In vitro examination of the effect of Orlistat on the ability of the faecal microbiota to utilize dietary lipids

Orlistat is an anti-obesity treatment with which several gastrointestinal (GI) side-effects are commonly associated in the initial stages of therapy. There is no physiological explanation as to why two-thirds of those who take the drug experience one or more side-effects. It has been hypothesized that the GI microbiota may protect from or contribute to these GI disturbances. Using in vitro batch culture and human gut model systems, studies were conducted to determine whether increased availability of dietary lipids and/or orlistat affect the composition and/or activity of the faecal microbiota. Results from 24-h batch culture fermentation experiments demonstrated no effect of orlistat in the presence or absence of a dietary lipid (olive oil) on the composition of bacterial communities [as determined by fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses], but did show there was great variability in the lipolytic activities of the microbiotas of individuals, as determined by gas chromatography analysis of long-chain fatty acids in samples. Subsequent studies focused on the effect of orlistat in the presence and absence of lipid in in vitro human gut model systems. Systems were run for 14 days with gut model medium (GMM) only (to steady state, SS), then fed at 12-h intervals with 50 mg orlistat, 2 g olive oil or a mixture of both for 14 days. FISH and DGGE were used to monitor changes in bacterial populations. Bacteria were cultivated from the GMM only (control) systems at SS. All strains isolated were screened for lipolytic activity using tributyrin agar. FISH and DGGE demonstrated that none of the compounds (singly or in combination) added to the systems had any notable effect on microbial population dynamics for any of the donors, although Subdoligranulum populations appeared to be inhibited by orlistat in the presence or absence of lipid. Orlistat had little or no effect on the metabolism of indigenous and added lipids in the fermentation systems, but there was great variability in the way the faecal microbiotas of the donors were able to degrade added lipids. Variability in lipid degradation could be correlated with the number and activity of isolated lipolytic bacteria. The mechanism by which orlistat and the GI microbiota cause side-effects in individuals is unknown, but several hypotheses have been proposed to account for their manifestation. The demonstration of great variability in the lipolytic activity of microbiotas to degrade lipids led to a large-scale cultivation-based study of lipolytic/lipase-positive bacteria present in the human faecal microbiota. Of 4,000 colonies isolated from 15 donors using five different agars, 378 strains were identified that had lipase activity. Molecular identification of strains isolated from five donors demonstrated that lipase activity is more prevalent in the human GI microbiota than previously thought, with members of the phyla Firmicutes, Bacteroidetes and Actinobacteria identified. Molecular identification and characterization of the substrate specificities of the strains will be carried out as part of ongoing work.

A double-blind, placebo-controlled, cross-over study to establish the bifidogenic effect of a very-long-chain inulin extracted from globe artichoke (Cynara scolymus) in healthy human subjects

There is growing interest in the use of inulins as substrates for the selective growth of beneficial gut bacteria such as bifidobacteria and lactobacilli because recent studies have established that their prebiotic effect is linked to several health benefits. In the present study, the impact of a very-long-chain inulin (VLCI), derived from globe artichoke (Cynara scolymus), on the human intestinal microbiota compared with maltodextrin was determined. A double-blind, cross-over study was carried out in thirty-two healthy adults who were randomised into two groups and consumed 10 g/d of either VLCI or maltodextrin, for two 3-week study periods, separated by a 3-week washout period. Numbers of faecal bifidobacteria and lactobacilli were significantly higher upon VLCI ingestion compared with the placebo. Additionally, levels of Atopobium group significantly increased, while Bacteroides–Prevotella numbers were significantly reduced. No significant changes in faecal SCFA concentrations were observed. There were no adverse gastrointestinal symptoms apart from a significant increase in mild and moderate bloating upon VLCI ingestion. These observations were also confirmed by in vitro gas production measurements. In conclusion, daily consumption of VLCI extracted from globe artichoke exerted a pronounced prebiotic effect on the human faecal microbiota composition and was well tolerated by all volunteers.

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.

