In vitro evaluation of the fermentation properties of galactooligosaccharides synthesised by alpha-galactosidase from Lactobacillus reuteri

Stirred, pH-controlled anaerobic batch cultures were used to evaluate the in vitro utilisation by canine gut microflora of novel alpha-galactooligosaccharides synthesised with an enzyme extract from a canine Lactobacillus reuteri strain. Fructooligosaccharides (FOS), melibiose and raffinose were used as reference carbohydrates for the prebiotic properties of the synthesised oligosaccharide (galactosyl melibiose mixture-GMM). Addition of Lactobacillus acidophilus was used as control for the evaluation of the synbiotic properties of the oligosaccharide with L. reuteri. Populations of predominant gut bacterial groups were monitored over 48 h of batch culture by fluorescent in situ hybridisation, and short-chain fatty acid (SCFA) production was measured. GMM showed a higher increase in bifidobacteria and lactobacilli population number and size as well as a higher decrease in clostridia population number and size compared to the commercial prebiotics (FOS, melibiose, raffinose). This prebiotic effect was further increased by the addition of L. reuteri followed by a change in the SCFA production pattern compared to GMM alone or GMM with L. acidophilus. The observed change in SCFA production was in accordance with the fermentation properties of L. reuteri, suggesting that the novel synbiotic had a significant effect on the canine gut microflora fermentation.

Biodiversity of human faecal bacteria isolated from phytic acid enriched chemostat fermenters

Background: Myo-inositol hexaphosphate (IP6) or phytic acid is found mostly in cereals and legumes and is thought to possess anti-carcinogenic properties. Aim: To isolate and identify faecal bacteria capable of phytic acid metabolism and to assess the effectiveness of prebiotics (dietary oligosaccharides, metabolised by selective colonic bacteria) in preserving the integrity of phytic acid. Methods: Faecal samples from three volunteers were used in continuous culture experiments under varying conditions of pH, substrate concentration and dilution rates, seventy three different isolates cultured at steady state were then screened for phytic acid metabolism and identified through partial sequencing of their 16S rRNA genes (16S ribosomal ribonucleic acid). Utilisation of phytic acid was also assessed in a continuous culture system enriched with prebiotic fructooligosaccharides (FOS). Results: Bacteroides spp., Clostridium spp. and facultatively anaerobic bacteria generally appeared to maintain viable counts in the presence of phytic acid. Bifidobacterium spp. and Lactobacillus spp. appeared less able to maintain viable counts in the presence of phytic acid. These results were confirmed by an increase in viable counts of Bacteroides spp., Clostridium spp. and a decrease in viable counts of Bifidobacterium spp. and Lactobacillus spp. once phytic acid was introduced to a FOS enriched continuous culture. Conclusions: The phytate metabolising biodiversity from the human large intestine does not appear to encompass major bacterial genera associated with beneficial or benign health effects (e.g. Lactobacillus spp. and Bifidobacterium spp).

Metabolism of the soyabean isoflavone glycoside genistin in vitro by human gut bacteria and the effect of prebiotics

The isoflavone genistein is found predominantly in soyabeans and is thought to possess various potent biological properties, including anticarcinogenic effects. Studies have shown that genistein is extensively degraded by the human gut microflora, presumably with a loss of its anti-carcinogenic action. The aim of the present study was to investigate the potential of a prebiotic to divert bacterial metabolism away from genistein breakdown: this may be of benefit to the host. Faecal samples were obtained from healthy volunteers and fermented in the presence of a source of soyabean isoflavones (Novasoy(TM) (10 g/l); ADM Neutraceuticals, Erith, Kent, UK). Bacterial genera of the human gut were enumerated using selective agars and genistein was quantified by HPLC. The experiment was repeated with the addition of glucose (10 g/l) or fructo-oligosaccharide (10 g/l; FOS) to the fermentation medium. The results showed most notably that counts of Bifidobacterium spp. and Lactobacillus spp. were significantly increased (P<0.05 and P<0.01 respectively) under steady-state conditions in the presence of FOS. Counts of Bacteroides spp. and Clostridium spp. were, however, both significantly reduced (P<0.05) during the fermentation. A decline in genistein concentration by about 52 and 56% over the 120h culture period was observed with the addition of glucose or FOS to the basal medium (P<0.01), compared with about 91% loss of genistein in the vessels containing Novasoy(TM) (ADM Neutraceuticals) only. Similar trends were obtained using a three-stage chemostat (gut model), in which once again the degradation of genistein was about 22% in vessel one, about 24% in vessel two and about 26% in vessel three in the presence of FOS, compared with a degradation of genistein of about 67% in vessel one, about 95% in vessel two and about 93% in vessel three in the gut model containing Novasoy(TM) (ADM Neutraceuticals) only. The present study has shown that the addition of excess substrate appeared to preserve genistein in vitro. In particular, the use of FOS not only augmented this effect, but also conferred an additional benefit in selectively increasing numbers of purportedly beneficial bacteria such as bifidobacteria and lactobacilli.

