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.

Selective fermentation of gentiobiose-derived oligosaccharides by human gut bacteria and influence of molecular weight

Gentiooligosaccharides and alternansucrase gentiobiose acceptor products were fractionated by their degree of polymerization (DP) on a Bio-Gel P2 column. Fractions were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, and incubated with human faecal bacteria under anaerobic conditions at 37 degrees C. The growth of predominant gut bacteria on the oligosaccharides was evaluated by fluorescence in situ hybridization and a prebiotic index (PI) was calculated. Lower DP gentiooligosaccharides (DP2-3) showed the highest selectivity (PI of 4.89 and 3.40, respectively), whereas DP4-5 alternansucrase gentiobiose acceptor products generated the greatest values (PI of 5.87). The production of short-chain fatty acids was also determined during the time course of the reactions. The mixture of DP6-10 alternansucrase gentiobiose acceptor products generated the highest levels of butyric acid but the lowest levels of lactic acid. Generally, for similar molecular weights, alternansucrase gentiobiose acceptor products gave higher PI values than gentiooligosaccharides.

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment wits determined in the human HT29 cell line by viable Count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coli and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected. but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 mu g ml(-1), respectively.

Bacteria in the gut: friends and foes and how to alter the balance

The activities of the bacteria resident in the colon of companion animals can have an impact upon the health of the host. Our understanding of this microbial ecosystem is presently increasing due to the development of DNA-based microbiological tools that allow identification and enumeration of nonculturable microorganisms. These techniques are changing our view of the bacteria that live in the gut, and they are facilitating dietary-intervention approaches to modulate the colonic ecosystem. This is generally achieved by the feeding of either live bacteria (probiotics) or nondigestible oligosaccharides (prebiotics) that selectively feed the indigenous probiotics. Feeding studies with a Lactobacillus acidophilus probiotic have shown positive effects on carriage of Clostridium spp. in canines and on recovery from Campylobacter spp. infection in felines. Immune function was improved in both species. Prebiotic feeding studies with lactosucrose and fructo-oligosaccharides in both cats and dogs have shown positive effects on the microflora balance. Recently synbiotic forms (a probiotic together with a prebiotic) targeted at canines have been developed that show promise as dietary-intervention tools.

In vitro studies on colonization resistance of the human gut microbiota to Candida albicans and the effects of tetracycline and Lactobacillus plantarum LPK

An anaerobic three-vessel continuous-flow culture system, which models the three major anatomical regions of the human colon, was used to study the persistence of Candida albicans in the presence of a faecal microbiota. During steady state conditions, overgrowth of C. albicans was prevented by commensal bacteria indigenous to the system. However antibiotics, such as tetracycline have the ability to disrupt the bacterial populations within the gut. Thus, colonization resistance can be compromised and overgrowth of undesirable microorganisms like C. albicans can then occur. In this study, growth of C. albicans was not observed in the presence of an established faecal microbiota. However, following the addition of tetracycline to the growth medium, significant growth of C. albicans occurred. A probiotic Lactobacillus plantarum LPK culture was added to the system to investigate whether this organism had any effects upon the Candida populations. Although C. albicans was not completely eradicated in the presence of this bacterium, cell counts were markedly reduced, indicating a compromised physiological function. This study shows that the normal gut flora can exert 'natural' resistance to C. albicans, however this may be diminished during antibiotic intake. The use of probiotics can help fortify natural resistance.

Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children

Children with autistic spectrum disorders (ASDs) tend to suffer from severe gastrointestinal problems. Such symptoms may be due to a disruption of the indigenous gut flora promoting the overgrowth of potentially pathogenic micro-organisms. The faecal flora of patients with ASDs was studied and compared with those of two control groups (healthy siblings and unrelated healthy children). Faecal bacterial populations were assessed through the use of a culture-independent technique, fluorescence in situ hybridization, using oligonucleotide probes targeting predominant components of the gut flora. The faecal flora of ASD patients contained a higher incidence of the Clostridium histolyticum group (Clostridium clusters I and 11) of bacteria than that of healthy children. However, the non-autistic sibling group had an intermediate level of the C. histolyticum group, which was not significantly different from either of the other subject groups. Members of the C. histolyticum group are recognized toxin-producers and may contribute towards gut dysfunction, with their metabolic products also exerting systemic effects. Strategies to reduce clostridial population levels harboured by ASD patients or to improve their gut microflora profile through dietary modulation may help to alleviate gut disorders common in such patients.

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.

Molecular methods for the analysis of gut microbiota

This review focuses on methodological approaches used to study the composition of human faecal microbiota. Gene sequencing is the most accurate tool for revealing the phylogenetic relationships between bacteria. The main application of fluorescence in situ hybridization (FISH) in both microscopy and flow cytometry is to enumerate faecal bacteria. While flow cytometry is a very fast method, FISH microscopy still has a considerably lower detection limit.

