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Effect of enzymatic hydrolysis on cholesterol-lowering activity of oat β-glucan
New Biotechnology Volume 27, Number 1 February 2010
RESEARCH PAPER
Research Paper
Effect of enzymatic hydrolysis on cholesterol-lowering activity of oat b-glucan In Young Bae1, Sung Mi Kim1, Suyong Lee2 and Hyeon Gyu Lee1 1 2
Department of Food and Nutrition, Hanyang University, Seoul, 133-791, Republic of Korea Department of Food Science and Technology, Sejong University, Seoul, 143-747, Republic of Korea
In this study, oat b-glucan hydrolysate, having average molecular weight of 730,000 g/mol which was previously shown to have great in vitro bile acid binding capacity, was prepared by enzymatic hydrolysis. Furthermore its in vivo hypocholestrolemic effects were evaluated in rats that were fed high-cholesterol diets. Supplements with b-glucan hydrolysate as well as native b-glucan significantly reduced the levels of LDL- and VLDL-cholesterol in serum and further improved the lipid profile in liver. When rats were fed high-cholesterol diets, supplemented with the b-glucan hydrolysate, greater fecal bile acid excretion was observed, which could be favorably correlated to in vitro bile acid binding capacity. In addition, the hydrolysate was more effective at increasing the excretion of fecal cholesterol and triglyceride than the native b-glucan, showing its effectiveness in improving the lipid profile.
Introduction Dietary fibers play a positive role in health promotion and prevention of diseases such as hypocholesterolemia and hypolipidemia [1,2], which have been extensively reported in human and animal studies [3–6]. Even though there is no clear consensus as to the mechanism by which dietary fibers lower the cholesterol level in the body, the gel formation and cholesterol excretion ability of dietary fibers are thought to be involved in their cholesterol- and lipid-lowering effects [7]. Especially, cereal b-glucan, one of the water-soluble dietary fibers which is rich in oat and barley endospermic cell walls, has caught public and scientific attention. This is primarily because of its beneficial health effects, including lowering postprandial blood glucose levels [8] and serum cholesterol levels [9–12] as well as modifying immune response [13]. It is recognized that high viscosity of b-glucan prevents the absorption of lipids or the reabsorption of bile acids and their metabolites, consequently bringing about the reduction of serum cholesterol level [2]. In addition, short-chain fatty acids (SCFA), which are produced by fermentation of b-glucan in the large intestine, prevent the absorption and conversion of cholesterol and bile acids by interfering with their Corresponding author:. Lee, H.G. ([email protected]) 1871-6784/$ - see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.11.003
diffusion [14] and by inhibiting the activity of cholesterol synthase [15]. More recently, our study [16] reported the biological properties of oat b-glucans with different average molecular weights throughout in vitro and in vivo experiments. In the study, b-glucans, with the average molecular weight 370,000 g/mol and 730,000 g/mol, exhibited great in vitro bile acid binding capacity even though a positive correlation between the average molecular weight and weight gain/ lipid profile of mice could not be observed. Therefore for further study, the fraction of oat b-glucan hydrolysate, which was previously proven to improve in vitro bile acid binding capacity, was used to evaluate its hypocholesterolemic effect in rats that were fed a high-cholesterol diet for 4 weeks in comparison with native oat b-glucan.
Materials and methods Materials Oat bran concentrate containing 43% b-glucan (Lot No. 050610-108) was purchased from Nature1 Advanced Oat Technologies (Missoula, MT, USA). Celluclast was obtained from Novozymes (Bagsvaerd, Denmark). Assay kits for total cholesterol (TC), highdensity lipoprotein cholesterol (HDL-C), triglyceride (TG), and bile acid were supplied by Asan Pharmaceutical Co. (Seoul, Korea) as well as Wako Chemical (Osaka, Japan), respectively. www.elsevier.com/locate/nbt
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New Biotechnology Volume 27, Number 1 February 2010
Preparation of oat b-glucan hydrolysate According to the method of Bae et al. [16], oat b-glucan hydrolysate was prepared by adding Celluclast (840 EGU/g) to oat bran concentrate suspension in distilled water (6.25% (w/v), 50 8C, pH 4.8). The solutions were placed 50 8C for 4 h and then heated to 100 8C for 30 min for the enzyme inactivation. After the addition of an equal volume of ethanol, it was centrifuged at 9700 g for 10 min to obtain the precipitate, which was then dried at 40 8C. Gel permeation chromatography was applied to make sure that the oat b-glucan hydrolysate had average molecular weight of 730,000 g/mol [16]. Research Paper
gation of the blood from the heart at 1500 g for 30 min at 4 8C. The liver was then removed, frozen in liquid nitrogen, and stored at 70 8C for further analysis. Serum lipid profile, such as total lipid, triglyceride, and total cholesterol was immediately measured by blood chemistry analyzer (Olympus AU 400, Tokyo, Japan). The lipid profile in the liver and feces, which were extracted using the mixed solution of chloroform and methanol (2:1, v/v) [17], was analyzed by an enzymatic colorimetric kit. Fecal bile acid was extracted according to the modified method of Uchida et al. [18] and determined with a bile acid assay kit.
