Abstract: In order to clarify the papain process for the preparation of chitooligosaccharides with high antioxidant activity, chitosan was used as raw material, the DPPH radical scavenging rate and total reducing power of the hydrolysates were used as detection indexes.The single factor experiment was used to explore the effects of enzyme substrate ratio, temperature, pH value and time on the antioxidant activity of the hydrolysates. The optimal conditions were determined as enzyme substrate ratio of 20%, temperature of 50. 00 ℃ and pH value of 4. 80. On the basis of single factor experiment, box Behnken response surface method was used to design and optimize the hydrolysis temperature, time and pH value. The optimal hydrolysis conditions were obtained as follows: temperature of 47. 74 ℃,pH of 4. 81 and time of 1 h. Under these conditions, the DPPH radical scavenging rate of chitosan hydrolysate was 65. 10% and the total reducing power was 0. 323. The experiment was repeated under the conditions of enzymolysis temperature of 48. 00 ℃, enzymolysis pH of 4. 80 and enzymolysis time of 1 h, the result showed that the DPPH radical scavenging rate of chitosan hydrolysates was 65. 43%±0. 71%, IC50 was 1. 86 mg / mL±0. 10 mg / mL, and the total reducing power was 0. 328 ± 0. 01, which was equivalent to the reducingpower of 39. 44 μg/ mL±2. 94 μg/ mL ascorbic acid. The average molecular weight of this chitooligosaccharide was 1 603 u and the yield of this chitooligosaccharide was 45. 07% ± 3. 52%. The result can provide a theoretical reference for the development of chitooligosaccharides with antioxidant activity.

Key words: papain, chitosan, chitooligosaccharides, DPPH radical scavenging rate, total reducing power