Effects of Cocoa Husk Feeding on the Composition of Swine Intestinal Microbiota.

A two-diet/two-period change over experiment was performed to investigate the effects of cocoa husks, as a source of dietary fiber and polyphenols, on pig intestinal microbial composition. Six pigs were fed a conventional cereal-based diet or a diet obtained by substitution of 7.5% of the conventional diet with cocoa husks for 3 weeks. Experimental diets were isoproteic and isoenergetic. At the end of each 3 week testing period, samples of fresh feces were collected and analyzed for microbial composition by fluorescence in situ hybridization. Cocoa husks did not affect feed intake, weight gain, and feed efficiency. Analysis of fecal microbial populations, grouped by phyla, showed a decrease of Firmicutes and an increase of Bacteroidetes in cocoa husk-fed pigs. Particularly, cocoa husks reduced fecal populations of the Lactobacillus-Enterococcus group and Clostridium histolyticum and increased the Bacteroides-Prevotella group and Faecalibacterium prausnitzii, suggesting a potential for cocoa husks in the improvement of intestinal microbial balance.

Rhizobacterial communities associated with spontaneous plant species in long-term arsenic contaminated soils.

The microbial community composition in three soil fractions (bulk soil, rhizosphere and rhizoplane) of the root-soil system of a thistle, Cirsium arvense, and of a tufted hair grass, Deschampsia caespitosa, was investigated. The two spontaneous wild plant species were predominant in two Italian lands contaminated since centuries by arsenic and at present show high levels of arsenic (from 215 to 12,500 mg kg(-1)). In order to better understand how the rhizobacterial ecosystem responds to a long-term arsenic contamination in term of composition and functioning, culture-independent techniques (DAPI counts, fluorescence in situ hybridization and denaturing gradient gel electrophoresis analysis) along with cultivation-based methods were applied. Microbial community structure was qualitatively similar in the two root-soil systems, but some quantitative differences were observed. Bacteria of the α-, ß-, and γ-subclasses of the Proteobacteria were dominant in all fractions, while the subdominant groups (Cytophagaceae, gram-positive spore-forming, and filamentous bacteria) were significantly more abundant in the root-soil system of D. caespitosa. As regards to arsenic resistant strains, Firmicutes, Actinobacteria, Enterobacteria and γ-Proteobacteria were isolated from soil system of both plants. Our results suggest that the response to a high level of arsenic contamination governed the rhizosphere microbial community structure together with the soil structure and the plant host type effects. Data from this study can provide better understanding of complex bacterial communities in metal-polluted soils, as well as useful information of indigenous bacterial strains with potential application to soil remediation.

R(-)-O-desmethylangolensin is the main enantiomeric form of daidzein metabolite produced by human in vitro and in vivo.

After ingestion, human intestinal bacteria transform daidzein into dihydrodaidzein, which can be further metabolised to O-desmethylangolensin. This metabolite, unlike daidzein, has a chiral centre and can therefore occur as two distinct enantiomers; however, it is unclear which enantiomer is present in humans. The aim of this study was to define in vitro and in vivo the structure of O-desmethylangolensin and then to evaluate its pharmacokinetic parameters. Daidzein metabolism was preliminarily investigated in anaerobic batch cultures inoculated with mixed faecal bacteria from O-desmethylangolensin producer volunteers. The transformation was monitored by liquid chromatography-mass spectrometry and a chiral column was used to distinguish dihydrodaidzein and O-desmethylangolensin enantiomers. These were purified, analysed by circular dichroism and the results established R(-)-O-desmethylangolensin as the main product (enantiomer excess 91%). However, both dihydrodaidzein enantiomers were detected. Similar results were obtained by in vivo trials. The in vitro formation of O-desmethylangolensin seems to be directly correlated with the number of transforming microorganisms. This correlation was found in vivo for tmax but not for other pharmacokinetic indexes. The pharmacokinetics of daidzein, dihydrodaidzein and O-desmethylangolensin were then evaluated in 11 healthy adult O-desmethylangolensin producers after the single administration of soy milk containing 100mg daidzein. The conjugated forms of daidzein, dihydrodaidzein and O-desmethylangolensin represent more than 90 and 95% of the plasmatic and urinary forms, respectively. The Cmax, tmax and half-life of O-desmethylangolensin in plasma were 62±53nM, 28±11 and 15±6h, respectively. Relevant inter-individual variations were observed as indicated by the high standard deviations.

Arsenite oxidation in Ancylobacter dichloromethanicus As3-1b strain: detection of genes involved in arsenite oxidation and CO2 fixation.

The aim of this study was to characterize a facultative chemolithotrophic arsenite-oxidizing bacterium by evaluating the growth and the rate of arsenite oxidation and to investigate the genetic determinants for arsenic resistance and CO(2) fixation. The strain under study, Ancylobacter dichloromethanicus As3-1b, in a minimal medium containing 3 mM of arsenite as electron donor and 6 mM of CO(2)-bicarbonate as the C source, has a doubling time (t(d)) of 8.1 h. Growth and arsenite oxidation were significantly enhanced by the presence of 0.01 % yeast extract, decreasing the t(d) to 4.3 h. The strain carried arsenite oxidase (aioA) gene highly similar to those of previously reported arsenite-oxidizing Alpha-proteobacteria. The RuBisCO Type-I (cbbL) gene was amplified and sequenced too, underscoring the ability of As3-1b to carry out autotrophic As(III) oxidation. The results suggest that A. dichloromethanicus As3-1b can be a good candidate for the oxidation of arsenite in polluted waters or groundwaters.