Animals and diets All animal protocols were approved by the Hanyang University Lab Animal Care Committee, which were in accordance with the Korean Guide for the Care and Use of Laboratory Animals. Four-week-old male Wistar rats (Central Lab. Animal Inc., Seoul, Korea) were acclimated by feeding a commercial standard rodent diet (Samyang Corp., Seoul, Korea) for 1 week. After the rats were divided into four groups (n = 10 per group), they received a normal diet, a high-cholesterol diet, and a high-cholesterol diet containing native b-glucan (1,450,000 g/mol) and its hydrolysate (730,000 g/mol), which were designated as ND, HCD, NBG, and BGH, respectively. Compositions of the experimental diets are shown in Table 1. The rats were housed in wire-bottom cages in a temperature-controlled room (23 8C; relative humidity 55%; 12 h light and dark cycle) and they received food and water ad libitum for the 4-week period. Food intakes and body weights were recorded every other day.
Sampling procedures and analysis The feces were collected during the final three days and were dried, grounded, and stored at 70 8C. Serum was obtained by centrifuTABLE 1
Compositions of the experimental diets (g/kg diet) Groupsa
Ingredients
ND Casein
HCD
NBG/BGH
200
200
180
3
3
3
Corn starch
150
150
100
Sucrose
DL-Methionine
Statistical analysis The results were statistically analyzed by using Statistical Package for the Social Science (SPSS, Version 12.0, 2004, SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was carried out to determine a significance difference among samples. Duncan’s multiple range test was then used for mean comparison at the level of 0.05.
Results and discussion Weight gain and feed efficiency Bile acids that are synthesized from cholesterol in the liver, move to intestines where most of the bile acids are reabsorbed and were then returned to the liver [19]. Therefore, a number of studies indicated that the binding and subsequent excretion of bile acids is thought to be a potent way to reduce cholesterol levels in the body [19,20]. Hence the oat b-glucan hydrolysate, having the average molecular weight of 730,000 g/mol which was proved to be great in vitro bile acid binding capacity in our preceding study [16], was chosen as a potent cholesterollowering material. Its effect on lipid profile was then investigated in rats that were fed high-fat diets in comparison with native oat b-glucan. As can be seen in Table 2, the addition of the b-glucan hydrolysate to the high-cholesterol diet significantly reduced the weight gain of the rats and improved feed efficiency; this is in comparison to the group that was fed the high-cholesterol diet. However, there was no significant difference in the initial body weight and the weight gain between native b-glucan and the hydrolysate.
500
500
500
Cellulose
50
50
50
Corn oil
50
50
44
Body weight and feed efficiency of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks
Mineral Mix S10022G
35
35
35
Groupsa
Initial weight (g)b
Vitamin Mix V10037
10
10
10
ND
Choline bitartrate
2
2
2
HCD
Cholesterol
–
10
10
NBG
Cholic acid
–
2
2
–
–
116
3792
3854
3854
b
Oat bran concentrate Total energy (kcal) a
ND, normal diet: AIN-76A purified rodent diet with 65% corn starch #111753 (Dyets Inc., Bethlehem, PA, USA); HCD, high-cholesterol diet: 1% cholesterol modified AIN-76A purified rodent diet #101556 (Dyets Inc., Bethlehem, PA, USA); BG, high-cholesterol diet contained with native b-glucan (NBG) and its hydrolysate (BGH). b Oat concentrates (NBG and BGH) were composed of 43% b-glucan, 44% starch, 13% protein and yielded energy 3.97 kcal/g.
