Bioinformatics-Guided Targeted Hydrolysis and Peptide Mass Fingerprinting Analysis Reveal the Mechanism of...

To achieve targeted enzymatic hydrolysis of whey protein and uncover the intrinsic mechanisms underlying the reduction in antigenicity during enzymatic hydrolysis, this study integrated bioinformatics prediction, structural characterization, and peptide mass fingerprinting to systematically analyze whey protein hydrolysates. A total of 17 and 24 linear antigenic epitopes were predicted, and five proteases were selected for hydrolysis based on the key amino acid composition of epitopes and protease cleavage preferences. Compared with other proteases, Alcalase and Papain demonstrated superior degradation capacity for α-LA and β-LG, significantly increasing the proportion of hydrolyzed fragments below 1 kDa and exhibiting lower residual antigenicity. Peptide mass fingerprinting analysis revealed that proteases achieve precise degradation of antigenic epitopes by specifically recognizing key amino acids such as leucine, lysine, and glutamic acid and cleaving peptide bonds, thereby reshaping the peptide profile through removal of long peptides associated with important epitopes and ultimately reducing antigenicity.