Abstract:Pigeon hemoglobin was used as raw material to simulate enzymolysis by using proteases in the online database ExPASy Peptide Cutter，and the potential biological activity，solubility，toxicity，and sensitization of peptides were filtered by in silico tools to select the most suitable proteases for preparing pigeon hemoglobin-derived antioxidant peptides.Different enzymolysis time，enzyme addition，and enzymolysis temperature were set as single factors，and the effects of different enzymolysis conditions on the hydrolysis degree and 1，1-diphenyl-2-picrylhydrazyl（DPPH）radical scavenging rate were studied.Based on the single-factor experiment，the optimal process conditions for preparing antioxidant peptides derived from pigeon hemoglobin by enzymolysis were explored by response surface methodology.The results indicated proteinase K was the optimal protease for the preparation of pigeon hemoglobin antioxidant peptides by enzymolysis，and the optimal conditions were pigeon hemoglobin concentration of 1 g/100 mL，pH8，enzymolysis time of 3 h，enzyme dosage of 415.54 U/g，and enzymolysis temperature of 65 ℃ .Under these conditions，through the validation test，the response value of the DPPH radical scavenging rate was（33.21±0.68）%，which was consistent with the predicted value.Therefore，the optimal processing conditions for enzymolysis of antioxidant peptides from pigeon hemoglobin were obtained.Moreover，the sodium laurylsulfonate-polyacrylamide gel electrophoresis（SDS-PAGE）test was used to find that the target band disappeared after the enzymolysis of pigeon hemoglobin under the optimal process conditions，which further confirmed this method as the optimal process for enzymolysis of pigeon hemoglobin-derived antioxidant peptides.