Abstract:Enzymatic hydrolysis technology was used to obtain enzymatic hydrolysis solution with high antioxidant activity and amino acid content to make full use of donkey bone resources.This study aimed to select types of enzymes and the combination of two enzymes by determining the hydrolysis degree and the yield of soluble peptide of donkey bone mud.The orthogonal experiment was used to optimize the process parameters of the two-enzyme segmented hydrolysis，and the free amino acid composition of the enzymatic hydrolysate was determined.The antioxidant activity was evaluated by determining the total reducing capacity，DPPH radical scavenging ability，superoxide anion free radical scavenging ability and hydroxyl radical scavenging ability of enzymatic hydrolysate.The results showed that the segmented hydrolysis of flavour protease combined with compound protease was optimum process and its enzymatic hydrolysis process were as follows：after enzymatic hydrolysis with flavour protease for 3 h under 1.5% enzyme at 50 ℃，pH 7.0，and solid-liquid ratio 1∶2（g/mL），the mixture was enzymatic hydrolyzed with compound proteinase for 4 h under 2.5% enzyme and solid-liquid ratio 1∶2（g/mL）.The hydrolysis degree of the enzymolysis solution and the yield of soluble peptides were 15.74% and 20.34%，respectively.The enzymolysis solution contained 17 kinds of amino acids.Besides，the content of alanine，phenylalanine，valine，leucine，tyrosine，lysine，arginine，proline and glycine were higher than 30 mg/100 mL.The enzymatic hydrolysate showed good scavenging effects on superoxide anion free radicals，hydroxyl free radicals and DPPH free radicals，and the highest scavenging rates were 31.65%，44.40% and 71.43%.This study showed that an enzymatic hydrolysate with high amino acid content and high oxidation resistance could be prepared with double enzyme enzymatic hydrolysis of the donkey bone mud protein.