1395 Research Article Received: 10 May 2011 Revised: 7 September 2011 Accepted: 17 Sepember 2011 Published online in Wiley Online Library: 8 November 2011 (wileyonlinelibrary.com) DOI 10.1002/jsfa.4713 Concentration-dependent displacement of cholesterol in micelles by hydrophobic rice bran protein hydrolysates Huijuan Zhang, a,b Wallace H Yokoyama b and Hui Zhang a* Abstract BACKGROUND: Rice bran, containing about 100–150 g kg -1 protein, is a by-product of rice milling that has only become an available ingredient in recent years owing to the centralisation of rice milling. Rice bran, but not its protein fraction or hydrolysates, has been shown to have a hypocholesterolaemic effect. Peptides from soy, milk and other foods have been proposed to have hypocholesterolaemic effects based on their ability to lower cholesterol solubility in bile acid/phosphotidyl choline micelles. RESULTS: Rice bran protein hydrolysates (RBPHs) were prepared and investigated for their potential to lower cholesterol concentration in micelles. The RBPHs were produced by digestion using four different peptidases, alcalase 2.4L  , neutrase 0.8L  , papaya latex papain and porcine pancreas trypsin, and then fractionated by hydrophobicity using styrene/divinylbeneze resins. Alcalase 2.4L  produced the highest degree of hydrolysis, and the resulting hydrolysates had the highest micellar cholesterol inhibition ability in an in vitro hypocholesterolaemic test. The adsorption dynamics of four different macroporous resins, DA201-C, Sepabeads SP207 and SP825 and Diaion HP20, were determined using the Langmuir isotherm model. DA201-C had the highest adsorption capacity with an equilibrium concentration of 220 mg g -1 . The hydrolysates eluted with 25, 50, 75 and 95% (v/v) ethanol lowered the micellar cholesterol concentration by 11.88, 14.76, 19.37 and 7.56% respectively. CONCLUSION: A hydrophobic fraction of RBPH had the highest inhibitory activity on micellar cholesterol, which suggests that it may have hypocholesterolaemic properties. c  2011 Society of Chemical Industry Keywords: rice bran protein (RBP); micelle; enzymatic hydrolysis; cholesterol inhibition; macroporous resins INTRODUCTION Hypercholesterolaemia is a risk factor for cardiovascular disease (CVD), 1 and reducing the level of serum cholesterol decreases the risk of CVD. Since cholesterol precipitates in aqueous environ- ments, it must be transported by micelles composed of bile acids and phospholipids to the intestinal epithelium for absorption. Compounds such as cholestyramine and β -sitosterol that disrupt the micellar structure by binding bile acids or displacing choles- terol from the micelle reduce the availability and absorption of the molecular form of cholesterol and result in lowered serum choles- terol levels. Many researchers have used in vitro measurements of reduced cholesterol solubilisation in micelles or animal studies to identify potential hypocholesterolaemic agents. The hypocholes- terolaemic effects of dietary proteins such as soy protein and its peptides 2–5 and milk whey protein and its peptides 6 were identified by both in vitro methods and animal studies. The hypoc- holesterolaemic effects of sunflower protein and its peptides, 7 clam protein and its peptides 8 and lupin protein and its peptides 9 were identified by in vitro methods alone. Micelle disruption is one possible mechanism of reduced cholesterol-carrying capacity of micelles by peptides. The observation that lower lysine/arginine or methionine/glycine ratios of dietary protein accompany low- ered cholesterol suggests that amino acids might also have some effects. 10,11 Rice bran is an attractive source of bioactive peptides because rice accounts for about 25% of world cereal grain production, 12 and in recent years China, Japan, Korea, India and the USA have produced food oil from rice bran, resulting in vast amounts of low-value defatted rice bran by-product. Defatted rice bran contains 120 – 200 g kg −1 protein and is high in lysine content, which is a limiting amino acid in most cereals. The quality of rice bran protein (RBP) is good, and its protein efficiency ratio value ranges from 2.0 to 2.5, compared with 2.5 for casein. RBP’s lysine/arginine ratio (0.53) is lower than that of soy protein (0.79), and its methionine/glycine ratio (0.33) is similar to that of soy protein (0.32). 13 The hypocholesterolaemic effect of white rice protein has been reported, 3,4,14 – 16 but to our knowledge the hypocholesterolaemic ability of RBP has not been reported. ∗ Correspondence to: Hui Zhang, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China. E-mail: cerealzhanghui@yahoo.com.cn a School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China b US Department of Agriculture, Albany, CA 94710, USA J Sci Food Agric 2012; 92: 1395–1401 www.soci.org c  2011 Society of Chemical Industry