Effects of Alcalase and Flavourzyme from different manufacturers on the antigenicity and mechanisms of β-Lactoglobulin

β-lactoglobulin (β-LG) is a major allergen in dairy, and enzymatic hydrolysis is an effective way to reduce its antigenicity. This research aimed to explore the differential mechanisms of the effects of Alcalase and Flavourzyme from different manufacturers, Novozymes (N) and Yuanye (Y), on the antigenicity of β-LG. Following optimization using Response Surface Methodology, the antigenicity reduction rates of β-LG by the two Alcalase enzymes were both higher than those of Flavourzyme. However, the degree of hydrolysis indicated that there was no linear relationship between the DH and antigenicity. The results of conformational changes indicated that compared with the other three enzymes, N-Alcalase treatment significantly altered the vibrational stretching of C=O and N-H groups, accompanied by a reduction in α-helix content, fluorescence intensity, and surface hydrophobicity. Peptidomics analysis further revealed that N-Alcalase effectively removed critical allergenic linear epitope regions of β-LG, including amino acids (AA) 16–22, 88–107, and 150–158, where AA 88–107 tends to exhibit higher allergenic potential. In contrast, Flavourzyme exhibited limited efficacy in this regard. The findings indicate that N-Alcalase holds greater potential for reducing β-LG antigenicity, providing a theoretical foundation and technical support for the production of hypoallergenic dairy-based materials.