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Antioxidant Properties of Breast Milk in a Novel In Vitro Digestion/Enterocyte Model

Yao, Li*; Friel, James K*; Suh, Miyoung*; Diehl-Jones, William L

Journal of Pediatric Gastroenterology and Nutrition: June 2010 - Volume 50 - Issue 6 - p 670–676
doi: 10.1097/MPG.0b013e3181bab2d3
Original Articles: Hepatology and Nutrition

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.

*Department of Human Nutritional Sciences, Canada

Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada.

Received 5 December, 2008

Accepted 18 April, 2009

Address correspondence and reprint requests to William Diehl-Jones, PhD, Associate Professor, Department of Biological Sciences and Faculty of Nursing, University of Manitoba, Winnipeg, MB R2M 1X9, Canada (e-mail:

This work was supported by Advanced Food and Material Network (AFMNet) grants to J.K.F. and W.L.D.-J., by a small research grant from the Manitoba Institute of Child Health, and through an operating grant from the Natural Sciences and Engineering Council (NSERC) (J.K.F.). Equipment was purchased through a Canada Foundation for Innovation New Opportunities Grant to W.L.D.-J.

The authors report no conflicts of interest.

© 2010 Lippincott Williams & Wilkins, Inc.