The potential role of the intestinal gut microbiota in obesity and the metabolic syndrome

The incidence of obesity has reached alarming levels worldwide, thus increasing the risk of development of metabolic disorders (e.g. type 2 diabetes, coronary heart disease (CHD) and cancer). Among the causes of obesity, diet and lifestyle play a central role. Although the treatment of obesity may appear quite straightforward, by simply re-addressing the balance between energy intake and energy expenditure, practically it has been very challenging. In the search for new therapeutic targets for treatment of obesity and related disorders, the gut microbiota and its activities have been investigated in relation to obesity. The human gut microbiota has already been shown to influence total energy intake and lipid metabolism, particularly through colonic fermentation of undigestible dietary constituents and production of short chain fatty acids (SCFA). Recent studies have highlighted the contribution of the gut microbiota to mammalian metabolism and energy harvested from the diet. A dietary modulation of the gut microbiota and its metabolic output could positively influence host metabolism and, therefore, constitute a potential coadjutant approach in the management of obesity and weight loss.

Functional petit-suisse cheese: measure of the prebiotic effect

Prebiotics and probiotics are increasingly being used to produce potentially synbiotic foods, particularly through dairy products as vehicles. It is well known that both ingredients may offer benefits to improve the host health. This research aimed to evaluate the prebiotic potential of novel petit-suisse cheeses using an in vitro fermentation model. Five petit-suisse cheese formulations combining candidate prebiotics (inulin. oligofructose. hone) and probiotics (Lactobacillus acidophilus, Bifidobacterium lactis) were tested in vitro using, sterile. stirred, batch culture fermentations with human faecal slurry. Measurement of prebiotic effect (MPE) values were generated comparing bacterial changes through determination of maximum growth rates of groups, rate of substrate assimilation and production of lactate and short chain fatty acids. Fastest fermentation and high lactic acid production, promoting increased growth rates of bifidobacteria and lactobacilli. were achieved with addition of prebiotics to a probiotic cheese (made using starter + probiotics). Addition of probiotic strains to control cheese (made using just a starter culture) also resulted in high lactic acid production. Highest MPE values were obtained with addition of prebiotics to a probiotic cheese, followed by addition of prebiotics and/or probiotics to a control cheese. Under the in vitro conditions used, cheese made with the combination of different prebiotics and probiotics resulted in the most promising functional petit-suisse cheese. The study allowed comparison of potentially functional petit-suisse cheeses and screening of preferred synbiotic potential for future market use. (c) 2007 Elsevier Ltd. All rights reserved.

Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine

Aims: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. Methods and Results: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. Conclusions: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. Significance and impact of the Study: Selected strains could be used as adjunct starters to make high quality Feta cheese.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

In vitro evaluation of the microbiota modulation abilities of different sized whole oat grain flakes.

Epidemiological studies and healthy eating guidelines suggest a positive correlation between ingestion of whole grain cereal and food rich in fibre with protection from chronic diseases. The prebiotic potential of whole grains may be related, however, little is known about the microbiota modulatory capability of oat grain or the impact processing has on this ability. In this study the fermentation profile of whole grain oat flakes, processed to produce two different sized flakes (small and large), by human faecal microbiota was investigated in vitro. Simulated digestion and subsequent fermentation by gut bacteria was investigated using pH controlled faecal batch cultures inoculated with human faecal slurry. The different sized oat flakes, Oat 23’s (0.53–0.63 mm) and Oat 25’s/26’s (0.85–1.0 mm) were compared to oligofructose, a confirmed prebiotic, and cellulose, a poorly fermented carbohydrate. Bacterial enumeration was carried out using the culture independent technique, fluorescent in situ hybridisation, and short chain fatty acid (SCFA) production monitored by gas chromatography. Significant changes in total bacterial populations were observed after 24 h incubation for all substrates except Oat 23’s and cellulose. Oats 23’s fermentation resulted in a significant increase in the Bacteroides–Prevotella group. Oligofructose and Oats 25’s/26’s produced significant increases in Bifidobacterium in the latter stages of fermentation while numbers declined for Oats 23’s between 5 h and 24 h. This is possibly due to the smaller surface area of the larger flakes inhibiting the simulated digestion, which may have resulted in increased levels of resistant starch (Bifidobacterium are known to ferment this dietary fibre). Fermentation of Oat 25’s/26’s resulted in a propionate rich SCFA profile and a significant increase in butyrate, which have both been linked to benefiting host health. The smaller sized oats did not produce a significant increase in butyrate concentration. This study shows for the first time the impact of oat grain on the microbial ecology of the human gut and its potential to beneficially modulate the gut microbiota through increasing Bifidobacterium population.

