Abstract: In order to develop and utilize alfalfa leaves as a good resource of protein, antioxidant peptides were prepared from alfalfa leaf protein by alkaline protease hydrolysis. Through one-factors-at-a-time experiments, hydrolysis time, enzyme concentration, hydrolysis temperature and pH were selected as independent variables for further optimization using response surface methodology, and the response variable was antioxidant activity. The optimum conditions that provided the maximum percentage scavenging of DPPH radical of 64.25% were found to be hydrolysis time of 240 minutes, enzyme concentration of 4.80%, hydrolysis temperature of 60 ℃ and pH of 11.60. Then the most potent antioxidant peptide was purified and collected by preparative liquid chromatography, having stronger antioxidant activity than the crude hydrolysate. The amino acid composition of the antioxidant peptide was determined by high performance chromatography (HPLC) to consist of Asp, Gly, His, Tyr, Val, Leu, Phe, Trp and Lys.

Key words: alfalfa leaf protein, optimization of enzymatic hydrolysis conditions, antioxidant peptide purification, amino acid composition, percentage scavenging, response surface methodology