Protease hydrolyses of an Okara protein yielded antioxidative activity against the peroxidation of linoleic acid in an aqueous system at pH 7.0. Four antioxidative peptides were isolated from the hydrolysate prepared with Protease N by size exclusion chromatography and reversed-phase HPLC. The peptides were composed of two and three amino acid residues, including aromatic amino acid at the C terminal end. Their amino acid sequences were determined to be Ala-Tyr, Gly-Tyr-Tyr, Ala-Asp-Phe, and Ser-Asp-Phe, respectively. The antioxidative activity of Gly-Tyr-Tyr is nearly equal to that of carnosine.Keywords: antioxidative activity, amino acid, peptide, hydrolysate, OkaraIn the manufacturing of soymilk and tofu, the soymilk residue, Okara, is produced as a by-product with little market value. However, Okara still contains about 25% protein (dry basis) with high nutritive quality. Studies on the utility of Okara (Takenaka et al., 1996;Miyamura et al., 1998;Tamura & Takenaka, 1999) have been done, but utilization of the substance is still in the developmental stage. Antioxidative activity has recently been detected in peptides from the proteolytic hydrolysis of many food proteins (Suetsuna et al., 2000;Chen et al., 1995). In this study, we examined the antioxidative effects of enzymatic hydrolysates of Okara protein with seven different proteases.
Materials and MethodsMaterials The Okara protein was prepared using the method of Yamauchi et al. (1984). Proteases were purchased from Amano Enzyme Co., Ltd (Nagoya, Japan): Protease A from Aspergillus oryzae, 10,000 u/g, pH 7.0; Protease M from Aspergillus oryzae, 150,000 u/g, pH 7.0; Protease N from Bacillus subtilus, 30,000 u/g, pH 7.0; Protease P from Aspergillus melleus, 30,000 u/g, pH 8.0; Protease S from Bacillus sp. 10,000 u/g, pH 7.0; Prolezer from Bacillus subtilus 10,000 u/g, pH 9.0; and Papain W-40 from Carica pancreas, 400 u/g, pH 8.0.Preparation of the Okara protein hydrolysate Okara protein (10 g) was dissolved in 200 ml of distilled water, and heated at 100˚C for 10 min. Protease (0.2 g) was added to the protein solution after the pH was properly adjusted. Enzymatic hydrolysis was performed under optimum pH conditions as the manufacturer recommended. After digestion, hydrolysates were heated in boiling water for 5 min to inactivate proteases, then neutralized and centrifuged (20,000¥g for 10 min). The supernatants were lyophilized and stored in 4˚C until use.Measurement of antioxidative activity Linoleic acid (10 mg) in 0.5 ml of ethanol, samples in 4.0 ml of 0.2 M phosphate buffer (pH 7.0), and 0.15 ml of 20 mM 2,2¢-azobis(2-amidinopropane) dihydrochloride (AAPH) were placed in a vial, which was then sealed tightly and kept at 50˚C in the dark. At regular intervals, an aliquot of the reaction mixture was withdrawn for measurement of the oxidation using the ferric thiocyanate method (Mitsuda et al., 1966). The time taken to attain an absorbance of 0.3 was defined as the induction period. The relative antioxidative activity was calculated by dividing the indu...