In vitro bioaccessibility and gut biotransformation of polyphenols present in the water-insoluble cocoa fraction

Scope: Cocoa, especially the water-insoluble cocoa fraction (WICF), is a rich source of polyphenols. In this study, sequential in vitro digestion of the WICF with gastrointestinal enzymes as well as its bacterial fermentation in a human colonic model system were carried out to investigate bioaccessibility and biotransformation of WICF polyphenols, respectively. Methods and results: The yield of each enzymatic digestion step and the total antioxidant capacity (TAC) were measured and solubilized phenols were characterized by MS/MS. Fermentation of WICF and the effect on the gut microbiota, SCFA production and metabolism of polyphenols was analyzed. In vitro digestion solubilized 38.6% of WICF with pronase and Viscozyme L treatments releasing 51% of the total phenols from the insoluble material. This release of phenols does not determine a reduction in the total antioxidant capacity of the digestion-resistant material. In the colonic model WICF significantly increased of bifidobacteria and lactobacilli as well as butyrate production. Flavanols were converted into phenolic acids by the microbiota following a concentration gradient resulting in high concentrations of 3-hydroxyphenylpropionic acid (3-HPP) in the last gut compartment. Conclusion: Data showed that WICF may exert antioxidant action through the gastrointestinal tract despite its polyphenols being still bound to macromolecules and having prebiotic activity.

Determination of the in vivo prebiotic potential of a maize based wholegrain breakfast cereal: a human feeding study

Epidemiological studies have shown an inverse relationship between risk of CVD and intake of whole grain (WG)-rich food. Regular consumption of breakfast cereals can provide not only an increase in dietary WG but also improvements to cardiovascular health. Various mechanisms have been proposed, including prebiotic modulation of the colonic microbiota. In the present study, the prebiotic activity of a maize-derived WG cereal (WGM) was evaluated in a double-blind, placebo-controlled human feeding study (n 32). For a period of 21 d, healthy men and women, mean age 32 (sd 8) years and BMI 23·3 (sd 0·58) kg/m2, consumed either 48 g/d WG cereal (WGM) or 48 g placebo cereal (non-whole grain (NWG)) in a crossover fashion. Faecal samples were collected at five points during the study on days 0, 21, 42, 63 and 84 (representing at baseline, after both treatments and both wash-out periods). Faecal bacteriology was assessed using fluorescence in situ hybridisation with 16S rRNA oligonucleotide probes specific for Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum/perfringens subgroup, Lactobacillus–Enterococcus subgroup and total bacteria. After 21 d consumption of WGM, mean group levels of faecal bifidobacteria increased significantly compared with the control cereal (P = 0·001). After a 3-week wash-out period, bifidobacterial levels returned to pre-intervention levels. No statistically significant changes were observed in serum lipids, glucose or measures of faecal output. In conclusion, this WG maize-enriched breakfast cereal mediated a bifidogenic modulation of the gut microbiota, indicating a possible prebiotic mode of action

Therapeutic modulation of microbiota-host metabolic interactions

The complex metabolic relationships between the host and its microbiota change throughout life and vary extensively between individuals, affecting disease risk factors and therapeutic responses through drug metabolism. Elucidating the biochemical mechanisms underlying this human supraorganism symbiosis is yielding new therapeutic insights to improve human health, treat disease, and potentially modify human disease risk factors. Therapeutic options include targeting drugs to microbial genes or co-regulated host pathways and modifying the gut microbiota through diet, probiotic and prebiotic interventions, bariatric surgery, fecal transplants, or ecological engineering. The age-associated co-development of the host and its microbiota provides a series of windows for therapeutic intervention from early life through old age

Gut microbial activity, implications for health and disease: the potential role of metabolite analysis

Microbial metabolism of proteins and amino acids by human gut bacteria generates a variety of compounds including phenol, indole, and sulfur compounds and branched chain fatty acids, many of which have been shown to elicit a toxic effect on the lumen. Bacterial fermentation of amino acids and proteins occurs mainly in the distal colon, a site that is often fraught with symptoms from disorders including ulcerative colitis (UC) and colorectal cancer (CRC). In contrast to carbohydrate metabolism by the gut microbiota, proteolysis is less extensively researched. Many metabolites are low molecular weight, volatile compounds. This review will summarize the use of analytical methods to detect and identify compounds in order to elucidate the relationship between specific dietary proteinaceous substrates, their corresponding metabolites, and implications for gastrointestinal health.

