Beyond purified dietary fibre supplements: Compositional variation between cell wall fibre from different plants influences human faecal microbiota activity and growth in vitro.

Dietary fibre is a major energy source for the human gut microbiota, but it is unclear to what extent the fibre source and complexity affect microbial growth and metabolite production. Cell wall material and pectin were extracted from five different dicotyledon plant sources, apples, beet leaves, beetroots, carrots and kale, and compositional analysis revealed differences in the monosaccharide composition. Human faecal batch incubations were conducted with 14 different substrates, including the plant extracts, wheat bran and commercially available carbohydrates. Microbial activity was determined for up to 72 h by measuring gas and fermentation acid production, total bacteria (by qPCR) and microbial community composition by 16S rRNA amplicon sequencing. The more complex substrates gave rise to more microbiota variation compared with the pectins. The comparison of different plant organs showed that the leaves (beet leaf and kale) and roots (carrot and beetroot) did not give rise to similar bacterial communities. Rather, the compositional features of the plants, such as high arabinan levels in beet and high galactan levels in carrot, appear to be major predictors of bacterial enrichment on the substrates. Thus, in-depth knowledge on dietary fibre composition should aid the design of diets focused on optimizing the microbiota.

Habitual consumption of high-fibre bread fortified with bean hulls increased plasma indole-3-propionic concentration and decreased putrescine and deoxycholic acid faecal concentrations in healthy volunteers.

Only 6 to 8 % of the UK adults meet the daily recommendation for dietary fibre. Fava bean processing lead to vast amounts of high-fibre by-products such as hulls. Bean hull fortified bread was formulated to increase and diversify dietary fibre while reducing waste. This study assessed the bean hull: suitability as a source of dietary fibre; the systemic and microbial metabolism of its components and postprandial events following bean hull bread rolls. Nine healthy participants (53·9 ± 16·7 years) were recruited for a randomised controlled crossover study attending two 3 days intervention sessions, involving the consumption of two bread rolls per day (control or bean hull rolls). Blood and faecal samples were collected before and after each session and analysed for systemic and microbial metabolites of bread roll components using targeted LC-MS/MS and GC analysis. Satiety, gut hormones, glucose, insulin and gastric emptying biomarkers were also measured. Two bean hull rolls provided over 85 % of the daily recommendation for dietary fibre; but despite being a rich source of plant metabolites (P = 0·04 v. control bread), these had poor systemic bioavailability. Consumption of bean hull rolls for 3 days significantly increased plasma concentration of indole-3-propionic acid (P = 0·009) and decreased faecal concentration of putrescine (P = 0·035) and deoxycholic acid (P = 0·046). However, it had no effect on postprandial plasma gut hormones, bacterial composition and faecal short chain fatty acids amount. Therefore, bean hulls require further processing to improve their bioactives systemic availability and fibre fermentation.

Immune Responsiveness to LPS Determines Risk of Childhood Wheeze and Asthma in 17q21 Risk Allele Carriers.

Rationale: In murine models, microbial exposures induce protection from experimental allergic asthma through innate immunity. Objectives: Our aim was to assess the association of early life innate immunity with the development of asthma in children at risk. Methods: In the PASTURE farm birth cohort, innate T-helper cell type 2 (Th2), Th1, and Th17 cytokine expression at age 1 year was measured after stimulation of peripheral blood mononuclear cells with LPS in n = 445 children. Children at risk of asthma were defined based on single-nucleotide polymorphisms at the 17q21 asthma gene locus. Specifically, we used the SNP rs7216389 in the GSDMB gene. Wheeze in the first year of life was assessed by weekly diaries and asthma by questionnaire at age 6 years. Measurements and Main Results: Not all cytokines were detectable in all children after LPS stimulation. When classifying detectability of cytokines by latent class analysis, carrying the 17q21 risk allele rs7216389 was associated with risk of wheeze only in the class with the lowest level of LPS-induced activation: odds ratio (OR), 1.89; 95% confidence interval [CI], 1.13-3.16; P = 0.015. In contrast, in children with high cytokine activation after LPS stimulation, no association of the 17q21 risk allele with wheeze (OR, 0.63; 95% CI, 0.29-1.40; P = 0.258, P = 0.034 for interaction) or school-age asthma was observed. In these children, consumption of unprocessed cow's milk was associated with higher cytokine activation (OR, 3.37; 95% CI, 1.56-7.30; P = 0.002), which was in part mediated by the gut microbiome. Conclusions: These findings suggest that within the 17q21 genotype, asthma risk can be mitigated by activated immune responses after innate stimulation, which is partly mediated by a gut-immune axis.