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TABLE 2
BGH
Weight gain (g)c
Feed efficiencyd
170.30 5.64
134.38 11.38
0.24 0.02
170.17 4.71ns
168.83 7.05a
0.29 0.01a
170.67 4.88
156.33 9.27ab
0.28 0.02a
170.67 5.47
b
157.67 5.16
0.27 0.01b
Values are means SD. (a and b) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. ns: not significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND, normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH). b Initial weight: mean body weight at the beginning of the experimental diets. c Weight gain (g) = final weight (g) initial weight. d Feed efficiency = mean body weight gain (g)/mean food consumption (g).
New Biotechnology Volume 27, Number 1 February 2010
RESEARCH PAPER
TABLE 3
Groupa
Total lipid (mg/dL)
ND
190.67 25.66
HCD
380.51 55.72ns
Triglyceride (mg/dL)
Total cholesterol (mg/dL)
59.52 18.70 140.80 34.58a a
HDL-C (mg/dL)
87.77 8.38
LDL-C (mg/dL)
52.20 5.49
167.95 24.37ns
33.30 10.15b
VLDL-C (mg/dL)
HTRb (%)
23.67 7.71
11.90 3.74
59.56 4.25
106.49 20.48a
28.16 8.59a
19.59 4.35c
b
a
a
NBG
380.43 34.96
140.45 15.38
154.40 18.20
47.20 9.85
79.71 20.86
28.09 4.65
30.99 7.11b
BGH
344.83 54.02
109.38 30.08b
149.384 16.39
54.10 7.65a
73.40 18.92b
21.68 5.72b
36.57 5.86a
Values are means SD. (a–c) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. ns: not significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND, normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH). b HTR = HDL-cholesterol/total cholesterol 100.
Serum lipid profile
the coronary heart disease risk [22]. However, no significant difference in the levels of total lipid and cholesterol was found between native b-glucan and the hydrolysate.
Table 3 details the effect of native b-glucan and its hydrolysate on the serum lipid profile of rats. Overall, the serum lipid profile of the rats fed the b-glucan hydrolysate had improved while the significant changes between native b-glucan and the hydrolysate were not observed except in triglyceride and VLDL-C. The level of triglyceride in serum was significantly lower in the rats that were fed the diet supplemented with the b-glucan hydrolysate (P < 0.05). In comparison to the HCD group, the HDL-cholesterol content in the serum that has a positive effect on coronary heart disease [21], increased up to 42–62% in the b-glucan groups. Similar HDL cholesterol level (54.10 mg/dL) to the normal diet group (52.20 mg/dL) was observed in the b-glucan hydrolysate group. Furthermore, the diet containing b-glucans substantially reduced the LDL- and VLDL-cholesterol contents by 25–31% and 0.2–23%, respectively (P < 0.05). More importantly, the group treated with the b-glucan hydrolysate significantly showed the lowest VLDL-cholesterol and the highest HTR. Thereby the bglucan hydrolysate seemed to have beneficial effects on the serum lipid profile of rats since a higher content of HDL-cholesterol and a lower level of LDL- and VLDL-cholesterol are positively related to
Liver and fecal lipid profile Lipid profile in the liver exhibited a similar tendency as that of serum, showing the effectiveness of b-glucan hydrolysate in improving the lipid profile in the liver. As shown in Table 4, both the native b-glucan and the hydrolysate were significantly effective at lowering total lipid, triglyceride, and cholesterol levels in the liver by more than 25%, 42%, and 30%, respectively at the end of the study. However, the lipid profile change in liver did not reach a statistical significance between native b-glucan and its hydrolysate. Supplementation with b-glucans caused significant differences in the dry fecal weight, fecal lipid profile, and bile acid (Table 5). Both b-glucans promoted the excretion of fecal lipid, cholesterol, and bile acid by more than 28%, 50%, and 61% as compared with the high-cholesterol diet group, respectively (P < 0.05). The discharge of bile acids was more efficient in the rats that were fed the b-glucan hydrolysate, which could be correlated to the result of in
TABLE 4
Liver lipid profile of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks Groupa
Total lipid (mg/g liver)
ND
Triglyceride (mg/g liver)
Total cholesterol (mg/g liver)
37.95 4.71
2.61 0.31
9.90 2.86
HCD
155.02 14.03a
11.09 1.48a
25.34 4.06a
NBG
118.85 17.56b
7.13 0.71b
18.17 4.27b
b
17.73 3.58b
b
BGH
116.45 8.06
6.48 0.72
Values are means SD. (a and b) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND, normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH).
