Novel human milk-derived peptide β-casein 65 repairs intestinal injury via modulating gut microbiota

Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in preterm infants, and effective pharmacologic options are scarce. Studies have demonstrated that human milk peptides can improve NEC, β-casein 65 is a novel peptide derived from preterm human milk, yet its specific function and mechanism remain unclear. Here we evaluated the therapeutic potential role of β-casein 65 in a mouse model of NEC. β-casein 65 improved survival and mitigated abdominal distension and hematochezia in experimental NEC models. Concomitantly, β-casein 65 significantly reduced pro-inflammatory cytokines (TNF-α and IL-6 by approximately 56% and 71%, respectively) and increased intestinal barrier proteins compared with the NEC group. Using 16S rRNA sequencing and untargeted metabolomics, β-casein 65 altered gut microbiota profiling by lowering pathogenic Proteobacteria and promoting beneficial bacteria like Bifidobacterium and Akkermansia . Metabolomics analysis showed notable changes in immune-regulating metabolites, especially enriched in the IgA immune network. This finding not only provides a mechanistic understanding but also highlights β-casein 65 as a promising candidate for the development of preventative therapeutics for this devastating neonatal disease.