Structure-activity relationship analysis of the ACE inhibitory activity of three walnut protein hydrolysates and elucidation of the ACE inhibitory mechanism of its derived peptides

Abstract

Walnut protein, a high-quality plant protein, shows potential as a source of ACE inhibitory peptides. Digestive enzymes can effectively hydrolyze walnut protein to release peptide fragments with ACE inhibitory activity. However, existing research has focused on the overall digestive process, with limited exploration into how digestive enzymes hydrolyze walnut protein and the biological characteristics of the hydrolysates, particularly their ACE inhibitory activity. In this study, walnut protein was hydrolyzed using pepsin, trypsin, and chymotrypsin. Our results showed that chymotrypsin efficiently hydrolyzed walnut protein, with the chymotrypsin hydrolysate exhibiting the highest ACE inhibitory activity, demonstrated by an IC₅₀ value of 1.725 mg/mL. Further analysis revealed that the chymotrypsin hydrolysate was rich in hydrophobic and aromatic amino acids, which correlated positively with ACE inhibition. The highly active peptide YHP was identified through molecular docking and exhibited competitive inhibition properties, with an IC₅₀ value of 0.248 mg/mL. Molecular dynamics simulations, isothermal titration calorimetry, and atomic force microscopy analyses confirmed the stability and affinity of YHP for binding to ACE. Additionally, YHP demonstrated high stability during simulated gastrointestinal digestion. This study provides a significant foundation for the advanced processing of walnut protein and the development of novel ACE inhibitors.