TABLE 5
Fecal weight, lipid profile and bile acid excretion in fecal of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks Groupa
Weight (g/day)
Total lipid (mg/day)
ND
2.36 0.15
35.07 5.52
1.48 0.36
HCD
1.11 0.13c
39.31 5.39c
6.75 1.87c
1.44 0.35b
1.09 0.23c
NBG
1.31 0.15b
47.31 5.67b
9.94 2.23b
1.83 0.44a
1.42 0.17b
BGH
a
a
a
a
1.75 0.31a
1.46 0.20
50.07 7.99
Total cholesterol (mg/day)
10.10 1.52
Triglyceride (mg/day) 1.583 0.35
1.82 0.30
Bile acid (mmol/day) 0.40 0.05
Values are means SD. (a–c) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND: normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH). www.elsevier.com/locate/nbt
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Research Paper
Serum lipid profile of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks
RESEARCH PAPER
Research Paper
vitro bile acid binding [16]. The hypocholesterolemic effects of bglucan hydrolysate could be due in part to higher fecal excretion of cholesterol and bile acids as shown in Table 5. These results indicate that some factors, including structural and physicochemical properties other than viscosity, may influence the bile acid/fat binding capacities of soluble dietary fiber. Also Bae et al. [16] suggested the optimum average molecular weight range of bglucan to be most suitable for the improvement of its bile acid/ fat binding capacities. Thus, from an overall point of view, oat bglucan hydrolysate was as effective as native b-glucan in lowering cholesterol level in the serum and liver, increasing fecal bile acid excretion, and presumably reducing the risk of coronary heart disease.
New Biotechnology Volume 27, Number 1 February 2010
Conclusions Oat b-glucan hydrolysates, with different average molecular weights, were prepared in our previous work where the average molecular weight of 730,000 g/mol provided great in vitro bile acid binding capacity. Thus in this study, the oat b-glucan hydrolysate, with the average molecular weight of 730,000 g/mol, was chosen and its hypocholesterolemic effect was tested in an animal model system with rats. Supplementation with the b-glucan hydrolysate was significantly effective in improving the lipid profile and bile acid excretion of the experimental rats, probably contributing to the reduction of the cardiovascular disease risk. Furthermore, it would be worthwhile to carry out more in vivo experiments with bglucans from various other sources.
References 1 Anderson, J.W. et al. (1984) Hypocholesterolemic effects of oat bran or bran intake for hypercholesterolemic men. Am. J. Clin. Nutr. 40, 1146–1155 2 Kahlon, T.S. et al. (1998) Effect of extrusion on hypocholesterolemic properties of rice, oat, corn, and wheat bran diets in hamsters. Cereal Chem. 75, 897–903 3 Fernandez, M.L. et al. (1995) Guar gum effects on plasma low-density lipoprotein and hepatic cholesterol metabolism in guinea pigs fed low- and high-cholesterol diets: a dose-response study. Am. J. Clin. Nutr. 61, 127–134 4 Glore, S.R. et al. (1994) Soluble fiber and serum lipids: a literature review. J. Am. Diet. Assoc. 94, 425–436 5 Moundras, C. et al. (1997) Fecal losses of sterols and bile acids induced by feeding rats guar gum are due to a greater pool size and liver bile acid secretion. J. Nutr. 127, 1068–1076 6 Nishina, P.M. et al. (1991) Effects of dietary fibers on nonfasting plasma lipoprotein and apolipoprotein levels in rats. J. Nutr. 121, 431–437 7 Stark, A. and Madar, Z. (1994) Dietary fiber. In Functional Foods (Goldberg, I., ed.), p. 189, Chapman and Hall 8 Wood, P.J. et al. (1994) Effect of dose and modification of viscous properties of oat gum on plasma glucose and insulin following an oral glucose load. Br. J. Nutr. 72, 731–743 9 Chen, W.L. et al. (1981) Effects of oat bran, oat gum and pectin on lipid metabolism of cholesterol-fed rats. Nutr. Rep. Int. 24, 1093–1098 10 Hsing-Hsien, C. and Ming-Hoang, L. (2000) Fermentation of resistant rice starch produces propionate reducing serum and hepatic cholesterol in rats. J. Nutr. 130, 1991–1995 11 Jennings, C.D. et al. (1988) A comparison of lipid-lowering and intestinal morphological effects of cholestyramine, chitosan and oat gum in rats. Proc. Soc. Exp. Biol. Med. 189, 13–20
