Abstract: In this study, single factor experiment and response surface methodology (RSM) based on Box-Behnken design (BBD) were used together to optimize the hydrolysis process of Porphyra haitanensis protein by acid protease. The antioxidant activity and molecular mass distribution of the hydrolysates at different hydrolysis times were measured. The results showed that under the optimized hydrolysis conditions (substrate concentration of 2 g/100 mL, enzyme/substrate of 4 240 U/g, pH value of 3.7, temperature of 54 ℃, and hydrolysis time of 4 h), the highest degree of hydrolysis (DH) of 11.40% was obtained. The relationship between DH and antioxidant activity of the hydrolysates was investigated. It was found that the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the 4 h hydrolysate (DH = 11.40%) was the strongest with a half maximum inhibition concentration (IC50) of 0.68 mg/mL, and that small molecular mass peptides (< 1 kDa) accounted for 73.46% of the total peptides in the hydrolysate. This hydrolysate was fractionated by gel filtration chromatography, yielding an antioxidant peptide (named PHAP). PHAP at 1 mg/mL concentration scavenged DPPH and hydroxyl radicals by 73.32% and 30.15%, respectively. It was confirmed that PHAP could repair H2O2-induced oxidative damage in HepG2 cells by enhancing the activity of superoxide dismutase and decreasing the content of malondialdehyde.

Key words: Porphyra haitanensis; enzymatic hydrolysis; preparation; purification; antioxidant peptide