Microbial liberation of N-methylserotonin from orange fiber in gnotobiotic mice and humans.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin "mining" conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

A distinct clade of Bifidobacterium longum in the gut of Bangladeshi children thrives during weaning.

The gut microbiome has an important role in infant health and development. We characterized the fecal microbiome and metabolome of 222 young children in Dhaka, Bangladesh during the first two years of life. A distinct Bifidobacterium longum clade expanded with introduction of solid foods and harbored enzymes for utilizing both breast milk and solid food substrates. The clade was highly prevalent in Bangladesh, present globally (at lower prevalence), and correlated with many other gut taxa and metabolites, indicating an important role in gut ecology. We also found that the B. longum clades and associated metabolites were implicated in childhood diarrhea and early growth, including positive associations between growth measures and B. longum subsp. infantis, indolelactate and N-acetylglutamate. Our data demonstrate geographic, cultural, seasonal, and ecological heterogeneity that should be accounted for when identifying microbiome factors implicated in and potentially benefiting infant development.

Evaluating microbiome-directed fibre snacks in gnotobiotic mice and humans.

Changing food preferences brought about by westernization that have deleterious health effects1,2-combined with myriad forces that are contributing to increased food insecurity-are catalysing efforts to identify more nutritious and affordable foods3. Consumption of dietary fibre can help to prevent cardiovascular disease, type 2 diabetes and obesity4-6. A substantial number of reports have explored the effects of dietary fibre on the gut microbial community7-9. However, the microbiome is complex, dynamic and exhibits considerable intra- and interpersonal variation in its composition and functions. The large number of potential interactions between the components of the microbiome makes it challenging to define the mechanisms by which food ingredients affect community properties. Here we address the question of how foods containing different fibre preparations can be designed to alter functions associated with specific components of the microbiome. Because a marked increase in snack consumption is associated with westernization, we formulated snack prototypes using plant fibres from different sustainable sources that targeted distinct features of the gut microbiomes of individuals with obesity when transplanted into gnotobiotic mice. We used these snacks to supplement controlled diets that were consumed by adult individuals with obesity or who were overweight. Fibre-specific changes in their microbiomes were linked to changes in their plasma proteomes indicative of an altered physiological state.

An approach for evaluating the effects of dietary fiber polysaccharides on the human gut microbiome and plasma proteome.

Increases in snack consumption associated with Westernized lifestyles provide an opportunity to introduce nutritious foods into poor diets. We describe two 10-wk-long open label, single group assignment human studies that measured the effects of two snack prototypes containing fiber preparations from two sustainable and scalable sources; the byproducts remaining after isolation of protein from the endosperm of peas and the vesicular pulp remaining after processing oranges for the manufacture of juices. The normal diets of study participants were supplemented with either a pea- or orange fiber-containing snack. We focused our analysis on quantifying the abundances of genes encoding carbohydrate-active enzymes (CAZymes) (glycoside hydrolases and polysaccharide lyases) in the fecal microbiome, mass spectrometric measurements of glycan structures (glycosidic linkages) in feces, plus aptamer-based assessment of levels of 1,300 plasma proteins reflecting a broad range of physiological functions. Computational methods for feature selection identified treatment-discriminatory changes in CAZyme genes that correlated with alterations in levels of fiber-associated glycosidic linkages; these changes in turn correlated with levels of plasma proteins representing diverse biological functions, including transforming growth factor type ß/bone morphogenetic protein-mediated fibrosis, vascular endothelial growth factor-related angiogenesis, P38/MAPK-associated immune cell signaling, and obesity-associated hormonal regulators. The approach used represents a way to connect changes in consumer microbiomes produced by specific fiber types with host responses in the context of varying background diets.

A multi-glycomic platform for the analysis of food carbohydrates.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.

Microbial communities in placentas from term normal pregnancy exhibit spatially variable profiles.

The placenta is the principal organ nurturing the fetus during pregnancy and was traditionally considered to be sterile. Recent work has suggested that the placenta harbours microbial communities, however the location and possible function of these microbes remain to be confirmed and elucidated. Here, we employed genomic DNA sequencing of multiple variable (V) regions of the bacterial 16S ribosomal gene, to interrogate microbial profiles in term pregnancies, from the basal plate, which is in direct contact with maternal uterine, endothelial, and immune cells; placental villi, which are bathed in maternal blood, and fetal membranes, which encapsulate the amniotic cavity. QIIME, R package "Phyloseq" analysis was used to assess alpha and beta diversity and absolute abundance of the 16S rRNA gene per location. We demonstrate that (1) microbiota exhibit spatially distinct profiles depending on the location within the placenta and (2) "semi-composite" 16S profiles using multiple V regions validated by quantitative PCR analysis confirmed that distinct bacterial taxa dominate in different placental niches. Finally, profiles are not altered by mode of delivery. Together these findings suggest that there is niche-specificity to the placental microbiota and placental microbiome studies should consider regional differences, which may affect maternal, fetal, and/or neonatal health and physiology.