In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria. In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism. Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied. The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.

Insight into the prebiotic concept: lessons from an exploratory, double blind intervention study with inulin-type fructans in obese women

Objective To highlight the contribution of the gut microbiota to the modulation of host metabolism by dietary inulin-type fructans (ITF prebiotics) in obese women. Methods A double blind, placebo controlled, intervention study was performed with 30 obese women treated with ITF prebiotics (inulin/oligofructose 50/50 mix; n=15) or placebo (maltodextrin; n=15) for 3 months (16 g/day). Blood, faeces and urine sampling, oral glucose tolerance test, homeostasis model assessment and impedancemetry were performed before and after treatment. The gut microbial composition in faeces was analysed by phylogenetic microarray and qPCR analysis of 16S rDNA. Plasma and urine metabolic profiles were analysed by 1H-NMR spectroscopy. Results Treatment with ITF prebiotics, but not the placebo, led to an increase in Bifidobacterium and Faecalibacterium prausnitzii; both bacteria negatively correlated with serum lipopolysaccharide levels. ITF prebiotics also decreased Bacteroides intestinalis, Bacteroides vulgatus and Propionibacterium, an effect associated with a slight decrease in fat mass and with plasma lactate and phosphatidylcholine levels. No clear treatment clustering could be detected for gut microbial analysis or plasma and urine metabolomic profile analyses. However, ITF prebiotics led to subtle changes in the gut microbiota that may importantly impact on several key metabolites implicated in obesity and/or diabetes. Conclusions ITF prebiotics selectively changed the gut microbiota composition in obese women, leading to modest changes in host metabolism, as suggested by the correlation between some bacterial species and metabolic endotoxaemia or metabolomic signatures.

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.

Dietary quebracho tannins are not absorbed, but increase the antioxidant capacity of liver and plasma in sheep

A total of sixteen lambs were divided into two groups and fed two different diets. Of these, eight lambs were fed a control diet (C) and eight lambs were fed the C diet supplemented with quebracho tannins (C+T). The objective of the present study was to assess whether dietary quebracho tannins can improve the antioxidant capacity of lamb liver and plasma and if such improvement is due to a direct transfer of phenolic compounds or their metabolites, to the animal tissues. Feed, liver and plasma samples were purified by solid-phase extraction (SPE) and analysed by liquid chromatography–MS for phenolic compounds. Profisitinidin compounds were identified in the C+T diet. However, no phenolic compounds were found in lamb tissues. The liver and the plasma from lambs fed the C+T diet displayed a greater antioxidant capacity than tissues from lambs fed the C diet, but only when samples were not purified with SPE. Profisetinidin tannins from quebracho seem not to be degraded or absorbed in the gastrointestinal tract. However, they induced antioxidant effects in animal tissues.

Differential effects of two fermentable carbohydrates on central appetite regulation and body composition

BACKGROUND: Obesity is rising at an alarming rate globally. Different fermentable carbohydrates have been shown to reduce obesity. The aim of the present study was to investigate if two different fermentable carbohydrates (inulin and b-glucan) exert similar effects on body composition and central appetite regulation in high fat fed mice. METHODOLOGY/PRINCIPAL FINDINGS: Thirty six C57BL/6 male mice were randomized and maintained for 8 weeks on a high fat diet containing 0% (w/w) fermentable carbohydrate, 10% (w/w) inulin or 10% (w/w) b-glucan individually. Fecal and cecal microbial changes were measured using fluorescent in situ hybridization, fecal metabolic profiling was obtained by proton nuclear magnetic resonance (1H NMR), colonic short chain fatty acids were measured by gas chromatography, body composition and hypothalamic neuronal activation were measured using magnetic resonance imaging (MRI) and manganese enhanced MRI (MEMRI), respectively, PYY (peptide YY) concentration was determined by radioimmunoassay, adipocyte cell size and number were also measured. Both inulin and b-glucan fed groups revealed significantly lower cumulative body weight gain compared with high fat controls. Energy intake was significantly lower in b-glucan than inulin fed mice, with the latter having the greatest effect on total adipose tissue content. Both groups also showed an increase in the numbers of Bifidobacterium and Lactobacillus-Enterococcus in cecal contents as well as feces. b- glucan appeared to have marked effects on suppressing MEMRI associated neuronal signals in the arcuate nucleus, ventromedial hypothalamus, paraventricular nucleus, periventricular nucleus and the nucleus of the tractus solitarius, suggesting a satiated state. CONCLUSIONS/SIGNIFICANCE: Although both fermentable carbohydrates are protective against increased body weight gain, the lower body fat content induced by inulin may be metabolically advantageous. b-glucan appears to suppress neuronal activity in the hypothalamic appetite centers. Differential effects of fermentable carbohydrates open new possibilities for nutritionally targeting appetite regulation and body composition.

