Effects of extensive protein hydrolysate in supporting intestinal barrier function in vitro

Maternal breast milk is the optimal nutrition for preterm infants, supporting immune and gut maturation. When unavailable, alternatives include infant formulas or pasteurized donor milk, the latter conferring greater protection against feeding intolerance and necrotizing enterocolitis. This study examined the impact of cow's milk-derived extensively hydrolyzed proteins (eHP), widely used in formulas and human milk fortifiers, on intestinal barrier function and gene expression in fetal human intestinal organoid-derived monolayers, modeling premature epithelium.

Monolayers were exposed to free amino acids (AA), intact cow milk proteins (WP), or eHP at different concentrations, followed by inflammatory cytokines or commensal bacteria, to mimic physiologic gut conditions. Barrier integrity, permeability, and viability were measured using FITC-Dextran diffusion and LDH release. RNA-seq assessed transcriptional changes.

eHP at low and high concentrations decreased epithelial permeability at baseline, AA showed no effect, and WP only at high concentrations. Under inflammatory conditions, eHP significantly reduced epithelial barrier permeability; both eHP and AA improved cell viability at low concentrations. Transcriptomic analysis revealed modulation of proliferation- and regulation-related pathways, including NOTCH and WNT signaling.

In this gut model, eHP enhanced barrier function under baseline and inflammatory conditions, supporting their role in nutrition, though further validation is needed.

Extensively hydrolyzed proteins (eHP) derived from cow's milk improve intestinal barrier function and cell viability in a fetal organoid-derived model of premature human intestine. This is the first study to assess the functional and transcriptomic impact of eHP on primary intestinal epithelial cells derived from fetal organoids, a highly relevant model for preterm gut physiology. It demonstrates that eHP-unlike intact proteins or free amino acids-can mitigate inflammation-induced permeability and modulate pathways involved in epithelial proliferation and repair (e.g., NOTCH, WNT, E2F, G2M checkpoint). These findings highlight the potential of eHP as a nutritional strategy to enhance gut integrity and limit inflammatory damage in preterm infants, who are at high risk of intestinal complications such as NEC. This study paves the way for further mechanistic and clinical research on how peptide-based formulas might support immature gut function and reduce extraintestinal risks.