Nutrimetabonomics: applications for nutritional sciences, with specific reference to gut microbial interactions

Understanding the role of the diet in determining human health and disease is one major objective of modern nutrition. Mammalian biocomplexity necessitates the incorporation of systems biology technologies into contemporary nutritional research. Metabonomics is a powerful approach that simultaneously measures the low-molecular-weight compounds in a biological sample, enabling the metabolic status of a biological system to be characterized. Such biochemical profiles contain latent information relating to inherent parameters, such as the genotype, and environmental factors, including the diet and gut microbiota. Nutritional metabonomics, or nutrimetabonomics, is being increasingly applied to study molecular interactions between the diet and the global metabolic system. This review discusses three primary areas in which nutrimetabonomics has enjoyed successful application in nutritional research: the illumination of molecular relationships between nutrition and biochemical processes; elucidation of biomarker signatures of food components for use in dietary surveillance; and the study of complex trans-genomic interactions between the mammalian host and its resident gut microbiome. Finally, this review illustrates the potential for nutrimetabonomics in nutritional science as an indispensable tool to achieve personalized nutrition.

Neonatal environment exerts a sustained influence on the development of the intestinal microbiota and metabolic phenotype

The postnatal environment, including factors such as weaning and acquisition of the gut microbiota, has been causally linked to the development of later immunological diseases such as allergy and autoimmunity, and has also been associated with a predisposition to metabolic disorders. We show that the very early-life environment influences the development of both the gut microbiota and host metabolic phenotype in a porcine model of human infants. Farmpiglets were nursed by their mothers for 1 day, before removal to highly controlled, individual isolators where they received formula milk until weaning at 21 days. The experiment was repeated, to create two batches, which differed only in minor environmental fluctuations during the first day. At day 1 after birth, metabolic profiling of serum by 1H nuclear magnetic resonance spectroscopy demonstrated significant, systemic, inter-batch variation which persisted until weaning. However, the urinary metabolic profiles demonstrated that significant inter-batch effects on 3-hydroxyisovalerate, trimethylamine-N-oxide and mannitol persisted beyond weaning to at least 35 days. Batch effects were linked to significant differences in the composition of colonic microbiota at 35 days, determined by 16 S pyrosequencing. Different weaning diets modulated both the microbiota and metabolic phenotype independently of the persistent batch effects. We demonstrate that the environment during the first day of life influences development of the microbiota and metabolic phenotype and thus should be taken into account when interrogating experimental outcomes. In addition, we suggest that intervention at this early time could provide ‘metabolic rescue’ for at-risk infants who have undergone aberrant patterns of initial intestinal colonisation.

Lactobacillus acidophilus CRL 1014 improved gut health in the SHIME® reactor

Background: How to maintain gut health is a goal for scientists throughout the world. Therefore, microbiota management models for testing probiotics, prebiotics, and synbiotics have been developed.Methods: The SHIME® model was used to study the effect of Lactobacillus acidophilus 1014 on the fermentation pattern of the colon microbiota. Initially, an inoculum prepared from human feces was introduced into the reactor vessels and stabilized over 2-wk using a culture medium. This stabilization period was followed by a 2-wk control period during which the microbiota was monitored. The microbiota was then subjected to a 4-wk treatment period by adding 5 mL of sterile peptone water with L. acidophilus CRL1014 at the concentration of 108 CFU/mL to vessel one (the stomach compartment). Plate counts, Denaturing Gradient Gel Electrophoresis (DGGE), short-chain fatty acid (SCFA) and ammonium analyses were carried out for monitoring of the microbial community from the colon compartments.Results: A significant increase (p < 0.01) in the Lactobacillus spp. and Bifidobacterium spp. populations was observed during the treatment period. The DGGE obtained showed changes in the lactobacilli community from the colon compartments of the SHIME® reactor. The (SCFA) concentration increased (p < 0.01) during the treatment period, due mainly to significant increased levels of acetic, butyric, and propionic acids. However, ammonium concentrations decreased during the same period (p < 0.01).Conclusions: This study showed the beneficial influence of L. acidophilus CRL 1014 on microbial metabolism and lactobacilli community composition for improving human health. © 2013 Sivieri et al.; licensee BioMed Central Ltd.