Hypothesis about mechanisms through which nicotine might exert its effect on the interdependence of inflammation and gut barrier function in ulcerative colitis

Ulcerative colitis (UC) is characterized by impairment of the epithelial barrier and the formation of ulcer-type lesions, which result in local leaks and generalized alterations of mucosal tight junctions. Ultimately, this results in increased basal permeability. Although disruption of the epithelial barrier in the gut is a hallmark of inflammatory bowel disease and intestinal infections, it remains unclear whether barrier breakdown is an initiating event of UC or rather a consequence of an underlying inflammation, evidenced by increased production of proinflammatory cytokines. UC is less common in smokers, suggesting that the nicotine in cigarettes may ameliorate disease severity. The mechanism behind this therapeutic effect is still not fully understood, and indeed it remains unclear if nicotine is the true protective agent in cigarettes. Nicotine is metabolized in the body into a variety of metabolites and can also be degraded to form various breakdown products. It is possible these metabolites or degradation products may be the true protective or curative agents. A greater understanding of the pharmacodynamics and kinetics of nicotine in relation to the immune system and enhanced knowledge of out permeability defects in UC are required to establish the exact protective nature of nicotine and its metabolites in UC. This review suggests possible hypotheses for the protective mechanism of nicotine in UC, highlighting the relationship between gut permeability and inflammation, and indicates where in the pathogenesis of the disease nicotine may mediate its effect.

Effects of resistant starch type III polymorphs on human colon microbiota and short chain fatty acids in human gut models

This study probed the possible effects of type III resistant starch (RS) crystalline polymorphism on RS fermentability by human gut microbiota and the short chain fatty acids production in vitro. Human fecal pH-controlled batch cultures showed RS induces an ecological shift in the colonic microbiota with polymorph B inducing Bifidobacterium spp. and polymorph A inducing Atopobium spp. Interestingly, polymorph B also induced higher butyrate production to levels of 0.79 mM. In addition, human gut simulation demonstrated that polymorph B promotes the growth of bifidobacteria in the proximal part of the colon and double their relative proportion in the microbiota in the distal colon. These findings suggest that RS polymorph B may promote large bowel health. While the findings are limited by study constraints, they do raise the possibility of using different thermal processing to delineate differences in the prebiotic capabilities of RS, especially its butryrogenicity in the human colon.

Gut fermentation products of insulin-derived prebiotics beneficially modulate markers of tumour progression in human colon tumour cells

Insulin is a prebiotic food ingredient, which suppresses colon tumour growth and development in rats. In the gut lumen, it is fermented to lactic acid and short chain fatty acids (SCFA). Of these, butyrate has suppressing agent activities, but little is known concerning cellular responses to complex fermentation samples. To investigate the effects of fermentation products of insulin on cellular responses related to colon carcinogenesis. Fermentations were performed in anaerobic batch cultures or in a three-stage fermentation model that simulates conditions in colon-segments (proximal, transverse, distal). Substrate was insulin enriched with oligofructose (Raftilose® Synergy1), fermented with probiotics (Bifidobacterium lactis Bb12, Lactobacillus rhamnosus GG), and/or faecal inocula. HT29 or CaCo-2 cells were incubated with supernatants of the fermented samples (2.5%-25% v/v, 24-72 hours). Cellular parameters of survival, differentiation, tumour progression, and invasive growth were determined. Fermentation supernatants derived from probiotics and Synergy1 were more effective than with glucose. The additional fermentation with faecal slurries produced supernatants with lower toxicity, higher SCFA contents, and distinct cellular functions. The supernatant derived from the gut model vessel representing the distal colon, was most effective for all parameters, probably on account of higher butyrate-concentrations. Biological effects of insulin upon colon cells may be mediated not only by growth stimulation of the lactic acid-producing bacteria and/or production of butyrate, but also by other bacteria and products of the gut lumen. These newly reported properties of the supernatants to inhibit growth and metastases in colon tumour cells are important mechanisms of tumour suppression.

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

Dietary-based gut flora modulation against Clostridium difficile onset

Clostridium difficile infection is a frequent complication of antibiotic therapy in hospitalised patients, which today is attracting more attention than ever and has led to its classification as a 'superbug'. Disruption of the composition of the intestinal microflora following antibiotic treatment is an important prerequisite for overgrowth of C. difficile and the subsequent development of an infection. Treatment options for antibiotic-associated diarrhoea and C. difficile-induced colitis include administration of specific antibiotics (e.g. vancomycin), which often leads to high relapse rates. More importantly, both the rate and severity of C. difficile-associated diseases are increasing, with new epidemic strains of C. difficile often implicated. For the prevention and treatment of antibiotic-associated diarrhoea and C. difficile infection, several probiotic bacteria such as selected strains of lactobacilli (especially Lactobacillus rhamnosus GG), Bifidobacterium longum, and Enterococcus faecium and the non-pathogenic yeast Saccharomyces boulardii have been used. Controlled trials indicate a benefit of S. boulardii and L. rhamnosus GG as therapeutic agents when used as adjuncts to antibiotics. However, the need for more well designed controlled trials with probiotics is explicit.