A double-blind, placebo controlled human study investigating the effects of coffee derived manno-oligosaccharides on the faecal microbiota of a healthy adult population.

The aims of this study were to assess the impact of coffee derived mannooligosaccharides on the faecal microbiota of a healthy UK based population. Methods and Results: A double-blind, placebo-controlled, crossover human intervention study was conducted. Volunteers were assigned, 3g MOS, 5g MOS and placebo coffee preparations, to consume daily over a 3 wks, followed by a 2 wk washout period. Faecal samples were collected, and microbial population characterised using fluorescence in situ hybridization. Short-chain and branched-chain fatty acid profiles were obtained by gas chromatography. All treatments led to significant lactobacilli increases (placebo, p < 0.001; 3g, p = 0.04; 5g, p=0.04). The 3g treatment led to a significant bifidobacteria increase (p=0.001). Significantly less iso-valerate was found in faeces following 3g MOS daily (p=0.05). Conclusions: The 3g dose of MOS led to a potentially beneficial shift in the faecal microbiota. MOS was therefore confirmed to be a prebiotic at 3g dose. Significance and Impact of Study: This study provides confirmation of a new novel prebiotic, that can be considered for incorporation into a wider variety of food products, to provide different selective and nutritional properties.

Corynebacterium testudinoris sp. nov., from a tortoise, and Corynebacterium felinum sp. nov., from a Scottish wild cat

Two unknown gram-positive, rod-shaped bacteria isolated from a tortoise and a Scottish wild cat were subjected to a polyphasic taxonomic analysis. Chemical analysis revealed the presence of straight-chain and monounsaturated fatty acids and short-chain mycolic acids in the two isolates consistent with the genus Corynebacterium. Comparative 16S rRNA gene sequencing confirmed that the unknown isolates were members of the genus Corynebacterium, with the two organisms displaying greater than 3% sequence divergence from each other and from established species of the genus. The unknown Corynebacterium isolates were readily distinguished from each other and from all recognized species of the genus by biochemical tests. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown organisms from a tortoise and a cat be classified in the genus Corynebacterium as Corynebacterium testudinoris sp. nov. and Corynebacterium felinum sp. nov., respectively. The respective type strains of C. testudinoris and C. felinum are CCUG 41823T and CCUG 39943T.

Corynebacterium capitovis sp. nov., from a sheep

An unknown Gram-positive rod-shaped bacterium was isolated from skin scrapings from the infected head of a sheep and subjected to a polyphasic taxonomic analysis. Chemical analysis revealed the presence of straight-chain and monounsaturated fatty acids and short-chain (C32-C36) mycolic acids consistent with the genus Corynebacterium. Comparative 16S rRNA gene sequencing confirmed that the unknown rod was a member of the genus Corynebacterium, with the organism forming a distinct sub-line and displaying greater than 3% sequence divergence with established species. The unknown Corynebacterium isolate was readily distinguished from recognized species of the genus by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from a sheep be classified in the genus Corynebacterium, as Corynebacterium capitovis sp. nov. The type strain of Corynebacterium capitovis is CCUG 39779T (= CIP 106739T).

In vitro fermentation of rice bran combined with Lactobacillus acidophilus 14 150B or Bifidobacterium longum 05 by the canine faecal microbiota

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.

Constraint release in entangled binary blends of linear polymers: a molecular dynamics study

We present extensive molecular dynamics simulations of the dynamics of diluted long probe chains entangled with a matrix of shorter chains. The chain lengths of both components are above the entanglement strand length, and the ratio of their lengths is varied over a wide range to cover the crossover from the chain reptation regime to tube Rouse motion regime of the long probe chains. Reducing the matrix chain length results in a faster decay of the dynamic structure factor of the probe chains, in good agreement with recent neutron spin echo experiments. The diffusion of the long chains, measured by the mean square displacements of the monomers and the centers of mass of the chains, demonstrates a systematic speed-up relative to the pure reptation behavior expected for monodisperse melts of sufficiently long polymers. On the other hand, the diffusion of the matrix chains is only weakly perturbed by the diluted long probe chains. The simulation results are qualitatively consistent with the theoretical predictions based on constraint release Rouse model, but a detailed comparison reveals the existence of a broad distribution of the disentanglement rates, which is partly confirmed by an analysis of the packing and diffusion of the matrix chains in the tube region of the probe chains. A coarse-grained simulation model based on the tube Rouse motion model with incorporation of the probability distribution of the tube segment jump rates is developed and shows results qualitatively consistent with the fine scale molecular dynamics simulations. However, we observe a breakdown in the tube Rouse model when the short chain length is decreased to around N-S = 80, which is roughly 3.5 times the entanglement spacing N-e(P) = 23. The location of this transition may be sensitive to the chain bending potential used in our simulations.

