A new strategy to reduce casein antigenicity: Investigating immune response, gut microbiota dysbiosis, and metabolic changes in allergic mice.

As the primary allergen in cow's milk, casein (CN) has restricted the development of hypoallergenic infant formulas. This study proposes a targeted enzymatic hydrolysis strategy to eliminate the antigenic epitopes of CN by taking advantage of the hydrolytic characteristics of different proteases, and to evaluate its antigen-lowering effect from multiple perspectives with the help of an allergic mouse model. The results of LC-MS/MS and ELISA indicate that the SC group (subtilisin protease + chymotrypsin hydrolysate) could effectively remove antigen epitopes and significantly reduce the antigenicity of CN. In vivo experiments confirmed that the antibody and inflammatory factor levels of the SC group mice were comparable to those of the normal group, and the structural integrity of the spleen, lung and intestinal tissues was maintained. Compared with the CN group, the SC group significantly increased the abundance of beneficial bacteria (such as Ligilactobacillus and Carnobacterium), and effectively improved the metabolic disorders of fatty acid and bile acid caused by CN allergy. Spleen transcriptome analysis further revealed significant differences in gene expression profiles and related biological pathways between the CN and SC group, indicating that this enzymatic hydrolysis strategy can effectively alleviate immune dysregulation and metabolic disorders related to allergies. This study provides new insights into the mechanisms of milk allergy and lays a new methodological and theoretical foundation for targeted enzymatic strategy to reduce the allergenicity of milk proteins, especially CN.