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Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species were Pichia kudriavzevii and Pichia inanshurica, followed by Saccharomyces cerevisiae. Leuconostoc pseudomesenteroides (glucose and fructose fermenting), Fructobacillus tropaeoli-like (fructose fermenting), and Lactobacjllus fermenturn (citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates. Tatumella saanichensjs and Tatumella punctata were among the members of the family Enterobacteriaceae present during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely, Candida sorbosivorans-ljke. Acetic acid bacteria, whose main representative was Acetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans. [ABSTRACT FROM AUTHOR]

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of five butyrate- producing clostridial cluster XIVa colon bacteria grown on acetate plus fructose, oligofructose, inulin, or lactate were performed. A gas chromatography method was set up to assess H2 and CO2 production online and to ensure complete coverage of all metabolites produced. Method accuracy was confirmed through the calculation of electron and carbon recoveries. Fermentations with Anaerostipes caccae DSM 14662T, Roseburia faecis DSM 16840T, Roseburia hominis DSM 16839T, and Roseburia intestinalis DSM 14610T revealed similar patterns of metabolite production with butyrate, CO2, and H2 as the main metabolites. R. faecis DSM 16840T and R. intestinalis DSM 14610T were able to degrade oligofructose, displaying a nonpreferential breakdown mechanism. Lactate consumption was only observed with A. caccae DSM 14662T. Roseburia inulinivorans DSM 16841T was the only strain included in the present study that was able to grow on fructose, oligofructose, and inulin. The metabolites produced were lactate, butyrate, and CO2, without H2 production, indicating an energy metabolism distinct from that of other Roseburia species. Oligofructose degradation was nonpreferential. In a coculture of R. inulinivorans DSM 16841T with the highly competitive strain Bifidobacterium longum subsp. longum LMG 11047 on inulin, hardly any production of butyrate and CO2 was detected, indicating a lack of competitiveness of the butyrate producer. Complete recovery of metabolites during fermentations of clostridial cluster XIVa butyrate-producing colon bacteria allowed stoichiometric balancing of the metabolic pathway for butyrate production, including H2 formation. [ABSTRACT FROM AUTHOR]

Four bifidobacteria, each representing a cluster of strains with specific inulin type fructan degradation capacities, were grown in coculture fermentations with Bacteroides thetaiotaomicron LMG 11262, a strain able to metabolize both oligofructose and inulin. In a medium for colon bacteria with inulin as the sole added energy source, the ability of the bifidobacteria to compete fdr this substrate reflected phenotypical variation. Bifidobac terium breve Yakult, a strain that was not able to degrade oligofructose or inulin, was outcompeted by B. thetaiotaomicron LMG 11262. Bifidobacterium adoIescentis LMG 10734, a strain that could degrade oligofructose (displaying a preferential breakdown mechanism) but that did not grow on inulin, managed to become competitive when oligofructose and short fractions of inulin started to accumulate in the fermentation medium. Bifidobacterium angulatum LMG 11039[supT], a strain that was previously shown to degrade all oligofructose fractions simultaneously and to be able to partially break down inulin, was competitive from the beginning of the fermentation, consuming short fractions of inulin from the moment they appeared. Bifidobacterium longum LMG 11047, representing a cluster of bifidobacteria that shared both high fructose consumption and oligo fructose degradation rates and were able to perform partial breakdown of inulin, was the dominating strain in a coculture with B. thetaiotaornicron LMG 11262. These observations indicate that distinct subgroups within the large intestinal Bifidobacterium population will be stimulated by different groups of prebiotic inulin type fructans, a variation that could be reflected in differences concerning their health promoting effects. [ABSTRACT FROM AUTHOR]

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium strains grown on fructose, oligofructose, or inulin were performed. A principal component analysis of the data sets, expanded with the results of a genetic screen concerning the presence of a β-fructofuranosidase gene previously encountered in Bifidobacterium animalis subsp. lactis DSM 10140T, revealed the existence of four clusters among the bifidobacteria tested. Strains belonging to a first cluster could not degrade oligofructose or inulin. Strains in a second cluster could degrade oligofructose, displaying a preferential breakdown mechanism, but did not grow on inulin. Fructose consumption was faster than oligofructose degradation. A third cluster was composed of strains that degraded all oligofructose fractions simultaneously and could partially break down inulin. Oligofructose degradation was substantially faster than fructose consumption. A fourth, smaller cluster consisted of strains that shared high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin. For all strains, a metabolic shift toward more acetate, formate, and ethanol production, at the expense of lactate production, was observed during growth on less readily fermentable energy sources. No correlation between breakdown patterns and the presence of the f3-fructofuranosidase gene could be detected. These variations indicate niche-specific adaptation of bifidobacteria and could have in vivo implications on the strain specificity of the stimulatory effect of inulin-type fructans on bifidobacteria. [ABSTRACT FROM AUTHOR]

