cholesterol levels (32). Total adipose tissue was lower in the okara diet group in a RNA was extracted from the liver and the small intes- dose-dependent manner compared to the control diet tine using RNA extraction reagent (RNAiso Plus, Takara group while that of those tissues was not modified by Bio, Shiga, Japan) according to the manufacturer’s the soy diet (Table 2). As shown in Table 3, rats fed the instructions. Total RNA (1 mg) was reverse-transcribed okara diets had significantly lower levels of serum tri- to synthesize cDNA with a reverse-transcription kit glyceride compared to those fed the control diet at 2 wk. (PrimeScript® RT Master Mix, Takara Bio). RT-PCR was The triglyceride level was reduced by .50% by the 5% performed on a 7300 Real-Time PCR system (Applied okara diets. The phospholipid level was significantly Biosystems, Carlsbad, CA) using the manufacturer’s lower in the 5% okara diet group compared to the con- standard protocol. The PCR reaction mixture was pre- trol diet. However, the diets did not provide a significant pared using THUNDERBIRD SYBR qPCR Mix (Toyobo, difference in serum cholesterol levels among the groups Osaka, Japan). The levels of mRNA, measured relative although that in rats fed the okara diets tended to be to 36B4 mRNA levels used as an internal control, were lower. At 4 wk, a similar trend was found. The levels of determined using the 22DDCt method. Differences were serum triglyceride and phospholipid were significantly expressed as the fold-change over the control. Primers lower in okara-fed rats compared to the control diet-fed were designed using Primer Express® Software for Real- animals. The okara and soy diets also gave significantly Time PCR (Applied Biosystems), and the forward and lower insulin levels. However, the serum glucose level reverse primers used were described elsewhere (33). was not changed by the diets (data not shown). The Statistical analysis. All the values are expressed as okara diets showed lower hepatic cholesterol and triglyc- 166 Nagata Y et al.

Fig. 3. Effects of the diets on hepatic and intestinal mRNA expression of lipid metabolism-related proteins. Data are mean6SE (n56). Differences in mRNA expression were calculated after normalization to expression of 36B4 mRNA using the 22DDCt method and expressed as the fold-change over the control. ACAT, acyl-coenzyme A:cholesterol acyltransferase; CD36, fatty acid translocase; FAS, fatty acid synthase; HMG-CoAR, 3-hydroxyl-3-methylglutaryl-coenzyme A reductase; NPC1L1, Niemann-Pick C1-Like 1; SREBP-1c, sterol regulatory element binding protein-1c. eride levels without a significant difference. target gene of SREBP-1c, tended to be lower in rats fed Enzyme activity the okara diets without a significant difference. Gene To see how okara decreases the serum triglycer- expression of other proteins involved in intestinal and ide level, activities of enzymes involved in lipogenesis hepatic lipid trafficking was not significantly modified were determined in the liver and brown adipose tissue. after the 4-wk administration of the okara diets. As illustrated in Fig. 1, the okara diets significantly DISCUSSION decreased fatty acid synthesis-related enzymes, FAS, ME and G6PDH. These enzyme activities were dose-depen­ Okara has been known to be hypocholesterolemic dently affected by the okara diets. The soy diet also sig- (15, 16), while its hypotriglyceridemic action has not nificantly lowered these enzyme activities as much as been well determined. Our current study clearly showed the 5% okara diet did. However, CPT, the rate-limiting that okara has hypotriglyceridemic potential. enzyme in fatty acid oxidation, was not altered by the As shown in Table 3, the decrease in serum triglyceride diets either in the liver or brown adipose tissue. in the 5% okara diet-fed rats was more than 50% with Fecal excretion of lipids respect to control animals, whereas that in the liver was To see how the okara diet affects the intestinal func- moderate. A few studies have thus far shown the hypo- tion, fecal weight and fecal steroid excretion were mea- triglyceridemic action of okara. Okara exerted hypotri- sured (Fig. 2). Fecal weight was significantly and dose- glyceridemic action in Syrian hamsters only when added dependently increased by the okara diets. Rats fed the to diets at a high level as dietary fiber (20%), but not at okara diets tended to have higher fecal excretion of a lower level (13%) (17). A similar triglyceride-lowering acidic and total steroids (the 5% diet group vs the con- action was observed in mice fed the 20.5% okara fiber trol diet group, p50.1). However, fecal excretion of neu- diet, but not in those fed the 13% okara fiber diet (18). tral steroids was not influenced by the diets. In contrast, Lemes et al. did not observe hypotriglyceri- Gene expression of proteins related to lipid metabolism demic action of the okara diet containing ~16% dietary To get an insight into how lipid metabolism altered by fiber in rats irrespective of its hypocholesterolemic okara is modulated at the molecular level, gene expres- action (34). The discrepancy in hypotriglyceridemic sion of proteins involved in lipid metabolism was mea- action between our and their studies may be partly due sured as shown in Fig. 3. Gene expression of FAS, a to difference in animal species and diets (a low-fat diet in Okara and Triglyceride Metabolism 167 our rat study vs a high-fat diet in the hamster and mouse cochemical characterization of dietary fiber in okara studies). When okara with high fiber and low protein should be carried out. No clear consensus has thus far was given to mice via oral gavage in addition to conven- been reached in terms of the hypotriglyceridemic action tional rat chow, okara lowered serum triglyceride and of insoluble fiber. Dietary fiber in okara is not ferment- cholesterol levels (35). Because a conventional rat chow able (our unpublished data), has a bulking effect, and containing corn, puffed bean and soybean was given stimulates intestinal peristalsis. Thus, this supports to mice, the exact amount of dietary fiber the animals our result that fecal weight was significantly and dose- ingested was unknown. On the other hand, the study dependently increased by feeding okara (Fig. 2), indi- done by Préstamo et al. (36) did not observe a