The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota.

We have examined the gut bacterial metabolism of pomegranate by-product (POMx) and major pomegranate polyphenols, punicalagins, using pH-controlled, stirred, batch culture fermentation systems reflective of the distal region of the human large intestine. Incubation of POMx or punicalagins with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins. The time course profile confirmed the tetrahydroxylated urolithin D as the first product of microbial transformation, followed by compounds with decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A. POMx exposure enhanced the growth of total bacteria, Bifidobacterium spp. and Lactobacillus spp., without influencing the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group. In addition, POMx increased concentrations of short chain fatty acids (SCFA) viz. acetate, propionate and butyrate in the fermentation medium. Punicalagins did not affect the growth of bacteria or production of SCFA. The results suggest that POMx oligomers, composed of gallic acid, ellagic acid and glucose units, may account for the enhanced growth of probiotic bacteria.

Rifaximin modulates the colonic microbiota of patients with Crohn's disease: an in vitro approach using a continuous culture colonic model system.

Rifaximin, a rifamycin derivative, has been reported to induce clinical remission of active Crohn's disease (CD), a chronic inflammatory bowel disorder. In order to understand how rifaximin affects the colonic microbiota and its metabolism, an in vitro human colonic model system was used in this study. We investigated the impact of the administration of 1800 mg/day of rifaximin on the faecal microbiota of four patients affected by colonic active CD [Crohn's disease activity index (CDAI > 200)] using a continuous culture colonic model system. We studied the effect of rifaximin on the human gut microbiota using fluorescence in situ hybridization, quantitative PCR and PCR–denaturing gradient gel electrophoresis. Furthermore, we investigated the effect of the antibiotic on microbial metabolic profiles, using 1H-NMR and solid phase microextraction coupled with gas chromatography/mass spectrometry, and its potential genotoxicity and cytotoxicity, using Comet and growth curve assays. Rifaximin did not affect the overall composition of the gut microbiota, whereas it caused an increase in concentration of Bifidobacterium, Atopobium and Faecalibacterium prausnitzii. A shift in microbial metabolism was observed, as shown by increases in short-chain fatty acids, propanol, decanol, nonanone and aromatic organic compounds, and decreases in ethanol, methanol and glutamate. No genotoxicity or cytotoxicity was attributed to rifaximin, and conversely rifaximin was shown to have a chemopreventive role by protecting against hydrogen peroxide-induced DNA damage. We demonstrated that rifaximin, while not altering the overall structure of the human colonic microbiota, increased bifidobacteria and led to variation of metabolic profiles associated with potential beneficial effects on the host.

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.

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 1010.7 cells/g feces; antibiotic-treated, 108.4). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (109.1 cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.

Diallyl disulphide, a beneficial component of garlic oil, causes a redistribution of cell-cycle growth phases, induces apoptosis and enhances butyrate-induced apoptosis in colorectal adenocarcinoma cells (HT-29)

Colon cancer is a leading and expanding cause of death worldwide. A major contributory factor to this disease is diet composition; some components are beneficial (e.g. dietary fibre) whilst others are detrimental (e.g. alcohol). Garlic oil is a prominent dietary constituent that prevents the development of colorectal cancer. This effect is believed to be mainly due to diallyl disulphide (DADS), which selectively induces redox stress in cancerous (rather than normal) cells which leads to apoptotic cell death. However, the detailed mechanism by which DADS causes apoptosis remains unclear. We show that DADS-treatment of colonic adenocarcinoma cells (HT-29) initiates a cascade of molecular events characteristic of apoptosis. These include a decrease in cellular proliferation, translocation of phosphatidylserine to the plasma-membrane outer-layer, activation of caspase-3, genomic-DNA fragmentation and G2/M phase cell-cycle arrest. Short-chain fatty acids (SCFAs), particularly butyrate (abundantly produced in the gut by bacterial fermentation of dietary polysaccharides), enhance colonic cell integrity but, in contrast, inhibit colonic-cancer cell growth. Combining DADS with butyrate augmented the effect of butyrate on HT-29 cells. These results suggest that the anti-cancerous properties of DADS afford greater benefit when supplied with other favourable dietary factors (SCFA/polysaccharides) that likewise reduce colonic tumour development.