Summary: | <i>Pleurotus geesteranus</i> is a promising source of bioactive compounds. However, knowledge of the antioxidant behaviors <i>of P. geesteranus</i> protein hydrolysates (PGPHs) is limited. In this study, PGPHs were prepared with papain, alcalase, flavourzyme, pepsin, and pancreatin, respectively. The antioxidant properties and cytoprotective effects against oxidative stress of PGPHs were investigated using different chemical assays and H<sub>2</sub>O<sub>2</sub> damaged PC12 cells, respectively. The results showed that PGPHs exhibited superior antioxidant activity. Especially, hydrolysate generated by alcalase displayed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (91.62%), 2,2-azino-bis (3-ethylbenzothia zoline-6-sulfonic acid) (ABTS) radical scavenging activity (90.53%), ferric reducing antioxidant power, and metal ion-chelating activity (82.16%). Analysis of amino acid composition revealed that this hydrolysate was rich in hydrophobic, negatively charged, and aromatic amino acids, contributing to its superior antioxidant properties. Additionally, alcalase hydrolysate showed cytoprotective effects on H<sub>2</sub>O<sub>2</sub>-induced oxidative stress in PC12 cells via diminishing intracellular reactive oxygen species (ROS) accumulation by stimulating antioxidant enzyme activities. Taken together, alcalase hydrolysate of <i>P. geesteranus</i> protein can be used as beneficial ingredients with antioxidant properties and protective effects against ROS-mediated oxidative stress.
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