ß-Glucan is a major growth substrate for human gut bacteria related to Coprococcus eutactus.

A clone encoding carboxymethyl cellulase activity was isolated during functional screening of a human gut metagenomic library using Lactococcus lactis MG1363 as heterologous host. The insert carried a glycoside hydrolase family 9 (GH9) catalytic domain with sequence similarity to a gene from Coprococcus eutactus ART55/1. Genome surveys indicated a limited distribution of GH9 domains among dominant human colonic anaerobes. Genomes of C. eutactus-related strains harboured two GH9-encoding and four GH5-encoding genes, but the strains did not appear to degrade cellulose. Instead, they grew well on ß-glucans and one of the strains also grew on galactomannan, galactan, glucomannan and starch. Coprococcus comes and Coprococcus catus strains did not harbour GH9 genes and were not able to grow on ß-glucans. Gene expression and proteomic analysis of C. eutactus ART55/1 grown on cellobiose, ß-glucan and lichenan revealed similar changes in expression in comparison to glucose. On ß-glucan and lichenan only, one of the four GH5 genes was strongly upregulated. Growth on glucomannan led to a transcriptional response of many genes, in particular a strong upregulation of glycoside hydrolases involved in mannan degradation. Thus, ß-glucans are a major growth substrate for species related to C. eutactus, with glucomannan and galactans alternative substrates for some strains.

Comparative genetic and physiological characterisation of Pectinatus species reveals shared tolerance to beer-associated stressors but halotolerance specific to pickle-associated strains.

Obligate anaerobic bacteria from the genus Pectinatus have been known to cause beer spoilage for over 40 years. Whole genome sequencing was performed on eleven beer spoilage strains (nine Pectinatus frisingensis, one Pectinatus cerevisiiphilus and one Pectinatus haikarae isolate), as well as two pickle spoilage species (Pectinatus brassicae MB591 and Pectinatus sottacetonis MB620) and the tolerance of all species to a range of environmental conditions was tested. Exploration of metabolic pathways for carbohydrates, amino acids and vitamins showed little difference between beer spoilage- and pickle spoilage-associated strains. However, genes for certain carbohydrate- and sulphur-containing amino acid-associated enzymes were only present in the beer spoilage group and genes for specific transporters and regulatory genes were uniquely found in the pickle spoilage group. Transporters for compatible solutes, only present in pickle-associated strains, likely explain their experimentally observed higher halotolerance compared to the beer spoilers. Genes involved in biofilm formation and ATP Binding Cassette (ABC) transporters potentially capable of exporting hop-derived antimicrobial compounds were found in all strains. All species grew in the presence of alcohol up to 5% alcohol by volume (ABV) and hops extract up to 80 ppm of iso-α-acids. Therefore, the species isolated from pickle processes may pose novel hazards in brewing.

Formate cross-feeding and cooperative metabolic interactions revealed by transcriptomics in co-cultures of acetogenic and amylolytic human colonic bacteria.

