Flavour production by Saprochaete and Geotrichum yeasts and their close relatives.

In this study, a total of 30 yeast strains belonging to the genera Dipodascus, Galactomyces, Geotrichum, Magnusiomyces and Saprochaete were investigated for volatile organic compound production using HS-SPME-GC/MS analysis. The resulting flavour profiles, including 36 esters and 6 alcohols compounds, were statistically evaluated by cluster and PCA analysis. Two main groups of strains were extracted from this analysis, namely a group with a low ability to produce flavour and a group producing mainly alcohols. Two other minor groups of strains including Saprochaete suaveolens, Geotrichum marinum and Saprochaete gigas were diverging significantly from the main groups precisely because they showed a good ability to produce a large diversity of esters. In particular, we found that the Saprochaete genus (and their closed relatives) was characterized by a high production of unsaturated esters arising from partial catabolism of branched chain amino-acids. These esters were produced by eight phylogenetically related strains of Saprochaete genus.

Saccharomyces jurei sp. nov., isolation and genetic identification of a novel yeast species from Quercus robur.

Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain.

Complete Genome Sequence of Desulfovibrio piger FI11049.

The complete genome sequence of Desulfovibrio piger FI11049 was determined. The genome consists of a single circular chromosome of 2,807,531 bp encoding seven rRNA operons, 76 tRNA genes, and 2,535 coding genes.

Glucosinolate and Desulfo-glucosinolate Metabolism by a Selection of Human Gut Bacteria.

Glucosinolate (GSL) hydrolysis is mediated by the enzyme myrosinase which together with specifier proteins can give rise to isothiocyanates (ITCs), thiocyanates, and nitriles (NITs) in cruciferous plants. However, little is known about the metabolism of GSLs by the human gut flora. The aim of the work was to investigate the metabolic fates of sinigrin (SNG), glucotropaeolin (GTP), gluconasturtiin (GNT), and their corresponding desulfo-GSLs (DS-GSLs). Three human gut bacterial strains, Enterococcus casseliflavus CP1, Lactobacillus agilis R16, and Escherichia coli VL8, were chosen for this study. GNT was metabolized to completion within 24 h to phenethyl ITC and phenethyl NIT (PNIT) by all bacteria, except for L. agilis R16 which produced only PNIT. At least 80 % of GTP and SNG were metabolized by all bacteria within 24 h to the corresponding ITCs and NITs. The pH of media over time gradually became acidic for both L. agilis R16 and E. coli VL8, while for E. casseliflavus CP1 the media became slightly alkaline with NIT and ITC production occurring between pH 3.0 and 7.5. ITC production peaked between 4 and 10 h in most cases and gradually declined while NIT production increased and remained relatively constant over time. The total percentage products accounted for 3-53 % of the initial GSL. NITs were produced from DS-GSLs suggesting an alternative metabolism via desulfation for the food based GSLs. The metal ion dependency for NIT production for GNT and its DS form was investigated where it was shown that Fe(2+) increased NIT production, while Mg(2+) stimulated the formation of ITC.

Interactions of Salmonella enterica subspecies enterica serovar Typhimurium with gut bacteria.

The aim of this study was to evaluate the impact of the gut microbiota on the growth and survival of S. Typhimurium. This was tested in two-species co-cultures and in mixed cultures with a simplified gut model microbiota. Subsequently, interactions between S. Typhimurium and human faecal bacteria were quantified in both batch and continuous culture systems simulating the human colon. The exponential growth of S. Typhimurium was halted when the population of Escherichia coli reached the maximum population density in a two-compartment co-culture system where the two species were separated by a 0.45 µm pore membrane. Furthermore, the growth of some gut bacteria such as Lactobacillus gasseri and Bifidobacterium bifidum was inhibited by the presence of S. Typhimurium in the other compartment. The survival of S. Typhimurium was severely affected in mixed batch cultures with human faecal samples; a reduction of 10(3)-10(4) cfu/ml in the concentration of S. Typhimurium was observed in these cultures. However, no effect on S. Typhimurium survival was observed in mixed batch cultures with a simplified gut model microbiota under the same conditions. The effect of human faecal samples on S. Typhimurium in a three-stage continuous culture was different to that obtained in batch cultures; its growth rather than survival was affected under these conditions. S. Typhimurium growth was inhibited, and the bacterium was therefore eliminated by the continuous flow of the medium. Depending upon culturing conditions, the gut microbiota caused either growth inhibition, inactivation or did not affect S. Typhimurium.

A comparative study on the potential of epiphytic yeasts isolated from tropical fruits to produce flavoring compounds.

