Abstract: In order to achieve high-valued utilization of algal residue, the present study investigated the preparation and activity of antioxidant peptides from Haematococcus pluvialis. Antioxidant peptides were prepared through 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity-guided fractionation of an enzymatic protein hydrolysate from H. pluvialis by using ultrafiltration, preparative high performance liquid chromatography (HPLC) and analytical HPLC. Amino acid sequence was identified by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The antioxidant peptides were screened and confirmed by molecular docking, and the mechanism of action of the antioxidant peptides was clarified. Furthermore, the in vivo antioxidant activity of the antioxidant peptides was evaluated by using Caenorhabditis elegans. The results showed that the antioxidant capacity of the protein hydrolysate from H. pluvialis was enhanced by 2.96-fold after purification, and fraction i, with the strongest free radical scavenging ability (which scavenged (96.97 ± 2.00)% of ABTS radical cation at 0.25 mg/mL) was selected for structural identification. Ten peptide sequences i-1 to i-10 were obtained. The molecular docking results showed that the minimum binding free energy between i-1 and ABTS radical cation was lowest, indicating that i-1 had the strongest antioxidant capacity. Furthermore, the novel antioxidant peptide was identified as KFTPAP. In vivo experiments showed that the peptide significantly improved antioxidant capacity in C. elegans (P < 0.05) in terms of the activities of superoxide dismutase (SOD) and catalase (CAT) and malondialdehyde (MDA) content, the effect being most pronounced at a concentration of 100 μmol/L. The potent antioxidant activity of this peptide may be due to the fact that hydrophobic amino acid residues concentrate at the C-terminus, and it contains phenylalanine residues and derives from chlorophyll proteins. This study provides a reference for the high value-added utilization of H. pluvialis residues and the development of food-derived antioxidants.

Key words: Haematococcus pluvialis; antioxidant peptide; purification; identification; molecular docking; Caenorhabditis elegans