Comparative effects of digested human milk and infant formulae on peptide transport and gut barrier function in infant- and adult-like Caco-2/HT29-MTX models

Early postnatal nutrition is critical for gut barrier maturation, yet infant formulae only partially replicate the functional benefits of human milk. Using infant- and adult-like Caco-2/HT29-MTX models, this in vitro study compared the effects of gastrointestinal digested human milk, dairy-, soya-, and amino acid–based infant formulae on gut barrier integrity and peptide transport. Barrier function was assessed by transepithelial electrical resistance and occludin–actin colocalization, while amino acid and peptide transport were profiled by liquid chromatography plus mass spectrometry. In the infant-like model, human milk uniquely enhanced occludin–actin co-localization ( P < 0.05) and increased the abundance of basolateral bioactive peptides. Amino acid–based infant formula significantly increased occludin fluorescence intensity in infant-like monolayers but reduced transepithelial electrical resistance (TEER) in adult-like monolayers. For all digested foods, peptide transport and diversity were significantly greater in infant-like than in adult-like monolayers, with human milk promoting the highest basolateral peptide load. These findings demonstrate distinct functional differences between human milk and infant formulas in their interaction with the gut barrier. They also underscore the importance of using age-appropriate in vitro models for accurately evaluating dietary effects during early life, highlighting the need for further research to optimize infant nutrition strategies. • Human milk enhances occludin–actin co-localization in infant-like epithelium. • Amino acid–based formula increased occludin signal in the infant-like epithelium. • Peptide transport is higher in infant- vs. adult-like model. • Bioactive human milk peptides show higher translocation in the infant-like epithelium. • Monolayer brush border enzymes further digest dietary peptides.