Abstract: Black shark skin was enzymatically hydrolyzed by protease to produce antioxidant peptides. The mechanism of action of the peptides was explored. The hydrolysis process was optimized based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity and also by taking into account degree of hydrolysis. The antioxidant peptides were separated and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The amino acid sequences were identified by electrospray ionization quadrupole-time of flight mass (ESI Q-TOF MS), and the in vitro antioxidant activity of the purified peptides was analyzed. The optimal hydrolysis parameters were determined as follows: use of alkaline protease as the best enzyme, enzyme dosage 311 U/mL, substrate concentration 3%, temperature 45 ℃, pH 8.0 and time 4.9 h. The hydrolysate obtained with a degree of hydrolysis of 14.21% under the optimized conditions scavenged 77.61% of DPPH radical. The hydrolysate was separated into 26 fractions. F19 was found to possess the strongest DPPH radical scavenging activity. The total ion current peak with higher antioxidant activity (at 10.02 min retention time) was selected for mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analysis. The amino acid sequence of F19 was determined as Pro-Gly-Gly-Thr-Met. F19 at 1.0 mg/mL scavenged 75.01% of DPPH radical, and its oxygen radical absorption capacity (ORAC, calculated as Trolox equivalent) was 2 490.01 μmol/g. Pro and Met residues played a leading role in the antioxidant activity of F19. This study provides a theoretical basis for understanding the antioxidant mechanism and the structureactivity relationship of the pentapeptide derived from black shark skin.

Key words: black shark skin, antioxidant peptide, isolation and purification, sequence