Abstract: This study aimed to systematically elucidate the mechanism by which walnut pellicle polyphenols modulate alkaline protease hydrolysis of walnut protein. First, by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we identified nine characteristic polyphenolic components in the pellicle, including ellagic acid, gallic acid, and catechin. Subsequently, enzymatic kinetic analysis using the Lineweaver-Burk plot revealed that the polyphenol extract acted as an uncompetitive inhibitor of alkaline protease, that is, polyphenols bound selectively to the enzyme-substrate complex, forming an inactive ternary complex, which resulted in a decreased Michaelis constant (Km) and a reduced maximum reaction velocity (Vmax). Finally, Fourier transform infrared spectroscopy (FTIR) was employed to analyze the changes in protein secondary structure induced by polyphenol-protease interaction. The analysis revealed that the phenolic hydroxyl groups of the polyphenols formed new hydrogen bonds with the protease backbone, thereby reconstructing the protein’s hydrogen-bonding network. This reconstruction led to a blue shift in the amide I band and an increase in the proportion of random coil, consequently altering the enzyme’s conformation and catalytic microenvironment. This study comprehensively elucidates the molecular mechanism by which walnut pellicle polyphenols regulate protein hydrolysis through uncompetitive inhibition from an integrated perspective of chemical composition, enzyme kinetics and protein structure. It provides a crucial theoretical foundation for the targeted optimization of walnut protein enzymatic hydrolysis and the enhancement of walnut peptide yield.

Key words: walnut meal; walnut pellicle polyphenols; alkaline protease; uncompetitive inhibition; polyphenol-protein interaction