In vitro investigation into the potential prebiotic activity of honey oligosaccharides

The effect of honey oligosaccharides on the growth of fecal bacteria was studied using an in vitro fermentation system. Prior to treatment, glucose and fructose (31.73 and 21.41 g/100 g of product, respectively) present in honey, which would be digested in the upper gut, were removed to avoid any influence on bacterial populations in the fermentations. Nanofiltration, yeast (Saccharomyces cerevisiae) treatment, and adsorption onto activated charcoal were used to remove monosaccharides. Prebiotic (microbial fermentation) activities of the three honey oligosaccharide fractions and the honey sample were studied and compared with fructooligosaccharide (FOS), using 1% (w/v) fecal bacteria in an in vitro fermentation system (10 mg of carbohydrate, 1.0 mL of basal medium). A prebiotic index (PI) was calculated for each carbohydrate source. Honey oligosaccharides seem to present potential prebiotic activity (PI values between 3.38 and 4.24), increasing the populations of bifidobacteria and lactobacilli, although not to the levels of FOS (PI of 6.89).

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

Carbohydrate preferences of Bifidobacterium species isolated from the human gut

The growth of nine species of Bifidobacterium on media containing glucose, xylose, xylooligosaccharides (XOS), xylan or fructooligosaccharides (FOS) as the sole carbon source were compared in pure culture. The bifidobacteria differed in fermentation profiles when tested on different carbohydrates. All species grew to their highest final optical density (OD) on a glucose containing medium, with the exception of B. catenulatum which demonstrated a preference for xylose over glucose, and XOS over FOS. B. bifidum grew to the highest OD on XOS compared to xylose suggesting a specific transport system for the oligosaccharide over the monomer. This is consistent with a lack of β-xylosidase activity present in the culture medium. Lactate, formate and acetate levels were determined and the ratios of these metabolites altered between and within species growing on different carbohydrates. In general, high lactate production correlated with low formate production and low lactate concentrations were obtained at higher levels of formate. Bifidobacteria may alter their metabolic pathways based upon the carbohydrates that are available for their use.

Effect of pH and dose on the growth of gut bacteria on prebiotic carbohydrates in vitro

The effect of pH and substrate dose on the fermentation profile of a number of commercial prebiotics was analysed in triplicate using stirred, pH and temperature controlled anaerobic batch culture fermentations, inoculated with a fresh faecal slurry from one of three healthy volunteers. Bacterial numbers were enumerated using fluorescence in situ hybridisation. The commercial prebiotics investigated were fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), isomaltooligosaccharides (IMO) and lactulose. Two pH values were investigated, i.e. pH 6 and 6.8. Doses of 1% and 2% (w/v) were investigated, equivalent to approximately 4 and 8 g per day, respectively, in an adult diet. It was found that both pH and dose altered the bacterial composition. It was observed that FOS and inulin demonstrated the greatest bifidogenic effect at pH 6.8 and 1% (w/v) carbohydrate, whereas GOS, IMO and lactulose demonstrated their greatest bifidogenic effect at pH 6 and 2% (w/v) carbohydrate. From this we can conclude that various prebiotics demonstrate differing bifidogenic effects at different conditions in vitro. (C) 2003 Elsevier Science Ltd. All rights reserved.

Profiling of composition and metabolic activities of the colonic microflora of growing pigs fed diets supplemented with prebiotic oligosaccharides

It is evident that quantitative information on different microbial groups and their contribution in terms of activity in the gastrointestinal (GI) tract of humans and animals is required in order to formulate functional diets targeting improved gut function and host health. In this work, quantitative information on levels and spatial distributions of Bacteroides spp, Eubacterium spp, Clostridium spp, Escherichia coli, Bifidobacterium spp and Lactobacillus/Enterococcus spp. along the porcine large intestine was investigated using 16S rRNA targeted probes and fluorescent in situ hybridisation (FISH). Caecum, ascending colon (AC) and rectum luminal digesta from three groups of individually housed growing pigs fed either a corn-soybean basal diet (CON diet) or a prebiotic diet containing 10 g/kg oligofructose (FOS diet) or trans-galactooligosaccharides (TOS diet) at the expense of cornstarch were analysed. DAPI staining was used to enumerate total number of cells in the samples. Populations of total cells, Bacteroides, Eubacterium, Clostridium and Bifidobacterium, declined significantly (P < 0.05) from caecum to rectum, and were not affected by dietary treatments. Populations of Lactobacillus/ Enterococcus and E coli did not differ throughout the large intestine. The relative percent (%) contribution of each bacterial group to the total cell count did not differ between caecum and rectum, with the exception of Eubacterium that was higher in the AC digesta. FISH analysis showed that the sum of all bacterial groups made up a small percentage of the total cells, which was 12.4%, 21.8% and 10.3% in caecum, AC and rectum, respectively. This supports the view that in swine, the diversity of GI microflora might be higher compared to other species. In terms of microflora metabolic activity, the substantially higher numerical trends seen in FOS and TOS treatments regarding total volatile fatty acid, acetate concentrations and glycolytic activities, it could be postulated that FOS and TOS promoted saccharolytic activities in the porcine colon. (c) 2006 Elsevier Ltd. All rights reserved.

