Abstract:A two-step enzymatic hydrolysis method was used to extract the polypeptides from crayfish by-products.Alcalase was used to prepare the protein from cray fish by-products.Single-factor and orthogonal array design revealed that the optimal preparation efficiency parameters were 2% alcalase，hydrolysis time 2 h，1∶20（g/mL）solid/liquid ratio，pH8.5，and 55 ℃.The protein extraction efficiency using the optimized conditions was 64.32%.In the second step，the protein from crayfish by-products was hydrolyzed by trypsin to prepare polypeptides with proteolysis as the standard.The optimum conditions were 2% trypsin concentration，hydrolysis duration of 4 h，1∶15（g/mL）solid/liquid ratio，pH8.0，and 37 ℃.The protein hydrolysis degree was up to 45.2% under the optimized conditions.The total antioxidant capacity of secondary hydrolysates were stronger than the primary ones.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the relative molecular weight of most secondary hydrolysates was below 3.3 kDa.The two-step enzymatic hydrolysis resulted in 18 amino acids in the polypeptides.Essential amino acids comprised 64.36% of the total amino acids.The polypeptides could be used in functional foods and condiments.