Davis Jasmine C C, Totten Sarah M, Huang Julie O, Nagshbandi Sadaf, Kirmiz Nina, Garrido Daniel A, Lewis Zachery T, Wu Lauren D, Smilowitz Jennifer T, German J Bruce, Mills David A, Lebrilla Carlito B
From the ‡Department of Chemistry, University of California, Davis, California 95616; §Foods for Health Institute, University of California, Davis, California 95616;
From the ‡Department of Chemistry, University of California, Davis, California 95616;
Mol Cell Proteomics. 2016 Sep;15(9):2987-3002. doi: 10.1074/mcp.M116.060665. Epub 2016 Jul 19.
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.
母乳中的聚糖含量丰富，以游离寡糖形式存在，或与蛋白质和脂质结合。游离人乳寡糖（HMOs）通过刺激有益细菌生长，同时防止有害细菌与肠道上皮细胞结合，发挥益生元的作用。细菌通过开发分解聚糖的酶，适应了肠道中富含聚糖的环境。HMOs的减少和聚糖消化产物的增加表明了微生物群体中活性酶的存在。在本研究中，我们定量了完整HMOs的消失情况，并对肠道中由微生物酶作用于HMOs和母乳中的糖缀合物所产生的聚糖消化产物进行了表征。采用纳升液相色谱-质谱联用技术对纯母乳喂养婴儿粪便样本中的寡糖进行提取和分析。在粪便样本中发现了完整的HMOs，此外，还发现了与降解的HMOs和非HMO基化合物相对应的其他寡糖。后一类化合物是通过与天冬酰胺残基的连接断裂以及N-聚糖的壳二糖核心断裂而释放的N-聚糖片段。粪便样本的标记基因测序显示双歧杆菌是婴儿胃肠道中的主要菌群。然后表达了来自长双歧杆菌亚种的一种糖苷酶，用于体外消化HMOs，结果表明粪便中消化的寡糖与糖苷酶对HMOs的作用相对应。内切糖苷酶的类似表达也表明细菌酶可释放N-聚糖。尽管双歧杆菌可能在肠道中占主导地位，但特定的少数菌种也可能是粪便中观察到的主要产物的产生原因。尽管如此，酶活性与各细菌中已知的糖苷酶密切相关，表明微生物丰度与分解代谢活性之间存在直接关系。