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.

In vitro fermentation of commercial α-gluco-oligosaccharide by faecal microbiota from lean and obese human subjects

The fermentation selectivity of a commercial source of α-gluco-oligosaccharides (BioEcolians; Solabia) was investigated in vitro. Fermentation by faecal bacteria from four lean and four obese healthy adults was determined in anaerobic, pH-controlled faecal batch cultures. Inulin was used as a positive prebiotic control. Samples were obtained at 0, 10, 24 and 36 h for bacterial enumeration by fluorescent in situ hybridisation and SCFA analyses. Gas production during fermentation was investigated in non-pH-controlled batch cultures. α-Gluco-oligosaccharides significantly increased the Bifidobacterium sp. population compared with the control. Other bacterial groups enumerated were unaffected with the exception of an increase in the Bacteroides–Prevotella group and a decrease in Faecalibacterium prausnitzii on both α-gluco-oligosaccharides and inulin compared with baseline. An increase in acetate and propionate was seen on both substrates. The fermentation of α-gluco-oligosaccharides produced less total gas at a more gradual rate of production than inulin. Generally, substrates fermented with the obese microbiota produced similar results to the lean fermentation regarding bacteriology and metabolic activity. No significant difference at baseline (0 h) was detected between the lean and obese individuals in any of the faecal bacterial groups studied.

New insights into probiotic mechanisms: a harvest from functional and metagenomic studies

There has been continued and expanding recognition of probiotic approaches for treating gastrointestinal and systemic disease, as well as increased acceptance of probiotic therapies by both the public and the medical community. A parallel development has been the increasing recognition of the diverse roles that the normal gut microbiota plays in the normal biology of the host. This advance has in turn has been fed by implementation of novel investigative technologies and conceptual paradigms focused on understanding the fundamental role of the microbiota and indeed all commensal bacteria, on known and previously unsuspected aspects of host physiology in health and disease. This review discusses current advances in the study of the host-microbiota interaction, especially as it relates to potential mechanisms of probiotics. It is hoped these new approaches will allow more rational selection and validation of probiotic usage in a variety of clinical conditions.

Fermentable carbohydrate alters hypothalamic neuronal activity and protects against the obesogenic environment

Obesity has become a major global health problem. Recently, attention has focused on the benefits of fermentable carbohydrates on modulating metabolism. Here, we take a system approach to investigate the physiological effects of supplementation with oligofructose-enriched inulin (In). We hypothesize that supplementation with this fermentable carbohydrate will not only lead to changes in body weight and composition, but also to modulation in neuronal activation in the hypothalamus. Male C57BL/6 mice were maintained on a normal chow diet (control) or a high fat (HF) diet supplemented with either oligofructose-enriched In or corn starch (Cs) for 9 weeks. Compared to HF+Cs diet, In supplementation led to significant reduction in average daily weight gain (mean ± s.e.m.: 0.19 ± 0.01 g vs. 0.26 ± 0.02 g, P < 0.01), total body adiposity (24.9 ± 1.2% vs. 30.7 ± 1.4%, P < 0.01), and lowered liver fat content (11.7 ± 1.7% vs. 23.8 ± 3.4%, P < 0.01). Significant changes were also observed in fecal bacterial distribution, with increases in both Bifidobacteria and Lactobacillius and a significant increase in short chain fatty acids (SCFA). Using manganese-enhanced MRI (MEMRI), we observed a significant increase in neuronal activation within the arcuate nucleus (ARC) of animals that received In supplementation compared to those fed HF+Cs diet. In conclusion, we have demonstrated for the first time, in the same animal, a wide range of beneficial metabolic effects following supplementation of a HF diet with oligofructose-enriched In, as well as significant changes in hypothalamic neuronal activity.