Interspecies cross-feeding is a fundamental factor in anaerobic microbial communities. In the human colon, formate is produced by many bacterial species but is normally detected only at low concentrations. Ruminococcus bromii produces formate, ethanol and acetate in approximately equal molar proportions in pure culture on RUM-RS medium with 0.2% Novelose resistant starch (RS3) as energy source. Batch co-culturing on starch with the acetogen Blautia hydrogenotrophica however led to the disappearance of formate and increased levels of acetate, which is proposed to occur through the routing of formate via the Wood Ljungdahl pathway of B. hydrogenotrophica. We investigated these inter-species interactions further using RNAseq to examine gene expression in continuous co-cultures of R. bromii and B. hydrogenotrophica. Transcriptome analysis revealed upregulation of B. hydrogenotrophica genes involved in the Wood-Ljungdahl pathway and of a 10 gene cluster responsible for increased branched chain amino acid fermentation in the co-cultures. Cross-feeding between formate-producing species and acetogens may be a significant factor in short chain fatty acid formation in the colon contributing to high rates of acetate production. Transcriptome analysis also indicated competition for the vitamin thiamine and downregulation of dissimilatory sulfate reduction and key redox proteins in R. bromii in the co-cultures, thus demonstrating the wide-ranging consequences of inter-species interactions in this model system.

A Diet Low in FODMAPs Reduces Symptoms in Patients With Irritable Bowel Syndrome and A Probiotic Restores Bifidobacterium Species: A Randomized Controlled Trial.

BACKGROUND & AIMS: Dietary restriction of fermentable carbohydrates (a low FODMAP diet) has been reported to reduce symptoms in some patients with irritable bowel syndrome (IBS). We performed a randomized, placebo-controlled study to determine its effects on symptoms and the fecal microbiota in patients with IBS. METHODS: We performed a 2×2 factorial trial of 104 patients with IBS (18-65 years old), based on the Rome III criteria, at 2 hospitals in the United Kingdom. Patients were randomly assigned (blinded) to groups given counselling to follow a sham diet or diet low in FODMAPs for 4 weeks, along with a placebo or multistrain probiotic formulation, resulting in 4 groups (27 receiving sham diet/placebo, 26 receiving sham diet/probiotic, 24 receiving low FODMAP diet /placebo, and 27 receiving low FODMAP diet/probiotic). The sham diet restricted a similar number of staple and non-staple foods as the low FODMAP diet; the diets had similar degrees of difficulty to follow. Dietary counselling was given to patients in all groups and data on foods eaten and compliance were collected. The incidence and severity of 15 gastrointestinal symptoms and overall symptoms were measured daily for 7 days before the study period; along with stool frequency and consistency. At baseline, global and individual symptoms were measured, along with generic and disease-specific health-related quality of life, using standard scoring systems. All data were collected again at 4 weeks, and patients answered questions about adequate symptom relief. Fecal samples were collected at baseline and after 4 weeks and analyzed by quantitative PCR and 16S rRNA sequencing. The co-primary endpoints were adequate relief of symptoms and stool Bifidobacterium species abundance at 4 weeks. RESULTS: There was no significant interaction between the interventions in adequate relief of symptoms (P = .52) or Bifidobacterium species (P = .68). In the intention-to-treat analysis, a higher proportion of patients in the low FODMAP diet had adequate symptom relief (57%) than in the sham diet group (38%), although the difference was not statistically significant (P = .051). In the per-protocol analysis, a significantly higher proportion of patients on the low FODMAP diet had adequate symptom relief (61%) than in the sham diet group (39%) (P = .042). Total mean IBS-Severity Scoring System score was significantly lower for patients on the low FODMAP diet (173 ± 95) than the sham diet (224 ± 89) (P = .001), but not different between those given probiotic (207 ± 98) or placebo (192 ± 93) (P = .721) Abundance of Bifidobacterium species was lower in fecal samples from patients on the low FODMAP diet (8.8 rRNA genes/g) than patients on the sham diet (9.2 rRNA genes/g) (P = .008), but higher in patients given probiotic (9.1 rRNA genes/g) than patients given placebo (8.8 rRNA genes/g) (P = .019). There was no effect of the low FODMAP diet on microbiota diversity in fecal samples. CONCLUSIONS: In a placebo-controlled study of patients with IBS, a low FODMAP diet associates with adequate symptom relief and significantly reduced symptom scores compared with placebo. It is not clear whether changes resulted from collective FODMAP restriction or removal of a single component, such as lactose. Co-administration of the multistrain probiotic increased numbers of Bifidobacterium species, compared with placebo, and might be given to restore these bacteria to patients on a low FODMAP diet. Trial registration no: ISRCTN02275221.

