Human milk and mucosa-associated disaccharides impact on cultured infant fecal microbiota.

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods.

Extraction of the same novel homoglycan mixture from two different strains of Bifidobacterium animalis and three strains of Bifidobacterium breve.

Three strains of Bifidobacterium breve (JCM 7017, JCM 7019 and JCM 2258) and two strains of Bifidobacterium animalis subsp. lactis (AD011 and A1dOxR) were grown in broth cultures or on plates, and a standard exopolysaccharide extraction method was used in an attempt to recover exocellular polysaccharides. When the extracted materials were analysed by NMR it was clear that mixtures of polysaccharides were being isolated including exopolysaccharides (EPS) cell wall polysaccharides and intracellular polysaccharides. Treatment of the cell biomass from the B. breve strains, or the B. animalis subsp. lactis AD011 strain, with aqueous sodium hydroxide provided a very similar mixture of polysaccharides but without the EPS. The different polysaccharides were partially fractionated by selective precipitation from an aqueous solution upon the addition of increasing percentages of ethanol. The polysaccharides extracted from B. breve JCM 7017 grown in HBM media supplemented with glucose (or isotopically labelled D-glucose-1-13C) were characterised using 1D and 2D-NMR spectroscopy. Addition of one volume of ethanol generated a medium molecular weight glycogen (Mw=1×105 Da, yield 200 mg/l). The addition of two volumes of ethanol precipitated an intimate mixture of a low molecular weight ß-(1→6)-glucan and a low molecular weight ß-(1→6)-galactofuranan which could not be separated (combined yield 46 mg/l). When labelled D-glucose-1-13C was used as a carbon supplement, the label was incorporated into >95% of the anomeric carbons of each polysaccharide confirming they were being synthesised in situ. Similar 1H NMR profiles were obtained for polysaccharides recovered from the cells of B. animalis subsp. lactis AD011and A1dOxR (in combination with an EPS), B. breve JCM 7017, B. breve JCM 7019, B. breve JCM 2258 and from an EPS (-ve) mutant of B. breve 7017 (a non-EPS producer).

Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve.

Bifidobacterium breve represents one of the most abundant bifidobacterial species in the gastro-intestinal tract of breast-fed infants, where their presence is believed to exert beneficial effects. In the present study whole genome sequencing, employing the PacBio Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that genes encoding Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variable gene content (or variome). Using the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BS-seq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, we show that R/M systems typically impose a very significant barrier to genetic accessibility of B. breve strains, and that cloning of a methyltransferase-encoding gene may overcome such a barrier, thus allowing future functional investigations of members of this species.