Digestive and physiologic effects of a wheat bran extract, arabino-xylan-oligosaccharide, in breakfast cereal

Objective: We assessed whether a wheat bran extract containing arabino-xylan-oligosaccharide (AXOS) elicited a prebiotic effect and influenced other physiologic parameters when consumed in ready-to-eat cereal at two dose levels. Methods: This double-blind, randomized, controlled, crossover trial evaluated the effects of consuming AXOS at 0 (control), 2.2, or 4.8 g/d as part of ready-to-eat cereal for 3 wk in 55 healthy men and women. Fecal microbial levels, postprandial serum ferulic acid concentrations, and other physiologic parameters were assessed at the beginning and end of each condition. Results: The median bifidobacteria content of stool samples (log10/grams of dry weight [DW]) was found to be higher in the subjects consuming the 4.8-g/d dose (10.03) than in those consuming 2.2 g/d (9.93) and control (9.84, P < 0.001). No significant changes in the populations of other fecal microbes were observed, indicating a selective increase in fecal bifidobacteria. Postprandial ferulic acid was measured at 120 min at the start and end of each 3-wk treatment period in subjects at least 50 y old (n = 37) and increased in a dose-dependent manner (end-of-treatment values 0.007, 0.050, and 0.069 μg/mL for the control, AXOS 2.2 g/d, and AXOS 4.8 g/d conditions, respectively, P for trend < 0.001). Conclusion: These results indicate that AXOS has prebiotic properties, selectively increasing fecal bifidobacteria, and increases postprandial ferulic acid concentrations in a dose-dependent manner in healthy men and women.

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.

Comparative in vitro inhibition of urinary tract pathogens by single- and multi-strain probiotics

PURPOSE: Multi-species probiotic preparations have been suggested as having a wide spectrum of application, although few studies have compared their efficacy with that of individual component strains at equal concentrations. We therefore tested the ability of 4 single probiotics and 4 probiotic mixtures to inhibit the urinary tract pathogens Escherichia coli NCTC 9001 and Enterococcus faecalis NCTC 00775. METHODS: We used an agar spot test to test the ability of viable cells to inhibit pathogens, while a broth inhibition assay was used to assess inhibition by cell-free probiotic supernatants in both pH-neutralised and non-neutralised forms. RESULTS: In the agar spot test, all probiotic treatments showed inhibition, L. acidophilus was the most inhibitory single strain against E. faecalis, L. fermentum the most inhibitory against E. coli. A commercially available mixture of 14 strains (Bio-Kult(®)) was the most effective mixture, against E. faecalis, the 3-lactobacillus mixture the most inhibitory against E. coli. Mixtures were not significantly more inhibitory than single strains. In the broth inhibition assays, all probiotic supernatants inhibited both pathogens when pH was not controlled, with only 2 treatments causing inhibition at a neutral pH. CONCLUSIONS: Both viable cells of probiotics and supernatants of probiotic cultures were able to inhibit growth of two urinary tract pathogens. Probiotic mixtures prevented the growth of urinary tract pathogens but were not significantly more inhibitory than single strains. Probiotics appear to produce metabolites that are inhibitory towards urinary tract pathogens. Probiotics display potential to reduce the incidence of urinary tract infections via inhibition of colonisation.

Proportions of A1, A2, B and C β-casein protein variants in retail milk in the UK

The A1 variant protein of the β-casein family has been implicated in various disease states although much evidence is weak or contradictory. The primary objective was to measure, for the first time, the proportions of the key β-casein variant proteins in UK retail milk over the course of one year. In total, 55 samples of semi-skimmed milk were purchased from five supermarkets over the course of a year and the proportions of the A1, A2, B and C casein variant proteins were measured, using high resolution HPLC-MS. The results showed that β-casein in UK retail milk comprises approximately 0.58, 0.31, 0.07 and 0.03 A2, A1, B and C protein variants, respectively. The proportion of A2 is higher than some early studies would predict although the reasons for this and any implications for health are unclear