Beta-casein in human milk is associated with infant stool frequency: A pilot proteomics study

Breast milk proteins may regulate gut function and defecation frequency; however, their specific roles in these processes remain poorly understood. We hypothesized that variations in specific human milk proteins are associated with differences in stool frequency among exclusively breastfed infants. Infants were categorized into three groups based on defecation frequency: Group A (normal, 1-3 times per day), Group B (frequent, >3 times per day), and Group C (infrequent, ≤1 time every 3-4 days). Each group consisted of three biological replicates (total N = 9). This study was an exploratory pilot study. Breast-milk samples were analyzed using tandem mass tag (TMT)-based proteomics to identify differentially expressed proteins across groups. Gene Ontology (GO), KEGG pathway enrichment, and protein-protein interaction (PPI) analyses were conducted to explore functional pathways. A total of 2563 proteins were identified; β-casein, α_s1-casein, and κ-casein were significantly downregulated in infants with lower defecation frequency (Group C vs. Group A; fold change <0.6, p < 0.05). β-casein showed the most significant difference (p = 0.0003). Enrichment analysis revealed pathways related to glycolipid metabolism, fibroblast proliferation, and coagulation cascades, while PPI analysis identified fibrinogen-related proteins as key interaction nodes. These findings suggest that β-casein levels in breast milk are associated with infant stool frequency. This may help clarify the association between constipation, diarrhea, and breastfeeding in infants and young children and may provide a theoretical reference for the subsequent development of targeted breast milk fortifiers and improvements in the intestinal environment in infants.