Purification and characterization of antioxidant peptides from yak (Bos grunniens) bone hydrolysates and evaluation of cellular antioxidant activity

In this study, papain and alcalase were used to generate antioxidant peptides from yak bone protein. The antioxidant activities of hydrolysates in vitro were evaluated by 2,2′-azinobios-(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity, total reducing power, ferrous ion chelating ability and hydroxyl radical scavenging activity. The hydrolysates generated by alcalase possessed the best antioxidant activity among unhydrolyzed protein and samples treated by papain, but the antioxidant activity decreased after simulated gastrointestinal digestion in vitro. The products of simulated gastrointestinal digestion were separated by ultrafiltration and high performance liquid chromatography, and the amino acid sequences of peptides were identified by mass spectrometry. The digestion sites within peptides were predicted by a bioinformatics strategy, and ten peptides were selected for synthesis. Among 10 synthetic peptides, Gly-Phe-Hyp-Gly-Ala-Asp-Gly-Val-Ala, Gly-Gly-Pro-Gln-Gly-Pro-Arg and Gly-Ser-Gln-Gly-Ser-Gln-Gly-Pro-Ala possessed strong antioxidant activities, among which Gly-Phe-Hyp-Gly-Ala-Asp-Gly-Val-Ala had a significant cytoprotective effect in Caco-2 cells under oxidative stress induced by H2O2, which reduced the formation of reactive oxygen species and malondialdehyde, and improved the activity of antioxidant enzymes in cells. These results showed that yak bone peptides exhibited strong antioxidant activity and have a potential value as a new type of natural antioxidant.