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12 Braaten, J.T. et al. (1991) Oat gum, a soluble fiber which lowers glucose and insulin in normal individuals after an oral glucose load: Comparison with guar gum. Am. J. Clin. Nutr. 53, 1425–1430 13 Yang, J.-L. et al. (2008) b-Glucan suppresses LPS-stimulated NO production through the down-regulation of iNOS expression and NFkB transactivation in RAW 264.7 macrophages. Food Sci. Biotechnol. 17, 106–113 14 Chen, W.J.L. et al. (1984) Propionate may mediate the hypocholesteolemic effects of certain soluble plant fibers in cholesterol-fed rats. Proc. Soc. Exp. Biol. Med. 175, 215–218 15 Deckere, A.M.E. et al. (1993) Resistant starch decreases serum total cholesterol and triacylglycerol concentrations in rats. J. Nutr. 123, 2142–2151 16 Bae, I.Y. et al. (2009) Effect of partially hydrolyzed oat b-glucan on the weight gain and lipid profile of mice. Food Hydrocol. 23, 2016–2021 17 Folch, J. et al. (1956) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509 18 Uchida, K. et al. (1977) Effect of dietary cholesterol on cholesterol and bile acid metabolism in rats. Jpn. J. Phamacol. 27, 193–204 19 Kahlon, T.S. et al. (2005) In vitro binding of bile acids by kidney bean (Phaseolus vulgaris), black gram (Vigna mungo), bengal gram (Cicer arietinum) and moth bean (Phaseolus aconitifolins). Food Chem. 90, 241–246 20 Camire, M.E. and Dougherty, M.P. (2003) Raisin dietary fiber composition and in vitro bile acid binding. J. Agric. Food Chem. 51, 834–837 21 Manninen, V. et al. (1992) Joint effects of serum triglyceride and LDL cholesterol and HDL cholesterol concentrations on coronary heart disease risk in the Helsinki Heart Study. Implications for treatment. Circulation 85, 37–45 22 Martinez-Flores, H.E. et al. (2004) Effect of high fiber products on blood lipids and lipoproteins in hamsters. Nutr. Res. 24, 85–93
RESEARCH PAPER
Research Paper
Effect of enzymatic hydrolysis on cholesterol-lowering activity of oat b-glucan In Young Bae1, Sung Mi Kim1, Suyong Lee2 and Hyeon Gyu Lee1 1 2
Department of Food and Nutrition, Hanyang University, Seoul, 133-791, Republic of Korea Department of Food Science and Technology, Sejong University, Seoul, 143-747, Republic of Korea
In this study, oat b-glucan hydrolysate, having average molecular weight of 730,000 g/mol which was previously shown to have great in vitro bile acid binding capacity, was prepared by enzymatic hydrolysis. Furthermore its in vivo hypocholestrolemic effects were evaluated in rats that were fed high-cholesterol diets. Supplements with b-glucan hydrolysate as well as native b-glucan significantly reduced the levels of LDL- and VLDL-cholesterol in serum and further improved the lipid profile in liver. When rats were fed high-cholesterol diets, supplemented with the b-glucan hydrolysate, greater fecal bile acid excretion was observed, which could be favorably correlated to in vitro bile acid binding capacity. In addition, the hydrolysate was more effective at increasing the excretion of fecal cholesterol and triglyceride than the native b-glucan, showing its effectiveness in improving the lipid profile.
Introduction Dietary fibers play a positive role in health promotion and prevention of diseases such as hypocholesterolemia and hypolipidemia [1,2], which have been extensively reported in human and animal studies [3–6]. Even though there is no clear consensus as to the mechanism by which dietary fibers lower the cholesterol level in the body, the gel formation and cholesterol excretion ability of dietary fibers are thought to be involved in their cholesterol- and lipid-lowering effects [7]. Especially, cereal b-glucan, one of the water-soluble dietary fibers which is rich in oat and barley endospermic cell walls, has caught public and scientific attention. This is primarily because of its beneficial health effects, including lowering postprandial blood glucose levels [8] and serum cholesterol levels [9–12] as well as modifying immune response [13]. It is recognized that high viscosity of b-glucan prevents the absorption of lipids or the reabsorption of bile acids and their metabolites, consequently bringing about the reduction of serum cholesterol level [2]. In addition, short-chain fatty acids (SCFA), which are produced by fermentation of b-glucan in the large intestine, prevent the absorption and conversion of cholesterol and bile acids by interfering with their Corresponding author:. Lee, H.G. ([email protected]) 1871-6784/$ - see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.11.003
diffusion [14] and by inhibiting the activity of cholesterol synthase [15]. More recently, our study [16] reported the biological properties of oat b-glucans with different average molecular weights throughout in vitro and in vivo experiments. In the study, b-glucans, with the average molecular weight 370,000 g/mol and 730,000 g/mol, exhibited great in vitro bile acid binding capacity even though a positive correlation between the average molecular weight and weight gain/ lipid profile of mice could not be observed. Therefore for further study, the fraction of oat b-glucan hydrolysate, which was previously proven to improve in vitro bile acid binding capacity, was used to evaluate its hypocholesterolemic effect in rats that were fed a high-cholesterol diet for 4 weeks in comparison with native oat b-glucan.
