Glutamine and Isoleucine as the Key Amino Acids in Major IgE Linear Epitopes of α-Lactalbumin Identified by the Immune Peptide Microarray Assay For Chinese Bovine Milk Allergy

α-Lactalbumin (ALA) is the main allergen in bovine milk allergy. Due to differences in the principles of identification methods, variations in patient symptoms, and population characteristics, the identification of key amino acids in ALA epitopes is still incomplete. In this study, bioinformatics prediction was performed on key amino acids based on two potential IgE linear epitopes in ALA. Then, peptide AA66-77 was confirmed as the IgE linear epitope through alanine scanning mutagenesis and immunological analysis based on a peptide microarray, with glutamine (Q⁷³) and isoleucine (I⁷⁴) identified as key amino acids. Additionally, in vitro simulated digestion results indicated that the mutant peptide 73 (Q⁷³) and peptide 74 (I⁷⁴) were more easily digested, with the IgE binding capacity of digestion products significantly reduced at 60 or 90 min, while changes in the epitope were only observed at 120 min. Finally, the KU812 cell degranulation model showed that the mutant peptides induced significantly lower β-Hex release and IL-6 levels compared to the epitope, confirming the critical role of specific amino acids in allergenicity. These findings are significant for further research on the prevention and treatment of bovine milk allergy.