Abstract: This study explored optimal conditions for the enzymatic preparation of antioxidant peptides from tilapia skin collagen and the antioxidant activity of antioxidant peptides. Sequential hydrolysis with alkaline protease, trypsin and Orientase 20A was the best way to prepare antioxidant peptides from tilapia skin collagen, as shown by a comparison of the effects of single use and sequential combinations of proteases on DPPH radical scavenging activity of hydrolyzed tilapia skin collagen. In the final step (Orientase 20A) of the hydrolysis process, process parameters including temperature, time, pH and enzyme/substrate ratio were optimized using response surface methodology based on degree of hydrolysis (DH) and DPPH radical scavenging activity of hydrolyzed tilapia skin collagen to be 41.74 ℃, 3.97, 6 h and 1.5%, respectively. Under these hydrolysis conditions, the predicted DH and DPPH radical scavenging activity of hydrolyzed tilapia skin collagen were 9.42% and 35.01%, respectively, and the observed values were 9.57% and 35.21%, respectively. Thus, the developed response surface model is feasible and can be used for practical prediction. The tilapia skin collagen hydrolysate obtained under the optimized hydrolysis conditions had potent scavenging activity against DPPH and ABTS+ radicals with an IC50 of 10.78 mg/mL and 8.26 mg/mL, respectively.

Key words: three-step enzymatic hydrolysis response, surface methodology, hydrolysate, antioxidant activity, tilapia skin collagen, peptide