Materials and methods Materials Oat bran concentrate containing 43% b-glucan (Lot No. 050610-108) was purchased from Nature1 Advanced Oat Technologies (Missoula, MT, USA). Celluclast was obtained from Novozymes (Bagsvaerd, Denmark). Assay kits for total cholesterol (TC), highdensity lipoprotein cholesterol (HDL-C), triglyceride (TG), and bile acid were supplied by Asan Pharmaceutical Co. (Seoul, Korea) as well as Wako Chemical (Osaka, Japan), respectively. www.elsevier.com/locate/nbt
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New Biotechnology Volume 27, Number 1 February 2010
Preparation of oat b-glucan hydrolysate According to the method of Bae et al. [16], oat b-glucan hydrolysate was prepared by adding Celluclast (840 EGU/g) to oat bran concentrate suspension in distilled water (6.25% (w/v), 50 8C, pH 4.8). The solutions were placed 50 8C for 4 h and then heated to 100 8C for 30 min for the enzyme inactivation. After the addition of an equal volume of ethanol, it was centrifuged at 9700 g for 10 min to obtain the precipitate, which was then dried at 40 8C. Gel permeation chromatography was applied to make sure that the oat b-glucan hydrolysate had average molecular weight of 730,000 g/mol [16]. Research Paper
gation of the blood from the heart at 1500 g for 30 min at 4 8C. The liver was then removed, frozen in liquid nitrogen, and stored at 70 8C for further analysis. Serum lipid profile, such as total lipid, triglyceride, and total cholesterol was immediately measured by blood chemistry analyzer (Olympus AU 400, Tokyo, Japan). The lipid profile in the liver and feces, which were extracted using the mixed solution of chloroform and methanol (2:1, v/v) [17], was analyzed by an enzymatic colorimetric kit. Fecal bile acid was extracted according to the modified method of Uchida et al. [18] and determined with a bile acid assay kit.
Animals and diets All animal protocols were approved by the Hanyang University Lab Animal Care Committee, which were in accordance with the Korean Guide for the Care and Use of Laboratory Animals. Four-week-old male Wistar rats (Central Lab. Animal Inc., Seoul, Korea) were acclimated by feeding a commercial standard rodent diet (Samyang Corp., Seoul, Korea) for 1 week. After the rats were divided into four groups (n = 10 per group), they received a normal diet, a high-cholesterol diet, and a high-cholesterol diet containing native b-glucan (1,450,000 g/mol) and its hydrolysate (730,000 g/mol), which were designated as ND, HCD, NBG, and BGH, respectively. Compositions of the experimental diets are shown in Table 1. The rats were housed in wire-bottom cages in a temperature-controlled room (23 8C; relative humidity 55%; 12 h light and dark cycle) and they received food and water ad libitum for the 4-week period. Food intakes and body weights were recorded every other day.
Sampling procedures and analysis The feces were collected during the final three days and were dried, grounded, and stored at 70 8C. Serum was obtained by centrifuTABLE 1
Compositions of the experimental diets (g/kg diet) Groupsa
Ingredients
ND Casein
HCD
NBG/BGH
200
200
180
3
3
3
Corn starch
150
150
100
Sucrose
DL-Methionine
Statistical analysis The results were statistically analyzed by using Statistical Package for the Social Science (SPSS, Version 12.0, 2004, SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was carried out to determine a significance difference among samples. Duncan’s multiple range test was then used for mean comparison at the level of 0.05.