In vitro fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

In vitro study on gas generation and prebiotic effects of some carbohydrates and their mixtures

This study was carried out to examine the effect or inulin (IN), fructooligosaccharide (FOS), polydextrose (POL) and isomaltooligosaccharides (ISO), alone and in combination, on gas production, gas composition and prebiotic effects. Static batch culture fermentation was performed with faecal samples from three healthy volunteers to study the volume and composition of gas generated and changes in bacterial populations. Four carbohydrates alone or mixed with one another (50:50) were examined. Prebiotic index (PI) was calculated and used to compare the prebiotic effect. The high amount of gas produced by IN was reduced by mixing it with FOS. No reduction in gas generation was observed when POL and ISO mixed with other substrates. It was found that the mixture of IN and FOS was effective in reducing the amount of gas produced while augmenting or maintaining their potential to Support the growth of bifidobacteria in Faecal batch culture as the highest PI was achieved with FOS alone and a mixture of FOS and IN. It was also found that high volume of gas was generated in presence of POL and ISO and they had lower prebiotic effect. The results of this study imply that a Mixture of prebiotics could prove effective in reducing the amount of gas generated by the gut microflora. (c) 2007 Elsevier Ltd. All rights reserved.

Established and emerging prebiotics and their effects on the gut microflora

The use of probiotics combined with prebiotics (synbiotics) has been proved to be more and more interesting in the market of functional foods. The use of probiotics alone has a long history whereas the concept of prebiotics is rather new, introduced by Gibson & Roberfroid(1). Efficient prebiotics are considered the compounds that are not digested and selectively promote the growth of beneficial microorganisms (such as lactobacilli and bifidobacteria) in the colon. Some established prebiotics that are currently used in the European market are fructooligosaccharides (FOS), galactooligosaccharides (GOS) and inulin. However, there are more compounds considered as "emerging prebiotics" which have not been established yet, but there is a need of further investigation on them. Some of them are oligomers of soya & xylan, isomalto-oligosaccharides (IMO), polydextrose and possibly some oligosaccharides in honey. There is still an incomplete picture of their fermentation properties but according to the studies performed till now, it is quite possible that these molecules might have the same or more desirable properties than the established ones. In this review, the effects of the established and emerging prebiotics on the gut microflora are presented, based on in vitro and in vivo studies (healthy volunteers).

Differential induction of 12-O-tetradecanoylphorbol 13-acetate sequence gene expression in murine melanocytes and melanoma cells

We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281–3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) gene expression in Mel-ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel-ab cells to the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80-fold increase in steady-state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c-fos); TIS 10 expression was not affected). No induction of 115 gene expression was observed either in growing Mel-ab cells maintained in 324 nM phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways.

Physiological and bifidogenic effects of prebiotic supplements in infant formulae

This randomized controlled trial involving 110 healthy neonates studied physiological and bifidogenic effects of galactooligosaccharides (GOS), oligofructose and long-chain inulin (FOS) in formula. Subjects were randomized to Orafti Synergy1 (50 oligofructose: 50 FOS) 0.4g/dl or 0.8g/dl, GOS:FOS (90:10) 0.8g/dl or a standard formula according to Good Clinical Practise (GCP) guidelines. A breast-fed group was included for comparison. Outcome parameters were weight, length, intake, stool characteristics, crying, regurgitation, vomiting, adverse events and fecal bacterial population counts. Statistical analyses used non-parametric tests. During the first month of life weight, length, intake and crying increased significantly in all groups. Regurgitation and vomiting scores were low and similar. Stool frequency decreased significantly and similarly in all formula groups but was lower than in the breast-fed. All prebiotic groups maintained soft stools, only slightly harder than those of breast-fed infants. The standard group had significantly harder stools at wks 2 and 4 compared to 1 (P<0.001 & P=0.0279). The total number of fecal bacteria increased in all prebiotic groups (9.82, 9.73 and 9.91 to 10.34, 10.38 and 10.37, respectively, log10 cells/g feces, P=0.2298) and resembled more the breast-fed pattern. Numbers of lactic acid bacteria, bacteroides and clostridia were comparable. In the SYN1 0.8 g/dl and GOS:FOS groups Bifidobacterium counts were significantly higher at D14 & 28 compared to D3 and comparable to the breast-fed group. Tolerance and growth were normal. In conclusion, stool consistency and bacterial composition of infants taking SYN1 0.8 g/dl or GOS:FOS supplemented formula was closer to the breast-fed pattern. There was no risk for dehydration.

Regulation of expression of the rat orthologue of mouse double minute 2 (MDM2) by H2O2-induced oxidative stress in neonatal rat cardiac myocytes.

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress.