Abstract
Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity.
Highlights
From the ‡Department of Chemistry, University of California, Davis, California 95616; §Foods for Health Institute, University of California, Davis, California 95616; ¶Department of Food Science and Technology, University of California, Davis, California 95616; ʈDepartment of Viticulture and Enology, University of California, Davis, California 95616
Peaks from 25–26 min found in the milk sample were diminished in the fecal sample, whereas a peak at 8.5 min appeared in the fecal chromatogram that was absent in the milk (Fig. 2)
The bacteria are equipped with glycosidases that cleave Human milk oligosaccharides (HMOs) and other glycoconjugates, potentially leaving digestion products and free oligosaccharides to pass through the gut and exit as feces
Summary
Bif-TRFLP, Bifidobacterium-specific terminal restriction fragment length polymorphism; BLIR, Bifidobacterium longum/infantis ratio; ECC, extracted compound chromatogram; FA, formic acid; Fuc, Fucose; Gal, Galactose; GCC, graphitized carbon cartridge; Glc, Glucose; GlcNAc, N-acetylglucosamine; Hex, Hexose; HexNAc, N-acetylhexosamine; HMO, human milk oligosaccharide; IFLNH I, isomer 1 fucosyl-paralacto-N-hexaose; IFLNH III, isomer 3 fucosyl-paralacto-N-hexaose; LNH, lacto-N-hexaose; LNnH, lacto-N-neohexaose; LNT, lacto-N-tetraose; LNnT, lacto-Nneotetraose; MFLNH I, monofucosyllacto-N-hexaose I; Neu5Ac, N-acetylneuraminic acid; p-LNH, para-lacto-N-hexaose; 2ЈFL, 2Ј-fucosyllactose. Different microbial species and strains in the infant gastrointestinal tract have their own mechanisms for HMO catabolism leading to potentially diverse digested products in the feces. Infantis catabolizes the entire HMO internally, whereas B. bifidum secretes extracellular glycosidases for cleaving HMOs, leading to digested HMO products in the intestinal lumen [29, 30] These studies show that degraded glycans are created through the activity of key glycosyl hydrolases and can link relevant glycosidases with specific bacteria, providing insight into possible gut microbiota degradation pathways witnessed in feces. Intact and degraded oligosaccharides from milk glycoconjugates were identified and a fecal glycan library was created for the rapid and comprehensive analysis of postpartum alterations to the fecal glycome
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