In recent years, there has been an increasing interest in identifying and characterizing the yeast flora associated with diverse types of habitat because of the many potential desirable technological properties of these microorganisms, especially in food applications. In this study, a total of 101 yeast strains were isolated from the skins of tropical fruits collected in several locations in the South West Indian Ocean. Sequence analysis of the D1/D2 domains of the large subunit (LSU) ribosomal RNA gene identified 26 different species. Among them, two species isolated from the skins of Cape gooseberry and cocoa beans appeared to represent putative new yeast species, as their LSU D1/D2 sequence was only 97.1% and 97.4% identical to that of the yeasts Rhodotorula mucilaginosa and Candida pararugosa, respectively. A total of 52 Volatile Organic Compounds (VOCs) were detected by Head Space Solid Phase Micro Extraction coupled to Gas Chromatography and Mass Spectroscopy (HS-SPME-GC/MS) from the 26 yeast species cultivated on a glucose rich medium. Among these VOCs, 6 uncommon compounds were identified, namely ethyl but-2-enoate, ethyl 2-methylbut-2-enoate (ethyl tiglate), ethyl 3-methylbut-2-enoate, 2-methylpropyl 2-methylbut-2-enoate, butyl 2-methylbut-2-enoate and 3-methylbutyl 2-methylbut-2-enoate, making them possible yeast species-specific markers. In addition, statistical methods such as Principal Component Analysis allowed to associate each yeast species with a specific flavor profile. Among them, Saprochaete suaveolens (syn: Geotrichum fragrans) turned to be the best producer of flavor compounds, with a total of 32 out of the 52 identified VOCs in its flavor profile.

Kazachstania yasuniensis sp. nov., an ascomycetous yeast species found in mainland Ecuador and on the Galápagos.

Seven strains representing a novel yeast species belonging to the genus Kazachstania were found at several collection sites on both mainland Ecuador (Yasuní National Park) and the Galápagos (Santa Cruz Island). Two strains (CLQCA 20-132(T) and CLQCA 24SC-045) were isolated from rotten wood samples, two further strains (CLQCA 20-280 and CLQCA 20-348) were isolated from soil samples, and three strains (CLQCA 20-198, CLQCA 20-374 and CLQCA 20-431) were isolated from decaying fruits. Sequence analyses of the D1/D2 domains of the LSU rRNA gene and ribosomal internal transcribed spacer (ITS) region indicated that the novel species is most closely related to Kazachstania servazzii and Kazachstania unispora. Although the strains could not be distinguished from one another based upon their differing geographical origins, they could be differentiated according to their isolation source (fruit, soil or wood) by ITS sequencing. The species name Kazachstania yasuniensis sp. nov. is proposed to accommodate these strains, with CLQCA 20-132(T) ( = CBS 13946(T) = NCYC 4008(T)) designated the type strain.

Investigating flavour characteristics of British ale yeasts: techniques, resources and opportunities for innovation.

Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour-related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production.

The metabolism of methylsulfinylalkyl- and methylthioalkyl-glucosinolates by a selection of human gut bacteria.

SCOPE: Certain myrosinase-positive human gut bacteria can metabolize glucosinolates (GSLs) to produce isothiocyanates (ITC) as chemopreventive agents. We investigated glucoerucin, glucoiberin, and glucoraphanin (present in broccoli) metabolism by human gut strains. METHODS AND RESULTS: All tested bacteria metabolized glucoerucin to completion within 16 h to erucin and erucin nitrile (NIT). Lactobacillus agilis R16 metabolized only 10% of glucoiberin and glucoraphanin with no detectable products. Enterococcus casseliflavus CP1, however, metabolized 40-50% of glucoiberin and glucoraphanin producing relatively low concentrations of iberin and sulforaphane. Interestingly, Escherichia coli VL8 metabolized 80-90% of glucoiberin and glucoraphanin and also bioconverted glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively, producing erucin, erucin NIT, iberverin, and iberverin NIT from the two GSLs. The putative reductase enzyme in the cell-free extracts of this bacterium required both Mg(2+) and NAD(P)H as cofactors for bioconversion. The cell-free extract of E. coli VL8 containing the reductase enzyme was able to reduce both the GSL glucoraphanin and its hydrolysis product sulforaphane to glucoerucin and erucin/erucin NIT, respectively. CONCLUSION: The composition and metabolic activity of the human gut bacteria can indirectly impact on the potential chemopreventive effects of GSL-derived metabolites.

Validation of DM-Scan, a computer-assisted tool to assess mammographic density in full-field digital mammograms.

