Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion

To extract antioxidant peptide from hoki frame protein hydrolysate (APHPH), we employed six proteases (pepsin, trypsin, papain, α-chymotrypsin, Alcalase and Neutrase) for enzymatic hydrolysis, and the antioxidant activities of their hydrolysates were investigated using both lipid peroxidation inhibition assay and free radical scavenging assay by electron spin resonance spin-trapping technique. Among hydrolysates, peptic hydrolysate, having the highest antioxidant activity, further separated into four groups using ultrafiltration membranes and purified consecutive chromatographic methods. Finally, the purified peptide had a molecular mass of 1801 Da, and amino acid sequence was identified as Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-Ala-Cys-Pro-Asp-Phe-Asn. APHPH inhibited lipid peroxidation higher than that of α-tocopherol as positive control and efficiently quenched different sources of free radical: 1,1-diphenyl-2-pycryl-hydrazyl (IC50=41.37 μM), hydroxyl (IC50=17.77 μM), peroxyl (IC50=18.99 μM) and superoxide radicals (IC50=172.10 μM). Furthermore, APHPH decreased t-butylhydroperoxide-induced cytotoxicity on human embryonic lung fibroblasts and efficiently protected free-radical-induced DNA damage.