Mammalian milk is a source of antimicrobial compounds such as xanthine oxidase (XO). The interplay of infant saliva, which contains the substrates for XO activity, and human milk containing XO has been recently shown to inhibit the growth of pathogenic bacteria. Based on the complex and protective mechanism observed in human milk, we hypothesized that bovine milk XO operates similarly, thus representing an opportunity to investigate its functionality in broader health implications. We demonstrated that bovine milk-hypoxanthine mixture (0 to 400 μM) inhibited several Gram-negative and -positive bacterial pathogens in a dose-dependent manner. Kinetic experiments revealed that XO catalyzed hypoxanthine reduction (K_m, 58.0 μM; V_max, 5.1 μmol^-1 min^-1 mg) resulted in the production of antimicrobial hydrogen peroxide. These results demonstrate that the antimicrobial properties of bovine milk XO are similar to those of human milk XO with significant implications for the development of novel products targeting infant health.

Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants.

Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC-MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host-microbe associations and membrane protein activities.

A novel α1-2-fucosyltransferase from Thermosynechococcus elongatus BP-1 (Te2FT) with high fucosyltransferase activity and low donor hydrolysis activity was discovered and characterized. It was used in an efficient one-pot multienzyme (OPME) fucosylation system for the high-yield synthesis of human blood group H antigens containing β1-3-linked galactosides and an important human milk oligosaccharide (HMOS) lacto-N-fucopentaose I (LNFP I) on preparative and gram scales. LNFP I was shown to be selectively consumed by Bifidobacterium longum subsp. infantis but not Bifidobacterium animalis subsp. lactis and is a potential prebiotic.

Consumption of mothers' milk is associated with reduced incidence and severity of enteric infections, leading to reduced morbidity in breastfed infants. Fucosylated and sialylated human milk oligosaccharides (HMO) are important for both direct antimicrobial action - likely via a decoy effect - and indirect antimicrobial action through commensal growth enhancement. Bovine milk oligosaccharides (BMO) are a potential source of HMO-mimics as BMO resemble HMO; however, they have simpler and less fucosylated structures. BMO isolated at large scales from bovine whey permeate were modified by the addition of fucose and/or sialic acid to generate HMO-like glycans using high-yield and cost-effective one-pot multienzyme approaches. Quadrupole time-of-flight LC/MS analysis revealed that 22 oligosaccharides were synthesized and 9 had identical composition to known HMO. Preliminary anti-adherence activity assays indicated that fucosylated BMO decreased the uptake of enterohemorrhagic Escherichia coli O157:H7 by human intestinal epithelial Caco-2 cells more effectively than native BMO.