Sporulation capability and amylosome conservation among diverse human colonic and rumen isolates of the keystone starch-degrader Ruminococcus bromii.

Ruminococcus bromii is a dominant member of the human colonic microbiota that plays a 'keystone' role in degrading dietary resistant starch. Recent evidence from one strain has uncovered a unique cell surface 'amylosome' complex that organizes starch-degrading enzymes. New genome analysis presented here reveals further features of this complex and shows remarkable conservation of amylosome components between human colonic strains from three different continents and a R. bromii strain from the rumen of Australian cattle. These R. bromii strains encode a narrow spectrum of carbohydrate active enzymes (CAZymes) that reflect extreme specialization in starch utilization. Starch hydrolysis products are taken up mainly as oligosaccharides, with only one strain able to grow on glucose. The human strains, but not the rumen strain, also possess transporters that allow growth on galactose and fructose. R. bromii strains possess a full complement of sporulation and spore germination genes and we demonstrate the ability to form spores that survive exposure to air. Spore formation is likely to be a critical factor in the ecology of this nutritionally highly specialized bacterium, which was previously regarded as 'non-sporing', helping to explain its widespread occurrence in the gut microbiota through the ability to transmit between hosts.

Porcine Small and Large Intestinal Microbiota Rapidly Hydrolyze the Masked Mycotoxin Deoxynivalenol-3-Glucoside and Release Deoxynivalenol in Spiked Batch Cultures In Vitro.

Mycotoxin contamination of cereal grains causes well-recognized toxicities in animals and humans, but the fate of plant-bound masked mycotoxins in the gut is less well understood. Masked mycotoxins have been found to be stable under conditions prevailing in the small intestine but are rapidly hydrolyzed by fecal microbiota. This study aims to assess the hydrolysis of the masked mycotoxin deoxynivalenol-3-glucoside (DON3Glc) by the microbiota of different regions of the porcine intestinal tract. Intestinal digesta samples were collected from the jejunum, ileum, cecum, colon, and feces of 5 pigs and immediately frozen under anaerobic conditions. Sample slurries were prepared in M2 culture medium, spiked with DON3Glc or free deoxynivalenol (DON; 2 nmol/ml), and incubated anaerobically for up to 72 h. Mycotoxin concentrations were determined using liquid chromatography-tandem mass spectrometry, and the microbiota composition was determined using a quantitative PCR methodology. The jejunal microbiota hydrolyzed DON3Glc very slowly, while samples from the ileum, cecum, colon, and feces rapidly and efficiently hydrolyzed DON3Glc. No further metabolism of DON was observed in any sample. The microbial load and microbiota composition in the ileum were significantly different from those in the distal intestinal regions, whereas those in the cecum, colon and feces did not differ.IMPORTANCE Results from this study clearly demonstrate that the masked mycotoxin DON3Glc is hydrolyzed efficiently in the distal small intestine and large intestine of pigs. Once DON is released, toxicity and absorption in the distal intestinal tract likely occur in vivo This study further supports the need to include masked metabolites in mycotoxin risk assessments and regulatory actions for feed and food.

Formation of propionate and butyrate by the human colonic microbiota.

The human gut microbiota ferments dietary non-digestible carbohydrates into short-chain fatty acids (SCFA). These microbial products are utilized by the host and propionate and butyrate in particular exert a range of health-promoting functions. Here an overview of the metabolic pathways utilized by gut microbes to produce these two SCFA from dietary carbohydrates and from amino acids resulting from protein breakdown is provided. This overview emphasizes the important role played by cross-feeding of intermediary metabolites (in particular lactate, succinate and 1,2-propanediol) between different gut bacteria. The ecophysiology, including growth requirements and responses to environmental factors, of major propionate and butyrate producing bacteria are discussed in relation to dietary modulation of these metabolites. A detailed understanding of SCFA metabolism by the gut microbiota is necessary to underpin effective strategies to optimize SCFA supply to the host.

Modulation of the human gut microbiota by dietary fibres occurs at the species level.

BACKGROUND: Dietary intake of specific non-digestible carbohydrates (including prebiotics) is increasingly seen as a highly effective approach for manipulating the composition and activities of the human gut microbiota to benefit health. Nevertheless, surprisingly little is known about the global response of the microbial community to particular carbohydrates. Recent in vivo dietary studies have demonstrated that the species composition of the human faecal microbiota is influenced by dietary intake. There is now potential to gain insights into the mechanisms involved by using in vitro systems that produce highly controlled conditions of pH and substrate supply. RESULTS: We supplied two alternative non-digestible polysaccharides as energy sources to three different human gut microbial communities in anaerobic, pH-controlled continuous-flow fermentors. Community analysis showed that supply of apple pectin or inulin resulted in the highly specific enrichment of particular bacterial operational taxonomic units (OTUs; based on 16S rRNA gene sequences). Of the eight most abundant Bacteroides OTUs detected, two were promoted specifically by inulin and six by pectin. Among the Firmicutes, Eubacterium eligens in particular was strongly promoted by pectin, while several species were stimulated by inulin. Responses were influenced by pH, which was stepped up, and down, between 5.5, 6.0, 6.4 and 6.9 in parallel vessels within each experiment. In particular, several experiments involving downshifts to pH 5.5 resulted in Faecalibacterium prausnitzii replacing Bacteroides spp. as the dominant sequences observed. Community diversity was greater in the pectin-fed than in the inulin-fed fermentors, presumably reflecting the differing complexity of the two substrates. CONCLUSIONS: We have shown that particular non-digestible dietary carbohydrates have enormous potential for modifying the gut microbiota, but these modifications occur at the level of individual strains and species and are not easily predicted a priori. Furthermore, the gut environment, especially pH, plays a key role in determining the outcome of interspecies competition. This makes it crucial to put greater effort into identifying the range of bacteria that may be stimulated by a given prebiotic approach. Both for reasons of efficacy and of safety, the development of prebiotics intended to benefit human health has to take account of the highly individual species profiles that may result.

How to Manipulate the Microbiota: Prebiotics.

During the last century, human nutrition has evolved from the definition of our nutritional needs and the identification of ways to meet them, to the identification of food components that can optimise our physiological and psychological functions. This development, which aims to ensure the welfare, health and reduced susceptibility to disease during life, gave birth to the concept of "functional foods". In this context, there is an increasing interest in the physiological effects induced by the dense and diverse microbiota which inhabits the human colon and whose development depends on the fermentation of undigested food residues. Thus, much research aims at identifying ways to guide these impacts in order to benefit the health of the host. It is in this context that the concept of "prebiotics" was developed in the 1990s. Since then, prebiotics have stimulated extensive work in order to clarify their definition, their nature and their physiological properties in accordance with the evolution of knowledge on the intestinal microbiota. However many questions remain open about their specificities, their mechanism(s) of action and therefore the relevance of their current categorisation.

Modelling the emergent dynamics and major metabolites of the human colonic microbiota.

We present here a first attempt at modelling microbial dynamics in the human colon incorporating both uncertainty and adaptation. This is based on the development of a Monod-equation based, differential equation model, which produces computer simulations of the population dynamics and major metabolites of microbial communities from the human colon. To reduce the complexity of the system, we divide the bacterial community into 10 bacterial functional groups (BFGs) each distinguished by its substrate preferences, metabolic pathways and its preferred pH range. The model simulates the growth of a large number of bacterial strains and incorporates variation in microbiota composition between people, while also allowing succession and enabling adaptation to environmental changes. The model is shown to reproduce many of the observed changes in major phylogenetic groups and key metabolites such as butyrate, acetate and propionate in response to a one unit pH shift in experimental continuous flow fermentors inoculated with human faecal microbiota. Nevertheless, it should be regarded as a learning tool to be updated as our knowledge of bacterial groups and their interactions expands. Given the difficulty of accessing the colon, modelling can play an extremely important role in interpreting experimental data and predicting the consequences of dietary modulation.

Essential oils have different effects on human pathogenic and commensal bacteria in mixed faecal fermentations compared with pure cultures.