Prebiotic potential and gastrointestinal effects of immature wheat grain (IWG) biscuits.

In this study the effects of immature wheat grains (IWG), a natural source of fructo-oligosaccharides (FOS), on intestinal microbiota and gastrointestinal function were evaluated. Ileal effluents from three ileostomates were collected for 8 h after IWG-enriched or Control biscuit administration, and then fermented in vitro with human faecal inoculum. On fermentation broths, microbial counts and SCFA were measured. Moreover, we investigated the effect of IWG biscuits on gastric emptying. Twelve healthy volunteers underwent ultrasound measurement of gastric emptying of a standard meal consumed 5 h after a breakfast composed by lactose-free milk and IWG or Control biscuits; hunger and satiety sensations were also evaluated in this period. Bifidobacteria and lactic acid bacteria counts were higher (P < 0.05) in IWG than in Control cultures, supporting the prebiotic effects of IWG, probably linked to its FOS content; moreover IWG fermentation produced more acetate. In vivo IWG biscuits delayed gastric emptying of the next meal (P < 0.05), decreased hunger (P < 0.001) and increased satiety (P < 0.005). Therefore in vitro results substantiate the potential of IWG as a prebiotic ingredient and in vivo data suggest an effect of IWG on gastric emptying and on persistent satiety. These preliminary data tend to support the use of IWG in formulating functional prebiotic products suitable to promote satiety.

Arsenic-resistant bacteria associated with roots of the wild Cirsium arvense (L.) plant from an arsenic polluted soil, and screening of potential plant growth-promoting characteristics.

A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100mM arsenate and 10mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.

The role of diet in the metabolism of daidzein by human faecal microbiota sampled from Italian volunteers.

The intestinal microbial transformation of daidzein into equol is subject to a wide inter-individual variability. The aim of this study was to investigate in vitro this transformation and to evaluate possible correlations between individual diet and equol production. The transformation of daidzein was investigated in anaerobic batch cultures inoculated with mixed fecal bacteria from 90 volunteers. The daidzein metabolism was monitored by liquid chromatography-mass spectrometry, and a chiral column was used to distinguish equol and dihydrodaidzein enantiomers. The obtained results show that daidzein was unchanged ( approximately 27%) or degraded to equol ( approximately 28%), O-desmethylangolensin ( approximately 12%) or dihydrodaidzein ( approximately 31%). Furthermore, some subjects ( approximately 2%) are able to produce both equol and O-desmethylangolensin. Bacteria represent sub-dominant populations (10(5)-10(9) cell/g wet faeces) in "slow" equol producers, while higher counts of equol-producing microorganisms (10(10)-10(11) cell/g wet faeces) were found in "quick" equol producers. The in vitro test to evaluate equol-producing status is quick and not invasive, and the obtained results are comparable with those reported in vivo. Indeed, the only enantiomer present in the batch cultures containing equol was the S-form. No significant correlations between equol production, BMI, age and sex were found. It seems that the equol-producer group consumed less fibre, vegetables and cereals, and more lipids from animal sources.

Generation and comparison of bioluminescent and fluorescent Bacillus licheniformis.

The environmental bacterium Bacillus licheniformis was transformed with two different shuttle-vectors (pCSS810 and pGFPratiometric) containing insect luciferase and green fluorescent protein genes, respectively. The cells were treated with various antimicrobial agents and the emitted bioluminescence and fluorescence were measured. Plasmid harboring the green fluorescent protein gene was totally segregated without selective pressure, and fluorescent B. licheniformis showed a slower growth rate than the wild-type strain; those cells were bright green as visualized by epifluorescent microscopy. However, fluorescence was not correlated to the growth state of cells or affected by the antibiotic treatments. To the contrary, luminescent transformant was found to be stable without antibiotic selection and showed analogous growth behavior compared to non-plasmid-bearing cells. The luminescent strain functioned as a biosensor for the antibiotics employed. Bioluminescence measurements allowed one to determine the viability of the recombinant cells and the kinetics of the antibacterial action could be followed. Thus, the light emission was found to be a reliable, sensitive, and real-time indicator of the "well-being" of cells, whereas fluorescence allowed one to visualize both metabolically active and inactive cells.

Conditions affecting cell surface properties of human intestinal bifidobacteria.

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.

Metabolism of stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora.

Stevia rebaudiana standardized extracts (SSEs) are used as natural sweeteners or dietary supplements in different countries for their content of stevioside or rebaudioside A. These compounds possess up to 250 times the sweetness intensity of sucrose, and they are noncaloric and noncariogenic sweeteners. The aim of this study was to investigate the in vitro transformation of stevioside and rebaudioside A after incubation with human microflora, the influence of these sweeteners on human microbial fecal community and which specific groups metabolize preferentially stevioside and rebaudioside A. The experiments were carried out under strict anaerobic conditions in batch cultures inoculated with mixed fecal bacteria from volunteers. The hydrolysis was monitored by HPLC coupled to photodiode array and mass spectrometric detectors. Isolated bacterial strains from fecal materials incubated in selective broths were added to stevioside and rebaudioside A. These sweeteners were completely hydrolyzed to their aglycon steviol in 10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol. Furthermore, stevioside and rebaudioside A did not significantly influence the composition of fecal cultures; among the selected intestinal groups, bacteroides were the most efficient in hydrolyzing Stevia sweeteners to steviol.