In vitro coculture fermentations of Bifidobacterium longum BB536 and two acetate-converting, butyrate-producing colon bacteria, Anaerostipes caccae DSM 14662 and Roseburia intestinalis DSM 14610, with oligo-fructose as the sole energy source, were performed to study interspecies interactions. Two clearly distinct types of cross-feeding were identified. A. caccae DSM 14662 was not able to degrade oligofructose but could grow on the fructose released by B. longum BB536 during oligofructose breakdown. R. intestinalis DSM 14610 could degrade oligofructose, but only after acetate was added to the medium. Detailed kinetic analyses of oligofructose breakdown by the last strain revealed simultaneous degradation of the different chain length fractions, in contrast with the preferential degradation of shorter fractions by B. longum BB536. In a coculture of both strains, initial oligofructose degradation and acetate production by B. longum BB536 took place, which in turn also allowed oligofructose breakdown by R. intestinalis DSM 14610. These and similar cross-feeding mechanisms could play a role in the colon ecosystem and contribute to the combined bifidogenic/butyrogenic effect observed after addition of inulin-type fructans to the diet. [ABSTRACT FROM AUTHOR]

Virome and 16/18S analyses were performed on 304 longitudinal fecal samples of eight infants. The gut virota--the collection of all viruses present in the gut--was dominated by bacteriophages, which were nearly absent at birth and emerged rapidly within the first weeks after birth. Over 85% of phage reads correspond to 305 near-complete genomes, most of which (70.5%) were individual infant-specific, including two crAs-sphages, whereas 7.8% of phages were present in at least 50% of infants. Bacterial hosts could be predicted for 80% of phages, mainly infecting Firmicutes. Strong temporal correlations between phages and their predicted bacterial hosts were identified for >40% of our phages, and together with the observation of a decreasing fraction of phages with a temperate lifestyle further suggest that phages are induced from early-colonizing bacteria. The vast majority (>86%) of identified eukaryotic viruses, known to cause gastroenteritis, occurred without clinical signs, and an increase in the rate of infection occurred after day-care entrance. On average, 112 genomic contigs of distinct anelloviruses could be identified per infant, some of which were shed at > 1 y. The identified plant viruses reflected the infant diet. Finally, the sporadic identification of fungi and parasites argues against the presence of such stable communities in the study population. Overall, this work provides a very high temporal resolution on how the different members of the infant gut microbiota, and especially the virome, develop over time in the gut of healthy infants, and might serve as valuable baseline knowledge for further studies investigating the effect of perturbations in the infant gut microbiota. [ABSTRACT FROM AUTHOR]

First insights on the human gut microbiome have been gained from medium-sized, cross-sectional studies. However, given the modest portion of explained variance of currently identified covariates and the small effect size of gut microbiota modulation strategies, upscaling seems essential for further discovery and characterisation of the multiple influencing factors and their relative contribution. In order to guide future research projects and standardisation efforts, we here review currently applied collection and preservation methods for gut microbiome research. We discuss aspects such as sample quality, applicable omics techniques, user experience and time and cost efficiency. In addition, we evaluate the protocols of a large-scale microbiome cohort initiative, the Flemish Gut Flora Project, to give an idea of perspectives, and pitfalls of large-scale faecal sampling studies. Although cryopreservation can be regarded as the gold standard, freezing protocols generally require more resources due to cold chain management. However, here we show that much can be gained from an optimised transport chain and sample aliquoting before freezing. Other protocols can be useful as long as they preserve the microbial signature of a sample such that relevant conclusions can be drawn regarding the research question, and the obtained data are stable and reproducible over time. [ABSTRACT FROM AUTHOR]