A static batch culture system inoculated with human faeces was used to determine the influence of essential oil compounds (EOCs) on mixed faecal microbiota. Bacteria were quantified using quantitative PCR of 16S rRNA genes. Incubation for 24 h of diluted faeces from six individuals caused enrichment of Bifidobacterium spp., but proportions of other major groups were unaffected. Thymol and geraniol at 500 p.p.m. suppressed total bacteria, resulting in minimal fermentation. Thymol at 100 p.p.m. had no effect, nor did eugenol or nerolidol at 100 or 500 p.p.m. except for a slight suppression of Eubacterium hallii. Methyl isoeugenol at 100 or 500 p.p.m. suppressed the growth of total bacteria, accompanied by a large fall in the molar proportion of propionate formed. The relative abundance of Faecalibacterium prausnitzii was unaffected except with thymol at 500 p.p.m. The ability of EOCs to control numbers of the pathogen Clostridium difficile was investigated in a separate experiment, in which the faecal suspensions were amended by the addition of pure culture of C. difficile. Numbers of C. difficile were suppressed by thymol and methyl isoeugenol at 500 p.p.m. and to a lesser extent at 100 p.p.m. Eugenol and geraniol gave rather similar suppression of C. difficile numbers at both 100 and 500 p.p.m. Nerolidol had no significant effect. It was concluded from these and previous pure-culture experiments that thymol and geraniol at around 100 p.p.m. could be effective in suppressing pathogens in the small intestine, with no concern for beneficial commensal colonic bacteria in the distal gut.

Intestinal colonization: how key microbial players become established in this dynamic process: microbial metabolic activities and the interplay between the host and microbes.

In this review, we provide an overview of the dynamic changes within the microbiota and its metabolites that are implicated in establishing and maintaining gastrointestinal homeostasis during various stages of microbial colonization. The gradual conversion of the gut microbiota toward a mutualistic microbial community involves replacement of pioneer gut colonizers with bacterial taxa that are characteristic for the adult gut. An important microbial signature of homeostasis in the adult gut is the prevalence and activity of a diverse spectrum of bacterial species that produce beneficial metabolites through metabolic interactions between microbial groups. Deciphering these microbial signatures and their metabolites that govern short and long-term equilibrium, as well as imbalances in host-microbial relationships, may provide novel diagnostic tools and/or therapeutic targets for specific disorders associated with intestinal dysbiosis and loss of homeostasis.

Altered intestinal microbiota and blood T cell phenotype are shared by patients with Crohn's disease and their unaffected siblings.

OBJECTIVE: Crohn's disease (CD) is associated with intestinal dysbiosis, altered blood T cell populations, elevated faecal calprotectin (FC) and increased intestinal permeability (IP). CD-associated features present in siblings (increased risk of CD) but not in healthy controls, provide insight into early CD pathogenesis. We aimed to (1) Delineate the genetic, immune and microbiological profile of patients with CD, their siblings and controls and (2) Determine which factors discriminate between groups. DESIGN: Faecal microbiology was analysed by quantitative PCR targeting 16S ribosomal RNA, FC by ELISA, blood T cell phenotype by flow cytometry and IP by differential lactulose-rhamnose absorption in 22 patients with inactive CD, 21 of their healthy siblings and 25 controls. Subject's genotype relative risk was determined by Illumina Immuno BeadChip. RESULTS: Strikingly, siblings shared aspects of intestinal dysbiosis with patients with CD (lower concentrations of Faecalibacterium prausnitzii (p=0.048), Clostridia cluster IV (p=0.003) and Roseburia spp. (p=0.09) compared with controls). As in CD, siblings demonstrated a predominance of memory T cells (p=0.002) and elevated naïve CD4 T cell ß7 integrin expression (p=0.01) compared with controls. FC was elevated (>50 µg/g) in 8/21 (38%) siblings compared with 2/25 (8%) controls (p=0.028); whereas IP did not differ between siblings and controls. Discriminant function analysis determined that combinations of these factors significantly discriminated between groups (χ(2)=80.4, df=20, p<0.001). Siblings were separated from controls by immunological and microbiological variables. CONCLUSIONS: Healthy siblings of patients with CD manifest immune and microbiological abnormalities associated with CD distinct from their genotype-related risk and provide an excellent model in which to investigate early CD pathogenesis.

