Antioxidant properties of breast milk in a novel in vitro digestion/enterocyte model

OBJECTIVES: There is good evidence to suggest that human breast milk has antioxidant properties. Our primary goal was to investigate the antioxidant properties of human milk in a combined in vitro digestion/cell culture model that more closely replicates conditions in the gastrointestinal system of the preterm infant. MATERIALS AND METHODS: An in vitro digestion model was developed that incorporates both gastric and intestinal phases, based on reported luminal pH, digestive enzyme levels, and transit times observed in preterm infants. To mimic the human intestinal mucosa, 2 cell lines-Caco-2BBE and HT29-MTX-were cocultured on Matrigel, an artificial basement membrane substrate. Intracellular oxidative stress was measured with 2 broadly selective oxidant-sensitive dyes, and oxidative DNA damage was assessed by means of single-cell gel electrophoresis. RESULTS: Enterocyte differentiation and mucin secretion were observed by 14 seeding of cultures. Direct exposure to digested milk resulted in a loss of transepithelial electrical resistance; however, exogenous mucin mitigated this loss. Data suggested that both milk and digested milk alleviated oxidative stress in the coculture, and both reduced hydrogen peroxide-induced oxidative DNA damage, as demonstrated by the comet assay. CONCLUSIONS: Our results support the hypothesis that breast milk reduces oxidative stress in a cell culture model representative of the intestinal mucosa, and also confirmed the suitability of this combined in vitro digestion/cell culture system for investigating the physiologic effects of enteral nutrients such as breast milk, under conditions similar to those existing in the gastrointestinal system of the preterm infant.