Beneficial effects of fermented vegetal beverages on human gastrointestinal microbial ecosystem in a simulator

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prebiotic Effect of Fructooligosaccharide in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME (R) Model)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

P-215-Quantitative variation of some gut bacteria in Crohn's disease can be related to the patient's gender

Background: Imbalance in bacterial species composition of the gut microbiota is one of the factors associated with the cause or complication of the symptoms of Crohn's disease (CD). This disequilibrium consists in the reduction of biodiversity, decrease of genus such as Bifidobacterium and elevation of species such as Escherichia coli. Human microbiota varies among subjects of a same population irrespective of their health condition and among individuals living in distinct geographic locations. In animal models, sex related differences could also be observed in gut bacterial species composition under some pathological conditions. Experiments conducted with mice have demonstrated that the manifestation of type 1 diabetes (T1D) could be under the influence of the animal sex and its serum level of testosterone, which in turn could be modulated by a particular gut microbiota. Considering the existence of similar features between T1D and CD, such as strong genetic component and malfunctioning of the immune system, we investigated whether differences could be observed in the gut microbiota dysbiosis of male and female CD patients. Methods: Fifty and 5 gut mucosal biopsies from 25 adult CD patients (11 males and 14 females) and 43 specimens of an equivalent clinical material from 22 control subjects (11 males and 11 females) were screened for bacterial biodiversity by analyzing sequences of 16SrDNA V6 region. A number of 2-3 samples each from distinct gut segments (from ileum to rectum) were taken from each subject. The 16SrDNA sequences were obtained by sequencing PCR amplicons of the corresponding gene in the Ion torrent PGM sequencer. Identification and classification of the bacterial groups followed the Ribosomal Database Project (RDP) website pipeline. The relationships of the bacterial taxa with each of the study parameters was performed by compiling the data in a MS Excel and the level of statistical significance determined by the Chi-square test. Results: A total of 3203 16SrDNA sequences were detected in the 98 biopsies samples, the majority of which matching Proteobacteria, Firmicutes, Bacterioidetes, and Actinobacteria. The percentage of DNA sequences for each of these phyla found in Male control subjects/Male CD patients was 40.5/33, 32.7/32.4, 20.8/24.5, and 4.4/4,4 for Proteobacteria, Firmicutes, Bacterioidetes, and Actinobacteria, respectively. In Female comparisons, these values were 35.6/42, 39.2/26.3, 19.8/23.3, 5.2/7. Both Male and Female CD patients presented higher numbers of sequences of Actinobacteria and Bacterioidetes than those of control subjects of the same gender. Case-control differences for Firmicutes could be observed only in female comparisons and, for Proteobacteria, although case-control differences were observed in both genders, the nature of difference was distinct, since while in CD female patients a higher number of sequences matching this phylum was detected, in males a reduced number was observed, in comparison with controls. The species responsible for the Proteobacteria variation in both gender was Escherichia coli. Conclusions: The data presented above suggest that any analysis of dysbiosis in CD must take in account the patient's gender, an observation particularly relevant for Escherichia coli, whose association with CD has been most intensively investigated and for which the present study shows a reverse quantitative variation regarding the patients' gender.

Influência do suco de laranja na microbiota intestinal humana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.