Activity-Guided Identification of Prot2633 as a Major Proteolytic Contributor to the Reduced Allergenicity of Lacticaseibacillus paracasei XJ-003-Fermented Milk

Abstract Lactic acid bacterial fermentation offers a food-compatible strategy for reducing milk allergenicity, but the responsible proteolytic components remain poorly defined. We evaluated milk fermented with Lacticaseibacillus paracasei XJ-003 using pooled sera from milk-allergic donors and characterized its protease system. Activity-guided purification of cell pellet lysates yielded Prot2633, a native protease preparation dominated by S1 serine protease SP2685 and S9 oligopeptidase SP1714. Prot2633 showed maximal activity at pH 6.5 and 43 °C and reduced IgE-binding reactivity of casein and whey within defined hydrolysis windows. In sensitized BALB/c mice, XJ-003-fermented milk and Prot2633-hydrolyzed milk attenuated antibody responses, mast cell degranulation, Th2-associated inflammation, and intestinal barrier disruption, while producing treatment-associated changes in gut microbiota. These findings identify Prot2633 as an important, but not exclusive, proteolytic contributor to the reduced allergenic reactivity of XJ-003-fermented milk and support activity-guided development of protease-assisted dairy processing.