Recent metagenomic studies have demonstrated that the overall functional potential of the intestinal microbiome is rather conserved between healthy individuals. Here we assessed the biological processes undertaken in-vivo by microbes and the host in the intestinal tract by conducting a metaproteome analysis from a total of 48 faecal samples of 16 healthy adults participating in a placebo-controlled probiotic intervention trial. Half of the subjects received placebo and the other half consumed Lactobacillus rhamnosus GG for three weeks (1010 cfu per day). Faecal samples were collected just before and at the end of the consumption phase as well as after a three-week follow-up period, and were processed for microbial composition and metaproteome analysis. A common core of shared microbial protein functions could be identified in all subjects. Furthermore, we observed marked differences in expressed proteins between subjects that resulted in the definition of a stable and personalized microbiome both at the mass-spectrometry-based proteome level and the functional level based on the KEGG pathway analysis. No significant changes in the metaproteome were attributable to the probiotic intervention. A detailed taxonomic assignment of peptides and comparison to phylogenetic microarray data made it possible to evaluate the activity of the main phyla as well as key species, including Faecalibacterium prausnitzii. Several correlations were identified between human and bacterial proteins. Proteins of the human host accounted for approximately 14% of the identified metaproteome and displayed variations both between and within individuals. The individually different human intestinal proteomes point to personalized host-microbiota interactions. Our findings indicate that analysis of the intestinal metaproteome can complement gene-based analysis and contributes to a thorough understanding of the activities of the microbiome and the relevant pathways in health and disease. [ABSTRACT FROM AUTHOR]

Butyrate-producing bacteria are promising probiotic candidates to target microbial dysbiosis in gastrointestinal disorders like inflammatory bowel diseases. Butyricicoccus pullicaecorum 25-3T, a butyrate-producing clostridial cluster IV strain, is such a candidate. Little is known about its abundance in the colon microbiota and its butyrogenic properties. We used the M-SHIMER® , an in vitro simulator for the human intestinal microbial ecosystem, to study the effect of supplementing a single dose of B. pullicaecorum 25-3T on lumen- and mucus-associated microbiota of eight individuals. Butyricicoccus pullicaecorum was more abundant in mucus-associated microbiota compared with lumen microbiota. Supplementation with a single dose of B. pullicaecorum 25-3T resulted in a temporary increase in B. pullicaecorum bacteria in lumen compartment of all individuals. In two cases, the responders, an increased butyrate production was observed as compared with the control. 16S rRNA gene amplicon sequencing revealed the microbiota of responders to be different as compared to non-responder microbiota. We can conclude that B. pullicaecorum 25-3T is a mucus-associated bacterium whose potency to stimulate butyrate production is characterized by a large interindividual variability in terms of composition of the receiving microbial community. [ABSTRACT FROM AUTHOR]

FLEX i GUT is the first large-scale exposomics study focused on chronic low-grade inflammation. It aims to characterize human life course environmental exposure to assess and validate its impact on gut inflammation and related biological processes and diseases. The cumulative influences of environmental and food contaminants throughout the lifespan on certain biological responses related to chronic gut inflammation will be investigated in two Flemish prospective cohorts, namely the "ENVIRONAGE birth cohort", which provides follow-up from gestation to early childhood, and the "Flemish Gut Flora Project longitudinal cohort", a cohort of adults. The exposome will be characterised through biomonitoring of legacy and emerging contaminants, mycotoxins and markers of air pollution, by analysing the available metadata on nutrition, location and activity, and by applying state-of-the-art -omics techniques, including metagenomics, metabolomics and DNA adductomics, as well as the assessment of telomere length and measurement of inflammatory markers, to encompass both exposure and effect. Associations between exposures and health outcomes will be uncovered using an integrated -omics data analysis framework comprising data exploration, pre-processing, dimensionality reduction and data mining, combined with machine learning-based pathway analysis approaches. This is expected to lead to a more profound insight in mechanisms underlying disease progression (e.g. metabolic disorders, food allergies, gastrointestinal cancers) and/or accelerated biological ageing. [ABSTRACT FROM AUTHOR]

We are facing a global metabolic health crisis provoked by an obesity epidemic. Here we report the human gut microbial composition in a population sample of 123 non-obese and 169 obese Danish individuals. We find two groups of individuals that differ by the number of gut microbial genes and thus gut bacterial richness. They contain known and previously unknown bacterial species at different proportions; individuals with a low bacterial richness (23% of the population) are characterized by more marked overall adiposity, insulin resistance and dyslipidaemia and a more pronounced inflammatory phenotype when compared with high bacterial richness individuals. The obese individuals among the lower bacterial richness group also gain more weight over time. Only a few bacterial species are sufficient to distinguish between individuals with high and low bacterial richness, and even between lean and obese participants. Our classifications based on variation in the gut microbiome identify subsets of individuals in the general white adult population who may be at increased risk of progressing to adiposity-associated co-morbidities. [ABSTRACT FROM AUTHOR]