Enzymatic Preparation and Identification of Walnut Antioxidant Peptides and Their Inhibitory Mechanism on Lipoxygenase

Abstract:

To enhance the utilization of walnut meal, walnut protein was enzymatically hydrolyzed by trypsin and the purified hydrolysates were separated to analyze the antioxidant peptides. The preparation conditions were optimized by using superoxide anion free-radical scavenging as the main indicator. The purified components were sequenced by LC-MS/MS and the peptides were screened by docking binding energy and protein stability index. Under optimum hydrolysis conditions, the superoxide anion free-radical scavenging rate of hydrolysate was 26.57%. Altogether four hydroly- sate fractions were separated by Smartdex G-50, among which, the hydrolysate, namely D-4 displayed the highest scavenging rate at the same concentration. The sequence of D-4 component was identified and R.AGIQFPVG.R was presumed to be the main bearer of antioxidant activity, as determined by virtual computer screening. Through molecular docking with lipoxygenase, it was found that R.AGIQFPVG.R was able to form hydrogen bonds with inactive center of lipoxygenase with a binding energy of -21 933.85 J/mol. The docking results showed that R.AGIQFPVG.R bound to the lipoxygenase inactive site via Glu186, indicating that the peptide acted mainly as a non-competitive inhibitor. This study conducted an enzymatic hydrolysis of walnut meal to prepare antioxidant peptides to provide a reference research for the screening of natural antioxidants.