Results and discussion Weight gain and feed efficiency Bile acids that are synthesized from cholesterol in the liver, move to intestines where most of the bile acids are reabsorbed and were then returned to the liver [19]. Therefore, a number of studies indicated that the binding and subsequent excretion of bile acids is thought to be a potent way to reduce cholesterol levels in the body [19,20]. Hence the oat b-glucan hydrolysate, having the average molecular weight of 730,000 g/mol which was proved to be great in vitro bile acid binding capacity in our preceding study [16], was chosen as a potent cholesterollowering material. Its effect on lipid profile was then investigated in rats that were fed high-fat diets in comparison with native oat b-glucan. As can be seen in Table 2, the addition of the b-glucan hydrolysate to the high-cholesterol diet significantly reduced the weight gain of the rats and improved feed efficiency; this is in comparison to the group that was fed the high-cholesterol diet. However, there was no significant difference in the initial body weight and the weight gain between native b-glucan and the hydrolysate.
500
500
500
Cellulose
50
50
50
Corn oil
50
50
44
Body weight and feed efficiency of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks
Mineral Mix S10022G
35
35
35
Groupsa
Initial weight (g)b
Vitamin Mix V10037
10
10
10
ND
Choline bitartrate
2
2
2
HCD
Cholesterol
–
10
10
NBG
Cholic acid
–
2
2
–
–
116
3792
3854
3854
b
Oat bran concentrate Total energy (kcal) a
ND, normal diet: AIN-76A purified rodent diet with 65% corn starch #111753 (Dyets Inc., Bethlehem, PA, USA); HCD, high-cholesterol diet: 1% cholesterol modified AIN-76A purified rodent diet #101556 (Dyets Inc., Bethlehem, PA, USA); BG, high-cholesterol diet contained with native b-glucan (NBG) and its hydrolysate (BGH). b Oat concentrates (NBG and BGH) were composed of 43% b-glucan, 44% starch, 13% protein and yielded energy 3.97 kcal/g.
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TABLE 2
BGH
Weight gain (g)c
Feed efficiencyd
170.30 5.64
134.38 11.38
0.24 0.02
170.17 4.71ns
168.83 7.05a
0.29 0.01a
170.67 4.88
156.33 9.27ab
0.28 0.02a
170.67 5.47
b
157.67 5.16
0.27 0.01b
Values are means SD. (a and b) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. ns: not significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND, normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH). b Initial weight: mean body weight at the beginning of the experimental diets. c Weight gain (g) = final weight (g) initial weight. d Feed efficiency = mean body weight gain (g)/mean food consumption (g).
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RESEARCH PAPER
TABLE 3
Groupa
Total lipid (mg/dL)
ND
190.67 25.66
HCD
380.51 55.72ns
Triglyceride (mg/dL)
Total cholesterol (mg/dL)
59.52 18.70 140.80 34.58a a
HDL-C (mg/dL)
87.77 8.38
LDL-C (mg/dL)
52.20 5.49
167.95 24.37ns
33.30 10.15b
VLDL-C (mg/dL)
HTRb (%)
23.67 7.71
11.90 3.74
59.56 4.25
106.49 20.48a
28.16 8.59a
19.59 4.35c
b
a
a
NBG
380.43 34.96
140.45 15.38
154.40 18.20
47.20 9.85
79.71 20.86
28.09 4.65
30.99 7.11b
BGH
344.83 54.02
109.38 30.08b
149.384 16.39
54.10 7.65a
73.40 18.92b
21.68 5.72b
36.57 5.86a
Values are means SD. (a–c) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. ns: not significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND, normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH). b HTR = HDL-cholesterol/total cholesterol 100.
Serum lipid profile
the coronary heart disease risk [22]. However, no significant difference in the levels of total lipid and cholesterol was found between native b-glucan and the hydrolysate.
