Identification of a safe and effective antioxidant peptide from casein: <i>in vitro</i> and cellular studies

This study aimed to investigate the antioxidant potential of bovine casein hydrolysate and a pure peptide screened from it. The casein hydrolysate obtained by pepsin digestion exhibited obvious antioxidant activity <i>in vitro</i>, including potent Fe²⁺ chelating and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical scavenging activities. A novel tetrapeptide, VRYL, was predicted and successfully characterized using <i>in silico</i> techniques and showed excellent ABTS cation radical scavenging ability, with a half maximal inhibitory concentration of 0.36 mg/mL, which is significantly lower than that of the original hydrolysate. Moreover, at the cellular level, VRYL effectively protected against H₂O₂-induced oxidative stress damage in HepG2 cells and significantly reduced intracellular reactive oxygen species levels. The peptide VRYL not only exhibited excellent antioxidant capacity <i>in vitro</i> but also demonstrated its ability to mitigate oxidative stress damage in a cellular model. This study provides a new theoretical basis for the development of casein hydrolysates and their derived peptides in functional foods.