Peptidomics and molecular docking reveal digestion-resistant IgE-binding epitopes in bovine β-lactoglobulin and α-lactalbumin from simulated infant digestion

β-Lactoglobulin (BLG) and α-lactalbumin (ALA) are the primary allergens in whey protein. However, gastrointestinal digestion can induce structural rearrangements of their IgE-binding epitopes, complicating the precise evaluation of allergenicity. This study developed a novel approach to identify digestion-resistant antigenic epitopes of BLG and ALA through simulated in vitro infant digestion. A peptidomics approach based on mass spectrometry, combined with high-throughput molecular docking, was employed to predict peptide interactions with the IgE fragment of antigen binding. Seven antigenic epitopes and 22 key amino acids were identified from BLG-derived peptides, and five antigenic epitopes and 18 key amino acids were identified from ALA-derived peptides. The allergenicity of synthetic epitopes ranged from 40 % to 80 %, and the dissociation constant between epitopes and IgE ranged from 17.90 nM to 279.01 nM, confirming their strong IgE-binding potential. Notably, this synergistic integration of computational prediction and experimental validation establishes a paradigm for characterizing digestion-resistant antigenic epitopes.