Complete genome of a new Firmicutes species belonging to the dominant human colonic microbiota ('Ruminococcus bicirculans') reveals two chromosomes and a selective capacity to utilize plant glucans.

The recently isolated bacterial strain 80/3 represents one of the most abundant 16S rRNA phylotypes detected in the healthy human large intestine and belongs to the Ruminococcaceae family of Firmicutes. The completed genome sequence reported here is the first for a member of this important family of bacteria from the human colon. The genome comprises two large chromosomes of 2.24 and 0.73 Mbp, leading us to propose the name Ruminococcus bicirculans for this new species. Analysis of the carbohydrate active enzyme complement suggests an ability to utilize certain hemicelluloses, especially ß-glucans and xyloglucan, for growth that was confirmed experimentally. The enzymatic machinery enabling the degradation of cellulose and xylan by related cellulolytic ruminococci is however lacking in this species. While the genome indicated the capacity to synthesize purines, pyrimidines and all 20 amino acids, only genes for the synthesis of nicotinate, NAD+, NADP+ and coenzyme A were detected among the essential vitamins and co-factors, resulting in multiple growth requirements. In vivo, these growth factors must be supplied from the diet, host or other gut microorganisms. Other features of ecological interest include two type IV pilins, multiple extracytoplasmic function-sigma factors, a urease and a bile salt hydrolase.

Why does increased microbial fermentation in the human colon shift toward butyrate?

The microbial community of the human large intestine mainly ferments dietary fiber to short chain fatty acids (SCFAs), which are efficiently absorbed by the host. The three major SCFAs (acetate, propionate, and butyrate) have different fates within the body and different effects on health. A recent analysis of 10 human volunteer studies established that the proportions of these SCFA in fecal samples significantly shifted towards butyrate as the overall concentration of SCFA increased. Butyrate plays a key role in gut health and is preferentially utilized as an energy source by the colonic epithelium. Here we discuss possible mechanisms that underlie this 'butyrate shift'; these include the selection for butyrate-producing bacteria within the microbiota by certain types of fiber, and the possibility of additional butyrate formation from lactate and acetate by metabolite cross-feeding. However, a crucial factor appears to be the pH in the proximal colon, which decreases as the SCFA concentrations increase. A mildly acidic pH has been shown to have an important impact on microbial competition and on the stoichiometry of butyrate production. Understanding these complex interactions has been greatly aided by the refinement of theoretical models of the colonic microbiota that assume a small number (10) of microbial functional groups (MFGs).

Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes.

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL-1 to 12 ng mL-1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L-1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.

Phylogenetic distribution of genes encoding ß-glucuronidase activity in human colonic bacteria and the impact of diet on faecal glycosidase activities.

Bacterial ß-glucuronidase in the human colon plays an important role in cleaving liver conjugates of dietary compounds and xenobiotics, while other glycosidase activities are involved in the conversion of dietary plant glycosides. Here we detected an increase in ß-glucuronidase activity in faecal samples from obese volunteers following a high-protein moderate carbohydrate weight-loss diet, compared with a weight maintenance diet, but little or no changes were observed when the type of fermentable carbohydrate was varied. Other faecal glycosidase activities showed little or no change over a fivefold range of dietary NSP intake, although α-glucosidase increased on a resistant starch-enriched diet. Two distinct groups of gene, gus and BG, have been reported to encode ß-glucuronidase activity among human colonic bacteria. Degenerate primers were designed against these genes. Overall, Firmicutes were found to account for 96% of amplified gus sequences, with three operational taxonomic units particularly abundant, whereas 59% of amplified BG sequences belonged to Bacteroidetes and 41% to Firmicutes. A similar distribution of operational taxonomic units was found in a published metagenome dataset involving a larger number of volunteers. Seven cultured isolates of human colonic bacteria that carried only the BG gene gave relatively low ß-glucuronidase activity that was not induced by 4-nitrophenyl-ß-D-glucuronide. By comparison, in three of five isolates that possessed only the gus gene, ß-glucuronidase activity was induced.