Abstract: In this study, we prepared 15 hydrolysates from yak milk casein by enzymatic hydrolysis with neutral protease, papain, alkaline protease, trypsin, and pepsin alone or in combination, which were screened for their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical scavenging capacity and protective effects against H2O2-induced oxidative stress in AML12 hepatocytes. The results revealed that the hydrolysates obtained with neutral protease, trypsin + papain, and trypsin + neutral protease + pepsin (20 × dilution) exhibited significant antioxidant activity; they not only efficiently scavenged free radicals but also increased the viability of injured cells to 69.38%, 65.95% and 63.32%, respectively (P < 0.01). The hydrolysates were fractionated and purified by DEAE-52 anion exchange and Sephadex G50 column chromatography, obtaining five antioxidant peptides (Z1-C, Z3-B, YM3-B, YZW1-B, and YZW3-B), which elevated cell viability to 72.20%–82.19% (P < 0.000 1). Fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) confirmed that these fractions significantly reduced the levels of oxidative injury markers (reactive oxygen species, malondialdehyde, and​ nitric oxide) and pro-inflammatory cytokines (interleukin-1β and tumor necrosis factor-α) in cells. These results demonstrated that yak milk casein enzymatic hydrolysates effectively protected hepatocytes from oxidative stress damage through synergistic antioxidant and anti-inflammatory actions, providing a theoretical foundation for developing targeted functional foods for liver disease intervention.

Key words: casein; antioxidant peptides; oxidative stress injury; AML12 cells