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.

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.

Prebiotic fructans have greater impact on luminal microbiology and CD3+ T cells in healthy siblings than patients with Crohn's disease: A pilot study investigating the potential for primary prevention of inflammatory bowel disease.

BACKGROUND & AIMS: Siblings of people with Crohn's disease (CD) share aspects of the disease phenotype (raised faecal calprotectin, altered microbiota), which are markers of risk for their own development of CD. The aim was to determine whether supplementation with prebiotic oligofructose/inulin induces a prebiotic response and impacts the risk phenotype in CD patients and siblings. METHODS: Patients with inactive CD (n = 19, CD activity index <150) and 12 of their unaffected siblings (with calprotectin >50 µg/g) ingested oligofructose/inulin (15 g/day) for three weeks. Faecal microbiota (qPCR), intestinal permeability (lactulose-rhamnose test), blood T cells (flow-cytometry) and calprotectin (ELISA) were measured at baseline and follow-up. RESULTS: Following oligofructose/inulin, calprotectin did not significantly change in patients (baseline mean 537 SD 535 µg/g; follow-up mean 974 SD 1318 µg/g, p = 0.08) or siblings (baseline mean 73 SD 90 µg/g: follow up mean 58 SD 72 µg/g, p = 0.62). Faecal Bifidobacteria and Bifidobacterium longum increased in patients and siblings; Bifidobacterium adolescentis and Roseburia spp. increased only in siblings. Compared with patients, siblings had a greater magnitude change in Bifidobacteria (+14.6% vs +0.4%, p = 0.028), B. adolescentis (+1.1% vs 0.0% p = 0.006) and Roseburia spp. (+1.5% vs -0.1% p = 0.004). Intestinal permeability decreased significantly in patients after oligofructose/inulin to a level that was similar to siblings. Blood T cell abundance reduced in siblings but not patients following oligofructose/inulin. CONCLUSIONS: Oligofructose/inulin supplementation did not significantly impact calprotectin, but the prebiotic effect was more marked in healthy siblings compared with patients with inactive CD and was associated with alterations in other CD risk markers. Future research should focus on dietary intervention, including with prebiotics, in the primary prevention of CD.

The effect of prunes on stool output, gut transit time and gastrointestinal microbiota: A randomised controlled trial.

BACKGROUND & AIM: Prunes (dried plums) are perceived to maintain healthy bowel function, however their effects on gastrointestinal (GI) function are poorly researched and potential mechanisms of action are not clear. We aimed to investigate the effect of prunes on stool output, whole gut transit time (WGTT), gut microbiota and short-chain fatty acids (SCFA) in healthy adults METHODS: We conducted a parallel group, randomised controlled trial with three treatment arms in 120 healthy adults with low fibre intakes and stool frequency of 3-6 stools/wk. Subjects were randomised to 80 g/d prunes (plus 300 ml/d water); 120 g/d prunes (plus 300 ml/d water) or control (300 ml/d water) for 4 weeks. Stool weight was the primary outcome and determined by 7-day stool collection. Secondary outcomes included stool frequency and consistency (stool diary), WGTT (radio-opaque markers), GI symptoms (diary), microbiota (quantitative PCR) and SCFA (gas liquid chromatography). Group assignment was concealed from the outcome assessors. RESULTS: There were significantly greater increases in stool weight in both the 80 g/d (mean + 22.2 g/d, 95% CI -1-45.3) and 120 g/d (+32.8 g/d, 95% CI 13.9-51.7) prune groups compared with control (-0.8 g/d, 95% CI -17.2 to 15.6, P = 0.026). Stool frequency was significantly greater following 80 g/d (mean 6.8 bowel movements/wk, SD 3.8) and 120 g/d (5.6, SD 1.9) prune consumption compared with control (5.4, SD 2.1) (P = 0.023), but WGTT was unchanged. The incidence of flatulence was significantly higher after prune consumption. There were no significant differences in any of the bacteria measured, except for a greater increase in Bifidobacteria across the groups (P = 0.046). Prunes had no effect on SCFA or stool pH. CONCLUSIONS: In healthy individuals with infrequent stool habits and low fibre intake, prunes significantly increased stool weight and frequency and were well tolerated. Prunes may have health benefits in populations with low stool weight. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: ISRCTN42793297 http://www.isrctn.com/ISRCTN42793297.

Prebiotic potential of pectin and pectic oligosaccharides to promote anti-inflammatory commensal bacteria in the human colon.

Dietary plant cell wall carbohydrates are important in modulating the composition and metabolism of the complex gut microbiota, which can impact on health. Pectin is a major component of plant cell walls. Based on studies in model systems and available bacterial isolates and genomes, the capacity to utilise pectins for growth is widespread among colonic Bacteroidetes but relatively uncommon among Firmicutes. One Firmicutes species promoted by pectin is Eubacterium eligens. Eubacterium eligens DSM3376 utilises apple pectin and encodes a broad repertoire of pectinolytic enzymes, including a highly abundant pectate lyase of around 200 kDa that is expressed constitutively. We confirmed that certain Faecalibacterium prausnitzii strains possess some ability to utilise apple pectin and report here that F. prausnitzii strains in common with E. eligens can utilise the galacturonide oligosaccharides DP4 and DP5 derived from sugar beet pectin. Faecalibacterium prausnitzii strains have been shown previously to exert anti-inflammatory effects on host cells, but we show here for the first time that E. eligens strongly promotes the production of the anti-inflammatory cytokine IL-10 in in vitro cell-based assays. These findings suggest the potential to explore further the prebiotic potential of pectin and its derivatives to re-balance the microbiota towards an anti-inflammatory profile.

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.

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.

Influence of cabbage processing methods and prebiotic manipulation of colonic microflora on glucosinolate breakdown in man.

Glucosinolate consumption from brassica vegetables has been implicated in reduction of cancer risk. The isothiocyanate breakdown products of glucosinolates appear to be particularly important as chemoprotective agents. Before consumption, brassica vegetables are generally cooked, causing the plant enzyme, myrosinase, to be denatured, influencing the profile of glucosinolate breakdown products produced. Some human intestinal microflora species show myrosinase-like activity (e.g. bifidobacteria). We aimed to increase bifidobacteria by offering a prebiotic (inulin) in a randomised crossover study. Six volunteers consumed inulin (10 g/d) for 21 d followed by a 21 d control period (no inulin). Treatment periods were reversed for the remaining six volunteers. During the last 5 d of each period two cabbage-containing meals were consumed. Total urine output was collected for 24 h following each meal. Cabbage was microwaved for 2 min (lightly cooked) or 5.5 min (fully cooked). Faecal samples were collected at the start and after the inulin and control treatments. Bifidobacteria were enumerated by real-time PCR. Allyl isothiocyanate production was quantified by measuring urinary excretion of allyl mercapturic acid (AMA). Bifidobacteria increased following prebiotic supplementation (P < 0.001) but there was no impact of this increase on AMA excretion. AMA excretion was greater following consumption of lightly cooked cabbage irrespective of prebiotic treatment (P < 0.001). In conclusion, the most effective way to increase isothiocyanate production may be to limit the length of time that brassica vegetables are cooked prior to consumption.