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Bacillus subtilis CU1 is a recently described probiotic strain with beneficial effects on immune health in elderly subjects. The following work describes a series of studies supporting the safety of the strain for use as an ingredient in food and supplement preparations. Using a combination of 16S rDNA and gyrB nucleotide analyses, the species was identified as a member of the Bacillus subtilis complex (B. subtilis subsp. spizizenii). Further characterization of the organism at the strain level was achieved using random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) and pulsed field gel electrophoresis (PFGE) analyses. B. subtilis CU1 did not demonstrate antibiotic resistance greater than existing regulatory cutoffs against clinically important antibiotics, did not induce hemolysis or produce surfactant factors, and was absent of toxigenic activity in vitro. Use of B. subtilis CU1 as a probiotic has recently been evaluated in a 16-week randomized, double-blind, placebo-controlled, parallel-arm study, in which 2 × 109 spores per day of B. subtilis CU1 were administered for a total 40 days to healthy elderly subjects (4 consumption periods of 10 days separated by 18-day washouts). This work describes safety related endpoints not previously reported. B. subtilis CU1 was safe and well-tolerated in the clinical subjects without undesirable physiological effects on markers of liver and kidney function, complete blood counts, hemodynamic parameters, and vital signs.

Cranberry consumption has been demonstrated to improve features of the metabolic syndrome, therefore providing an alternative strategy to prevent obesity and type-2 diabetes. Moreover, gut dysbiosis is now considered as a key factor in metabolic disorders. In order to understand the involvement of phenolic compounds in the health-improving effects of cranberry, this study aimed to investigate their bioavailability after oral administration of a cranberry extract (CE) to high-fat high-sucrose (HFHS) fed mice, and to explore a possible modulation of gut microbiota composition following a co-supplementation with spores of Bacillus subtilis CU1 probiotic (CE/P). Phenolic metabolites were extracted and characterized from plasma using μSPE-UHPLC-MS/MS, and a metagenomic analysis was performed on feces to assess gut bacterial composition. 22 circulating metabolites were identified, mainly microbial degradation products of native cranberry phenolic compounds. Plasma concentration of 3 microbial metabolites was significantly increased with the CE/P co-treatment: p -coumaric acid, m -coumaric acid and p -hydroxybenzoic acid (+53%, +103% and +70%, respectively). Associated to this modulation, we reported significant differences in the proportion of Barnesiella and Oscillibacter genera in CE/P treated mice in comparison with control animals. This study thus highlights the impact of an altered gut microbiota on phenolic compounds degradation and bioavailability in mice.

Flavonoids are a group of polyphenolic dietary compounds that have been proposed to possess chemopreventive properties against lung cancer. In this work we analyzed the effect of a group of 20 structurally related flavonoids, including flavones, flavonols and isoflavones, on the production of vascular endothelial growth factor (VEGF) induced by hypoxia in NCI-H157 cells. VEGF is the main regulator of physiological and pathological angiogenesis and is highly stimulated by hypoxia-inducible factor 1 (HIF-1). We found that apigenin, luteolin, fisetin and quercetin inhibited hypoxia-induced VEGF expression in the low micromolar range. Structure-activity relationships demonstrated that flavone derivatives were the most active compounds and that hydroxylation of the A ring at the positions 5 and 7 and of the B ring at the 4' position were important for this activity. Interestingly, only a group of VEGF inhibitors, including apigenin, flavone and 4',7-dihydroxiflavone, reduced the expression of HIF-1alpha under these conditions, whereas others, such as fisetin, luteolin, galangin or quercetin, induced HIF-1alpha expression while reducing those of VEGF. When cells were exposed to hypoxia in the presence of these flavonoids, HIF-1alpha translocated to the nucleus and interacted with p300/CBP, but this complex was transcriptionally inactive. Taken together these findings indicate that flavonoids impair VEGF transcription by an alternative mechanism that did not depend on nuclear HIF levels. We also found that flavonoids suppressed hypoxia-induced STAT3 tyrosine phosphorylation and that this activity correlated with their potency as VEGF inhibitors, suggesting that inhibition of STAT3 function may play a role in this process.

The effect of tea on the fermentation and survival of yogurt microorganisms was studied. Green and black teas were added to milk at the beginning of fermentation. Acidity of yogurt products and survival of their microflora were studied during 42 days at 4 °C. Results showed that the presence of tea did not significantly (P