change in cating a well-known fiber action. In addition, it is well serum triglyceride when okara was added to the diets at known that dietary fiber and soy protein alter physiolog- the level of 5% as dietary fiber. The soy diet in the present ical function in the small intestine (38, 43). Collectively, study contained 2.3 g dietary fiber in addition to cellu- these results suggest that the hypotriglyceridemic action lose and did not show hypotriglyceridemic action (Table of okara at least in part results from intestinal function 3). Therefore, although dietary fiber may be responsible altered by dietary fiber, although the level of fiber at for okara’s hypotriglyceridemic action, the level of okara which the hypotriglyceridemic effect might be expected as dietary fiber in diets seems to be critical. In contrast, remains to be determined. dietary fiber-induced hypocholesterolemic activity has To gain insight into the mechanism responsible for been well characterized, and soluble fiber such as pec- okara’s hypotriglyceridemic action, activities of hepatic tin has an important role in lipid metabolism (37–39). enzymes involved in lipogenesis were measured (Fig. Furthermore, it appears that an effective level of dietary 2). The okara diets significantly reduced activities of fiber in most of the studies to exert hypocholesterolemic fatty acid-synthesizing enzymes, FAS, ME and G6PDH, action is greater than 5% (14, 38). Although cellulose whereas neither the okara diets nor the soy diet modi- was included in the diets at the level of 5% in the present fied those of CPT in the liver or brown adipose tissue. study, cellulose is known to be inert in terms of hypolip- These results imply that hypotriglyceridemic action is at idemic potential (40). In the present study, dietary fiber least partly due to decreased fatty acid synthesis in the derived from okara accounted for 5.7% and 11.5% in liver, but not to increased oxidation of fatty acid. This the 2.5% and 5.0% okara diets, respectively, in addition hypothesis was further supported by the fact that gene to 5% of cellulose. Collectively, dietary fiber in the okara expression of FAS, the rate-limiting enzyme of fatty acid diets at the level of more than 5% may be a good can- synthesis, tended to be down-regulated by the okara didate for hypotriglyceridemic action. Thus, to the best diets (Fig. 3). The okara diets significantly diminished of our knowledge, this is the first study showing that a serum insulin levels without affecting serum glucose relatively low amount of okara exerts hypotriglyceride- levels (Table 3). Although insulin is lipogenic and one mic action. In contrast, Matsuo and Hitomi (15) have of the factors regulating sterol regulatory element- suggested that the hypolipidemic action of okara is binding protein-1c (SREBP-1c), and one of its targets is attributable to both fiber and protein. Some studies with FAS, gene expression of SREBP-1c was not clearly influ- animals showed that protein in soy lowers serum triglyc- enced by the okara diets, indicating an involvement of a eride (41), but others did not (42, 43). The study show- SREBP-1c-independent pathway. These results indicate ing hypolipidemic action used diets containing ~16% of that serum levels of triglyceride may be controlled at the soy protein (40), while only 2.5–5% protein originated enzyme level in the liver. from the okara and soy diets in the present study. This Soy and soy products have been reported to be hypo- may corroborate the non-significant decrease in serum cholesterolemic (45). Our study also revealed a ten- triglyceride in the soy group in which 5% dietary pro- dency of lower serum and hepatic cholesterol levels in tein was provided by soy flour in our study (Table 3). the okara diet group compared to the control diet group Therefore, protein in okara, if any, may be only partly (Table 3). Enhanced bile acid secretion increases conver- responsible for the hypotriglyceridemic action of okara. sion of cholesterol into bile acids in the liver and impairs Dietary fiber used in the aforementioned studies was sol- micelle formation in the small intestine, thus interfering uble fiber while that in okara is insoluble fiber (11, 36). with cholesterol absorption and reducing the choles- In general, it appears that the more soluble fiber, the terol pool in the body. As shown in Fig. 2, in addition to more hypocholesterolemic effect (14). When three dif- increased fecal weight, the okara diets tended to increase ferent insoluble fibers derived from legumes were given acidic steroid excretion dose-dependently (p50.1), indi- to rats, all three fiber diets exerted significant hypocho- cating an involvement of intestinal events in altered lesterolemic action, but not hypotriglyceridemic action lipid metabolism. The discrepancy in okara’s hypocho- relative to the cellulose-based diet (44). The authors lesterolemic action between our and previous studies suggest that physical and chemical properties of dietary may be attributed to difference in rat strain (Sprague- fiber such as bulk density and cation-exchange capacity Dawley vs Wistar), animal species (rat vs mouse) and may be implicated in hypolipidemic action via impaired diet (AIN 93G vs AIN 76) (18, 42). It is also likely that micelle formation in the small intestine. In the current the low amount of dietary fiber and protein in our okara study, feeding okara significantly increased fecal output, and soy diets may not be high enough to exert hypo- indicating a bulking effect. To explain the role of okara cholesterolemic action through increased excretion of in regulating lipid metabolism, detailed studies on physi- fecal steroids. Further studies using more animals are 168 Nagata Y et al. definitely merited to explore the link between choles- 12) Hu X, Gao J, Zhang Q, Fu Y, Li K, Zhu S, Li D. 2013. Soy terol, bile acid metabolism and okara as, most likely due fiber improves weight loss and lipid profile in overweight to a large standard error, only the tendency toward an and obese adults: A randomized controlled trial. Mol increase in bile acid excretion was observed. Nutr Food Res 57: 2147–2154. 13) Mateos-Aparicio I, Mateos-Peinado C, Jiménez-Escrig A, In conclusion, our study shows that at a relatively Rupérez P. 2010. 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