Mechanistic insights into the preventive efficacy of 21-day prefeeding with β-galactosidase hydrolyzed milk in DSS-induced ulcerative colitis mice

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with limited preventive strategies. Here, we investigated the 21-day preventive effects of β -galactosidase hydrolyzed milk (Milk β -gal) against Dextran Sulfate Sodium (DSS)-induced UC in mice. C57BL/6 mice were randomly divided into control group (Control), DSS-induced UC model group (DSS), UC model group pretreated with regular milk group (Milk), and UC model group pretreated with β -galactosidase hydrolyzed milk group (Milk β -gal). After a 21-day pre-feeding period with milk and Milk β -gal, colitis was induced with DSS in the drinking water. We assessed disease severity through body weight change in mouse body weight, disease activity index (DAI) scores, and colon length. Assess colonic histopathology using hematoxylin and eosin (HE) staining. Detect changes in intestinal permeability using FITC. Use qPCR and Western blot analysis to measure the expression levels of inflammatory factors and cellular signaling pathway proteins in colonic tissue. Measure malondialdehyde (MDA) and superoxide dismutase (SOD) levels in colon tissue alongside liver and kidney injury markers in peripheral blood to assess physiological and biochemical indicators. 16S rRNA high-throughput sequencing was used to analyse changes in the gut microbiota of mouse faeces and observe microbial community structure and composition. Milk β -gal significantly attenuated colitis severity, as evidenced by reduced body weight loss, lower disease activity index (DAI), mitigated colon shortening, diminished histopathological damage, and restored colonic barrier integrity. Mechanistically, Milk β -gal suppressed pro-inflammatory cytokines (TNF- α , IL-6, IL-1 β , and IL-33) and oxidative mediators (iNOS, COX-2) while maintaining anti-inflammatory cytokines (IL-10, TGF- β ) at intermediate levels, suggesting a restored equilibrium between inflammatory and reparative pathways. Moreover, Milk β -gal reversed DSS-induced downregulation of ZO-1 and MUC-2, thereby counteracting epithelial barrier dysfunction. Following DSS administration, Milk β -gal restored Firmicutes/Bacteroidetes ratio, reduced Campilobacterota, and elevated mucosa-repairing Helicobacter and uncultured Oscillospiraceae . These coordinated improvements in host responses and microbial ecology suggest that the β -galactosidase hydrolyzed milk may represent a promising dietary strategy for mitigating colitis in a murine model, providing a foundation for further investigation.