Amicoumacins are natural products with potent anti-ulcerogenic and anti-bacterial activities, and have been isolated from different Bacillus genera. They belong to a family of 3,4-dihydroisocoumarin derivatives bearing hydroxylated amino acid side chains. The 3,4-dihydroisocoumarin moiety of Amicoumacins has been obtained in two steps from 2-methoxybenzoic acid by combining directed and benzylic metalation strategies. The use of s-BuLi in both steps gave satisfactory and reproducible yields. For the development of an immunoassay (ELISA) of Amicoumacin-related compounds in biological media, the deprotected 3,4-dihydroisocoumarin moiety has been coupled to the BSA carrier protein via a homobifunctional linker deriving from d-tartaric acid. This approach enabled to introduce the hydroxylated portion of Amicoumacin directly during the preparation of hapten-protein conjugates. The coupling ratio was evaluated by mass spectrometry. The hapten-protein conjugate showing the best coupling ratio was used to generate polyclonal immunosera in rabbits. After immunoserum titration, ELISA conditions were set up and specificity tests were performed on solutions of pure parent compounds, semi-purified Amicoumacin B as well as on culture supernatants of strains known for their Amicoumacin production. This immunoassay is suitable for a rapid and simple screening test for the production of Amicoumacins and its related compounds by bacterial strains.

A Lactobacillus plantarum strain producing exopolysaccharides (EPSs) was isolated from corn silage. When this strain, named L. plantarum EP56, was grown on a chemically defined medium, two EPS fractions were isolated. The cell-bound EPS fraction (EPS-b) was composed of a single high-molecular-mass polymer of 8.5x10(5) Da containing glucose, galactose and N-acetylgalactosamine in a molar ratio of approximately 3:1:1 and traces of glycerol and phosphoglycerol. The released EPS fraction (EPS-r) was composed of the high-molecular-mass bound polysaccharide and a second polymer of 4x10(4) Da containing glucose, galactose and rhamnose in a molar ratio of 3:1:1 and traces of glycerol and phosphoglycerol. EPS-b and EPS-r contained phosphate which contributes to their negative net charge. Studies on polysaccharide production and location showed that both polymers were synthesized during the exponential growth phase and that the EPS-b polymer was progressively released into the culture medium during the stationary growth phase. Carbon source and temperature influenced EPS synthesis when L. plantarum EP56 was grown in a chemically defined medium. Lactose was the most efficient carbon source among the five tested (glucose, galactose, fructose, lactose and sucrose). EPS production was also increased when the incubation temperature is lowered.

Nonculturable segmented filamentous bacteria (SFB) have been described in the gut of rats, mice and chickens, and 16S rRNA sequences for these organisms are available. These organisms, peripherically related to Clostridium phylogenetic group I, have been provisionally named 'Candidatus Arthromitus'. This work reports the observation of similar bacteria in the intestinal content of the distal intestine, preferentially, in the adult rainbow trout (Oncorhynchus mykiss) that exhibited episodic acute diarrhea, usually during the summer. Abdominal distension, intestinal fluid-mucus content and epithelium detachment were observed in trout. The demonstration that the observed microorganisms are bacteria and belong in the 'Candidatus Arthromitus' group was achieved by in situ hybridization with, respectively, a eubacterial probe and an oligonucleotide probe designed to react specifically with SFB 16S rRNA (encoded by the rrs gene) sequences. The sequenced rrs gene was compared with published sequences and found to be closely related to (although distinct from) other SFB sequences. Implication of these bacteria in trout diarrheic illness remains hypothetical.

A PCR-based method was developed to identify and detect Flavobacterium psychrophilum , the causative agent of “cold-water disease” and “rainbow trout fry syndrome” in salmonid fish. Two oligonucleotide primers were designed by comparing the 16S rRNA sequence of all taxa in the genus Flavobacterium and of representative species in most related genera within rRNA superfamily V. Purified chromosomal DNAs from all these bacterial species, from 25 F. psychrophilum isolates and from several other fish-pathogenic bacteria were used to assess the specificity of the reaction. Amplification products were generated only with F. psychrophilum DNA. The detection level, equivalent to approximately 10 to 100 bacterial cells, was increased 10-fold by hybridization with a radioactive probe. Preliminary experiments demonstrated that this procedure can also be applied to samples of infected tissue. This PCR assay is therefore a rapid, specific, and sensitive alternative to conventional plate culture methods for the identification and detection of F. psychrophilum .

In this paper we report a rapid method to screen yeast mutants exhibiting reduced viability directly on plates. This method avoids the need for replica plating and is based on the addition of the vital dye erythrosine B in nutrient medium. After 2 or 3 days of culture, colonies containing a large proportion of dead cells show a pink or a dark pink color whereas normal colonies are practically white.

The distribution of Vibrio species in water and seafood collected from Arcachon Bay (located in the southwest of France) was studied. All invertebrate animals collected were associated with one or more Vibrio species. Eighty strains corresponding to 14 species were precisely identified. The identification of strains with V. parahaemolyticus was checked by DNA/DNA hybridization. The most frequently recovered species were V. alginolyticus, V. parahaemolyticus (non-haemolytic strains), V. harveyi and V. metschnikovii. The three V. cholerae non-O1 strains isolated from water and crab did not produce immunologically detectable cholera toxin and had no DNA fragment hybridizing with a cholera-toxin-gene-specific probe. It is suggested that the sanitary surveillance of seafood in France should include the precise characterization of potentially pathogenic Vibrio species.