Table 3 details the effect of native b-glucan and its hydrolysate on the serum lipid profile of rats. Overall, the serum lipid profile of the rats fed the b-glucan hydrolysate had improved while the significant changes between native b-glucan and the hydrolysate were not observed except in triglyceride and VLDL-C. The level of triglyceride in serum was significantly lower in the rats that were fed the diet supplemented with the b-glucan hydrolysate (P < 0.05). In comparison to the HCD group, the HDL-cholesterol content in the serum that has a positive effect on coronary heart disease [21], increased up to 42–62% in the b-glucan groups. Similar HDL cholesterol level (54.10 mg/dL) to the normal diet group (52.20 mg/dL) was observed in the b-glucan hydrolysate group. Furthermore, the diet containing b-glucans substantially reduced the LDL- and VLDL-cholesterol contents by 25–31% and 0.2–23%, respectively (P < 0.05). More importantly, the group treated with the b-glucan hydrolysate significantly showed the lowest VLDL-cholesterol and the highest HTR. Thereby the bglucan hydrolysate seemed to have beneficial effects on the serum lipid profile of rats since a higher content of HDL-cholesterol and a lower level of LDL- and VLDL-cholesterol are positively related to
Liver and fecal lipid profile Lipid profile in the liver exhibited a similar tendency as that of serum, showing the effectiveness of b-glucan hydrolysate in improving the lipid profile in the liver. As shown in Table 4, both the native b-glucan and the hydrolysate were significantly effective at lowering total lipid, triglyceride, and cholesterol levels in the liver by more than 25%, 42%, and 30%, respectively at the end of the study. However, the lipid profile change in liver did not reach a statistical significance between native b-glucan and its hydrolysate. Supplementation with b-glucans caused significant differences in the dry fecal weight, fecal lipid profile, and bile acid (Table 5). Both b-glucans promoted the excretion of fecal lipid, cholesterol, and bile acid by more than 28%, 50%, and 61% as compared with the high-cholesterol diet group, respectively (P < 0.05). The discharge of bile acids was more efficient in the rats that were fed the b-glucan hydrolysate, which could be correlated to the result of in
TABLE 4
Liver lipid profile of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks Groupa
Total lipid (mg/g liver)
ND
Triglyceride (mg/g liver)
Total cholesterol (mg/g liver)
37.95 4.71
2.61 0.31
9.90 2.86
HCD
155.02 14.03a
11.09 1.48a
25.34 4.06a
NBG
118.85 17.56b
7.13 0.71b
18.17 4.27b
b
17.73 3.58b
b
BGH
116.45 8.06
6.48 0.72
Values are means SD. (a and b) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND, normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH).
TABLE 5
Fecal weight, lipid profile and bile acid excretion in fecal of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks Groupa
Weight (g/day)
Total lipid (mg/day)
ND
2.36 0.15
35.07 5.52
1.48 0.36
HCD
1.11 0.13c
39.31 5.39c
6.75 1.87c
1.44 0.35b
1.09 0.23c
NBG
1.31 0.15b
47.31 5.67b
9.94 2.23b
1.83 0.44a
1.42 0.17b
BGH
a
a
a
a
1.75 0.31a
1.46 0.20
50.07 7.99
Total cholesterol (mg/day)
10.10 1.52
Triglyceride (mg/day) 1.583 0.35
1.82 0.30
Bile acid (mmol/day) 0.40 0.05
Values are means SD. (a–c) Values with different superscripts within the same column are significantly different among samples at a = 0.05 level by Duncan’s multiple range test. a ND: normal diet; HCD, high-cholesterol diet; OBC, high-cholesterol diet contained with b-glucan (NBG) and its hydrolysate (BGH). www.elsevier.com/locate/nbt
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Research Paper
Serum lipid profile of rats fed high-cholesterol diet with/without oat b-glucan for 4 weeks
RESEARCH PAPER
Research Paper
vitro bile acid binding [16]. The hypocholesterolemic effects of bglucan hydrolysate could be due in part to higher fecal excretion of cholesterol and bile acids as shown in Table 5. These results indicate that some factors, including structural and physicochemical properties other than viscosity, may influence the bile acid/fat binding capacities of soluble dietary fiber. Also Bae et al. [16] suggested the optimum average molecular weight range of bglucan to be most suitable for the improvement of its bile acid/ fat binding capacities. Thus, from an overall point of view, oat bglucan hydrolysate was as effective as native b-glucan in lowering cholesterol level in the serum and liver, increasing fecal bile acid excretion, and presumably reducing the risk of coronary heart disease.
New Biotechnology Volume 27, Number 1 February 2010
Conclusions Oat b-glucan hydrolysates, with different average molecular weights, were prepared in our previous work where the average molecular weight of 730,000 g/mol provided great in vitro bile acid binding capacity. Thus in this study, the oat b-glucan hydrolysate, with the average molecular weight of 730,000 g/mol, was chosen and its hypocholesterolemic effect was tested in an animal model system with rats. Supplementation with the b-glucan hydrolysate was significantly effective in improving the lipid profile and bile acid excretion of the experimental rats, probably contributing to the reduction of the cardiovascular disease risk. Furthermore, it would be worthwhile to carry out more in vivo experiments with bglucans from various other sources.
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