Konjac glucomannan hydrolysate beneficially modulates bacterial composition and activity within the faecal microbiota

The prebiotic potential of a konjac glucomannan hydrolysate (GMH) was investigated in vitro using batch cultures inoculated with human faeces. Bacterial enumeration was carried out using the culture independent technique, fluorescent in situ hybridisation (FISH), and short chain fatty acid (SCFA) production was monitored by gas chromatography. The populations of Bifidobacterium genus, Lactobacillus–Enterococcus group and the Atopobium group all significantly increased after GMH and inulin fermentation. The Bacteroides–Prevotella group had a lower end population after GMH fermentation while inulin gave an increase, although these differences were not significant. No significant differences in SCFA concentrations were observed between inulin and GMH. As with inulin, GMH produced selective stimulation of beneficial gut microbiota and a favourable SCFA profile. In order to confirm a beneficial effect of GMH further in vivo studies involving healthy human volunteers should be considered.

Surface activity of surfactin recovered and purified from fermentation broth using a two-step ultrafiltration (UF) process

B. subtilis under certain types of media and fermentation conditions can produce surfactin, a biosurfactant which belongs to the lipopeptide class. Surfactin has exceptional surfactant activity, and exhibits some interesting biological characteristics such as antibacterial activity, antitumoral activity against ascites carcinoma cells, and a hypocholesterolemic activity that inhibits cAMP phosphodiesterase, as well as having anti-HIV properties. A cost effective recovery and purification of surfactin from fermentation broth using a two-step ultrafiltration (UF) process has been developed in order to reduce the cost of surfactin production. In this study, competitive adsorption of surfactin and proteins at the air-water interface was studied using surface pressure measurements. Small volumes of bovine serum albumin (BSA) and β-casein solutions were added to the air-water interface on a Langmuir trough and allowed to stabilise before the addition of surfactin to the subphase. Contrasting interfacial behaviour of proteins was observed with β-casein showing faster initial adsorption compared to BSA. On introduction of surfactin both proteins were displaced but a longer time were taken to displace β-casein. Overall the results showed surfactin were highly surface-active by forming a β-sheet structure at the air-water interface after reaching its critical micelle concentration (CMC) and were effective in removing both protein films, which can be explained following the orogenic mechanism. Results showed that the two-step UF process was effective to achieve high purity and fully functional surfactin.

Gut microbial catabolism of grape seed flavan-3-ols by human faecal microbiota. Targetted analysis of precursor compounds, intermediate metabolites and end-products.

In vitro batch culture fermentations were conducted with grape seed polyphenols and human faecal microbiota, in order to monitor both changes in precursor flavan-3-ols and the formation of microbial-derived metabolites. By the application of UPLC-DAD-ESI-TQ MS, monomers, and dimeric and trimeric procyanidins were shown to be degraded during the first 10 h of fermentation, with notable inter-individual differences being observed between fermentations. This period (10 h) also coincided with the maximum formation of intermediate metabolites, such as 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3′,4′-dihydroxyphenyl)-valeric acid, and of several phenolic acids, including 3-(3,4-dihydroxyphenyl)-propionic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxymandelic acid, and gallic acid (5–10 h maximum formation). Later phases of the incubations (10–48 h) were characterised by the appearance of mono- and non-hydroxylated forms of previous metabolites by dehydroxylation reactions. Of particular interest was the detection of γ-valerolactone, which was seen for the first time as a metabolite from the microbial catabolism of flavan-3-ols. Changes registered during fermentation were finally summarised by a principal component analysis (PCA). Results revealed that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone was a key metabolite in explaining inter-individual differences and delineating the rate and extent of the microbial catabolism of flavan-3-ols, which could finally affect absorption and bioactivity of these compounds.

Metabolism of anthocyanins by human gut microflora and their influence on gut bacterial growth

Consumption of anthocyanins has been related with beneficial health effects. However, bioavailability studies have shown low concentration of anthocyanins in plasma and urine. In this study, we have investigated the bacterial-dependent metabolism of malvidin-3-glucoside, gallic acid and a mixture of anthocyanins using a pH-controlled, stirred, batch-culture fermentation system reflective of the distal human large intestine conditions. Most anthocyanins have disappeared after 5 h incubation while gallic acid remained constant through the first 5 h and was almost completely degraded following 24 h of fermentation. Incubation of malvidin-3-glucoside with fecal bacteria mainly resulted in the formation of syringic acid, while the mixture of anthocyanins resulted in formation of gallic, syringic and p-coumaric acids. All the anthocyanins tested enhanced significantly the growth of Bif idobacterium spp. and Lactobacillus−Enterococcus spp. These results suggest that anthocyanins and their metabolites may exert a positive modulation of the intestinal bacterial population.

Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG

Background: The surface properties of probiotic bacteria influence to a large extent their interactions within the gut ecosystem. There is limited amount of information on the effect of the production process on the surface properties of probiotic lactobacilli in relation to the mechanisms of their adhesion to the gastrointestinal mucosa. The aim of this work was to investigate the effect of the fermentation pH and temperature on the surface properties and adhesion ability to Caco-2 cells of the probiotic strain Lactobacillus rhamnosus GG. Results: The cells were grown at pH 5, 5.5, 6 (temperature 37 °C) and at pH 6.5 (temperature 25 °C, 30 °C and 37 °C), and their surfaces analysed by X-ray photoelectron spectrometry (XPS), Fourier transform infrared spectroscopy (FT-IR) and gel-based proteomics. The results indicated that for all the fermentation conditions, with the exception of pH 5, a higher nitrogen to carbon ratio and a lower phosphate content was observed at the surface of the bacteria, which resulted in a lower surface hydrophobicity and reduced adhesion levels to Caco-2 cells as compared to the control fermentation (pH 6.5, 37 oC). A number of adhesive proteins, which have been suggested in previous published works to take part in the adhesion of bacteria to the human gastrointestinal tract, were identified by proteomic analysis, with no significant differences between samples however. Conclusions: The temperature and the pH of the fermentation influenced the surface composition, hydrophobicity and the levels of adhesion of L. rhamnosus GG to Caco-2 cells. It was deduced from the data that a protein rich surface reduced the adhesion ability of the cells.

Effects of fermentation products of pro- and prebiotics on trans-epithelial electrical resistance in an in vitro model of the colon

Evidence from in vivo and in vitro studies suggests that the consumption of pro- and prebiotics may inhibit colon carcinogenesis; however, the mechanisms involved have, thus far, proved elusive. There are some indications from animal studies that the effects are being exerted during the promotion stage of carcinogenesis. One feature of the promotion stage of colorectal cancer is the disruption of tight junctions, leading to a loss of integrity across the intestinal barrier. We have used the Caco-2 human adenocarcinoma cell line as a model for the intestinal epithelia. Trans-epithelial electrical resistance measurements indicate Caco-2 monolayer integrity, and we recorded changes to this integrity following exposure to the fermentation products of selected probiotics and prebiotics, in the form of nondigestible oligosaccharides (NDOs). Our results indicate that NDOs themselves exert varying, but generally minor, effects upon the strength of the tight junctions, whereas the fermentation products of probiotics and NDOs tend to raise tight junction integrity above that of the controls. This effect was bacterial species and oligosaccharide specific. Bifidobacterium Bb 12 was particularly effective, as were the fermentation products of Raftiline and Raftilose. We further investigated the ability of Raftilose fermentations to protect against the negative effects of deoxycholic acid (DCA) upon tight junction integrity. We found protection to be species dependent and dependent upon the presence of the fermentation products in the media at the same time as or after exposure to the DCA. Results suggest that the Raftilose fermentation products may prevent disruption of the intestinal epithelial barrier function during damage by tumor promoters.

In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: changes in microbial groups and phenolic metabolites

With the aim of investigating the potential of flavan-3-ols to influence the growth of intestinal bacterial groups, we have carried out the in vitro fermentation, with human faecal microbiota, of two purified fractions from grape seed extract (GSE): GSE-M (70% monomers and 28% procyanidins) and GSE-O (21% monomers and 78 % procyanidins). Samples were collected at 0, 5, 10, 24, 30 and 48 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and for analysis of phenolic metabolites. Both GSE-M and GSE-O fractions promoted growth of Lactobacillus/Enterococcus and decrease in the Clostridium histolyticum group during fermentation, although the effects were only statistically significant with GSE-M for Lactobacillus/Enterococcus (at 5 and 10 h of fermentation) and GSE-O for C. histolyticum (at 10 h of fermentation). Main changes in polyphenol catabolism also occurred during the first 10 h of fermentation, however no significant correlation coefficients (P>0.05) were found between changes in microbial populations and precursor flavan-3-ols or microbial metabolites. Together these data suggest that the flavan-3-ol profile of a particular food source could affect the microbiota composition and its catabolic activity, inducing changes that could in turn affect the bioavailability and potential bioactivity of these compounds.

Synergistic effects of mixing cocksfoot and sainfoin on in vitro rumen fermentation: role of condensed tannins

Some bioactive secondary metabolites in forage legumes can cause digestive interactions, so that the rumen fermentation pattern of a mixture of forages can differ from the average values of its components. The objective of this study was to investigate the potential role of condensed tannins (CT) on the synergistic effects between one grass species, cocksfoot, and one CT-containing legume species, sainfoin, on in vitro rumen fermentation characteristics. Cocksfoot and sainfoin in different proportions (in g/kg, 1000:0, 750:250, 500:500, 250:750 and 0:1000) were incubated under anaerobic conditions in culture bottles containing buffered rumen fluid from sheep. Incubations were carried out using artificial saliva with and without polyethylene glycol (PEG), which binds and thus inactivates CT. Rumen fermentation parameters describing the degradation and the fate of the energetic and nitrogenous substrates were measured at 3.5 and 24 h. At the early fermentation stage, when the sainfoin level increased from 0 to 1000 g/kg, the ammonia concentration in the medium quadratically decreased from 3.20 to 0.53 mmol/l in absence of PEG (P<0.01) but not in its presence. This result demonstrates that sainfoin CT decreased the rumen degradation of the proteins in the whole mixture, including the proteins in cocksfoot, rather than just the proteins in sainfoin. Interestingly, the total gas and methane productions were lower in mixtures incubated in absence of PEG than in presence of PEG (P<0.001) while no significant PEG effect was observed on digestibility. At the late fermentation stage, a positive quadratic effect on dry matter digestibility was detected without PEG (P<0.05), indicating a synergistic action of cocksfoot plus sainfoin on plant substrate degradation due to CT. The presence of PEG increased gas production (P<0.001) and NH3-N concentration in the medium (P<0.001). Our results suggest that CT could allow a better utilization of plant substrates in mixtures by the rumen ecosystem by improving the partitioning of degraded substrates toward lower gas losses, and decreasing the protein degradation.

Persistence of anticancer activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation.

Fruit and vegetable consumption is associated at the population level with a protective effect against colorectal cancer. Phenolic compounds, especially abundant in berries, are of interest due to their putative anticancer activity. After consumption, however, phenolic compounds are subject to digestive conditions within the gastrointestinal tract that alter their structures and potentially their function. However, the majority of phenolic compounds are not efficiently absorbed in the small intestine and a substantial portion pass into the colon. We characterized berry extracts (raspberries, strawberries, blackcurrants) produced by in vitro-simulated upper intestinal tract digestion and subsequent fecal fermentation. These extracts and selected individual colonic metabolites were then evaluated for their putative anticancer activities using in vitro models of colorectal cancer, representing the key stages of initiation, promotion and invasion. Over a physiologically-relevant dose range (0-50 µg/ml gallic acid equivalents), the digested and fermented extracts demonstrated significant anti-genotoxic, anti-mutagenic and anti-invasive activity on colonocytes. This work indicates that phenolic compounds from berries undergo considerable structural modifications during their passage through the gastrointestinal tract but their breakdown products and metabolites retain biological activity and can modulate cellular processes associated with colon cancer.

In vitro fermentation of potential prebiotic flours from natural sources: impact on the human colonic microbiota and metabolome

Scope: Fibers and prebiotics represent a useful dietary approach for modulating the human gut microbiome. Therefore, aim of the present study was to investigate the impact of four flours (wholegrain rye, wholegrain wheat, chickpeas and lentils 50:50, and barley milled grains), characterized by a naturally high content in dietary fibers, on the intestinal microbiota composition and metabolomic output. Methods and results: A validated three-stage continuous fermentative system simulating the human colon was used to resemble the complexity and diversity of the intestinal microbiota. Fluorescence in situ hybridization was used to evaluate the impact of the flours on the composition of the microbiota, while small-molecule metabolome was assessed by NMR analysis followed by multivariate pattern recognition techniques. HT29 cell-growth curve assay was used to evaluate the modulatory properties of the bacterial metabolites on the growth of intestinal epithelial cells. All the four flours showed positive modulations of the microbiota composition and metabolic activity. Furthermore, none of the flours influenced the growth-modulatory potential of the metabolites toward HT29 cells. Conclusion: Our findings support the utilization of the tested ingredients in the development of a variety of potentially prebiotic food products aimed at improving gastrointestinal health.