The Protection And Regulation Of Whey Protein Hydrolysates Against Oxidative Stress

Milk protein is abundant in China. Much attention has been given to rational exploitationof milk protein resource. Various bioactive peptides have been found in degradation productsof milk proteins. In particular, antioxidant peptides of whey protein hydrolysates became a hottopic because of its economy, safety and efficiency. The objective of this study was to explorebiological effects of WPHs as well as the connection between characteristics of WPHs andantioxidant abilities through evaluating the antioxidant abilities in vitro as well as studying theprotection against oxidative stress.WPHs were prepared with pepsin and trypsin, absorbed with DA201-C macroporousabsorption resin by dynamic adsorption and eluted using water and20%-60%ethanol. Gradedelution samples of WPHs named as M20，M40and M60were collected. Amino acidcomponent, molecular weight distribution, and antioxidant ability were tested. M20-M60have increasing hydrophobic Q value as well as increasing antioxidant capability.Graded elution samples of WPHs （M20-M60） were applied to PC12cell model to studytheir protective effect against H₂O₂-induced oxidative stress by investigating cells viabilityloss, the change of cell morphology, and apoptosis rate. The results indicated that M20-M60had good protective effect against oxidative stress. The protective effect of WPHs present atrend: M60>M40>M20. Among graded elution samples of WPHs, M60has the best protecteffect. M60increased19.3%PC12cells viability and inhibited28.6%apoptosis.The mechanism research was introduced in the last part of this paper. Flow cytometrywas used to assess the accumulation of reactive oxygen species （ROS）, Ca²⁺levels and themitochondrial membrane potential （MMP）. Western blot and test kit were used to investigateoxidative-stress-related proteins and enzyme. As illustrated, WPHs regulated themitochondrial apoptosis. What’s more, the ability of regulation presents a trend:M60>M40>M20. M60had the best ability to inhibit the accumulation of ROS, suppress Ca²⁺levels, stabilize MMP increased with the increasing hydrophobic and regulateoxidative-stress-related proteins and enzyme. At the same time, M60increased12.91%expression of anti-apoptosis protein Bcl-2while inhibiting25.12%expression of Bax. Wealso found WPHs could block H₂O₂-mediate activation of Caspase-3as well as degradation ofPARP.In conclusion, WPHs protect against H₂O₂-induced oxidative stress. In particular, whileWPHs were graded throught hydrophobicity, the graded elution sample with higher Q valueachieved a better ability to regulate apoptosis induced by oxidative stress via mitochondrialpathway.