Preparation, Purification and Identification of Antioxidant Peptides with Bienzyme Hydrolysis from Rice Bran Protein

The rice bran protein (RBP) was then hydrolyzed with various proteases (papain, flavorzyme, neutrase, protamex, and trypsin) to prepare antioxidant peptides. The rice bran protein hydrolysates (RBPH) were assessed using method of DPPH radical scavenging ability. Hydrolysate prepared with papain and flavorzyme (activity ratio 1:1) was found to have the highest antioxidant activity (IC50=6.778±0.21 mg/ml). This hydrolysate was purified using ultrafiltration, RBPH-III (Mw＜3KDa) had the highest DPPH and hydroxyl radical scavenging activity (IC50 value of 6.56±0.28, 5.43±0.22, respectively) and highest reducing power activity (1.02±0.18 at 4 mg/mL). Later, RBPH-III was fractionated by SP-SephadexC-25 cation-exchange column into six fractions (A–F), fraction F with the highest DPPH scavenging activity, was then separated by size exclusion chromatography on a SephadexG-25 into three major fractions (F1–F3). Fraction F2 exhibited the highest DPPH scavenging activity was choose to fractionate by reversed-phase high performance liquid chromatography (RP-HPLC), seven antioxidant peptides were isolated, The F2-5 peptide displayed the highest DPPH radical-scavenging activity (58.2±1.63%; at 250 μg/ml) among these peptides, the amino acids composition of F2-5 was determined, which might play an important role on its antioxidant activity. In addition, purified peptide did show remarkable inhibition rate on SGC-7901 cells proliferation, and it also revealed the dose-dependent relationship. The results of this study suggest that rice bran protein hydrolysates are good source of natural antioxidants.