We developed a semi-automated tool to assess mammographic density (MD), a phenotype risk marker for breast cancer (BC), in full-field digital images and evaluated its performance testing its reproducibility, comparing our MD estimates with those obtained by visual inspection and using Cumulus, verifying their association with factors that influence MD, and studying the association between MD measures and subsequent BC risk. Three radiologists assessed MD using DM-Scan, the new tool, on 655 processed images (craniocaudal view) obtained in two screening centers. Reproducibility was explored computing pair-wise concordance correlation coefficients (CCC). The agreement between DM-Scan estimates and visual assessment (semi-quantitative scale, 6 categories) was quantified computing weighted kappa statistics (quadratic weights). DM-Scan and Cumulus readings were compared using CCC. Variation of DM-Scan measures by age, body mass index (BMI) and other MD modifiers was tested in regression mixed models with mammographic device as a random-effect term. The association between DM-Scan measures and subsequent BC was estimated in a case-control study. All BC cases in screening attendants (2007-2010) at a center with full-field digital mammography were matched by age and screening year with healthy controls (127 pairs). DM-Scan was used to blindly assess MD in available mammograms (112 cases/119 controls). Unconditional logistic models were fitted, including age, menopausal status and BMI as confounders. DM-Scan estimates were very reliable (pairwise CCC: 0.921, 0.928 and 0.916). They showed a reasonable agreement with visual MD assessment (weighted kappa ranging 0.79-0.81). DM-Scan and Cumulus measures were highly concordant (CCC ranging 0.80-0.84), but ours tended to be higher (4%-5% on average). As expected, DM-Scan estimates varied with age, BMI, parity and family history of BC. Finally, DM-Scan measures were significantly associated with BC (p-trend=0.005). Taking MD<7% as reference, OR per categories of MD were: OR7%-17%=1.32 (95% CI=0.59-2.99), OR17%-28%=2.28 (95% CI=1.03-5.04) and OR>=29%=3.10 (95% CI=1.35-7.14). Our results confirm that DM-Scan is a reliable tool to assess MD in full-field digital mammograms.

Effect of garlic powder on the growth of commensal bacteria from the gastrointestinal tract.

Garlic (Allium sativum) is considered one of the best disease-preventive foods. We evaluated in vitro the effect of a commercial garlic powder (GP), at concentrations of 0.1% and 1% (w/v), upon the viability of representative gut bacteria. In pure culture studies, Lactobacillus casei DSMZ 20011 was essentially found to be resistant to GP whereas a rapid killing effect of between 1 and 3 log CFU/ml reduction in cell numbers was observed with Bacteroides ovatus, Bifidobacterium longum DSMZ 20090 and Clostridium nexile A2-232. After 6h incubation, bacterial numbers increased steadily and once the strains became resistant they retained their resistant phenotype upon sub-culturing. A colonic model was also used to evaluate the effect of GP on a mixed bacterial population representing the microbiota of the distal colon. Lactic acid bacteria were found to be more resistant to GP compared to the clostridial members of the gut microbiota. While for most bacteria the antimicrobial effect was transient, the lactobacilli showed a degree of resistance to garlic, indicating that its consumption may favour the growth of these beneficial bacterial species in the gut. Garlic intake has the potential to temporarily modulate the gut microbiota.

Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut.

A total of seventy lactic acid bacteria (LAB) were isolated from the faeces of healthy humans and their identities were confirmed by sequencing of their 16S rDNA genes. Of these only 5 isolates were found to resist bile salts and indicated survival in the simulated in vitro digestion assay which reproduces the stomach and intestinal digestion indicating their tolerance to gastric enzymes and the low pH conditions. Species that showed the best resistance to these conditions were: Lactobacillus casei, Lactobacillus sp., uncultured bifidobacteria, Enterococcus faecalis and Streptococcus anginosus. These strains were investigated further to study their capacity to adhere to human intestinal Caco-2 cells. E. faecalis was the most adherent strain. Examination of the virulence determinants for this strain indicated that it was positive for efaAfs, gelE, agg, cpd, cob, ccf and cad, a profile that is similar to that of many E. faecalis isolates from food sources. The cytolysin biosynthetic genes cylA, cylB and cylM that are more associated with the clinical isolates of E. faecium were not detected in this strain. The antibiotic susceptibility tests indicated that the strain was sensitive to vancomycin, tetracycline, rifampicin and erythromycin but resistant only to kanamycin and chloramphenicol. These data suggest that the strain E. faecalis CP58 may be tested further for beneficial properties and developed as a new probiotic.

Evaluation of the prebiotic properties of wheat arabinoxylan fractions and induction of hydrolase activity in gut microflora.

Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial bacteria such as lactobacilli and bifidobacteria in the human gastrointestinal tract. The effect of water-unextractable arabinoxylans (WU-AX) derived from wheat on the modulation of gut bacterial composition was investigated using a mixed culture fermentation system. A prebiotic index (PI) score of 2.03 was obtained after addition of 1% (w/v) WU-AX to a pH-controlled stirred anaerobic fermentation vessel. Pretreatment of the WU-AX with endo-beta-1,4-xylanase resulted in significantly higher PI value (3.48) indicating that pretreatment provided oligomers that were better utilised by the gut bacteria. The extracellular hydrolytic enzymes xylanase and ferulic acid esterase are both required for bacterial metabolism of WU-AX and both activities were present in supernatants derived from the mixed batch cultures. Addition of the WU-AX substrates to the batch cultures produced several fold increases of bacterial synthesis of both enzymes, and these increases were greater when the WU-AX substrate was pretreated with xylanase.

In vitro three-stage continuous fermentation of wheat arabinoxylan fractions and induction of hydrolase activity by the gut microflora.

In vitro fermentations were carried out by using a model of the human colon to stimulate microbial activities of gut bacteria. The model consisted of a three-stage culture system. Bacterial populations were evaluated under the effect of three types of arabinoxylan, a nonstarch polysaccharide derived from wheat, the water-unextractable arabinoxylan fraction (WU-AX), WU-AX pretreated with exogenous xylanase and the soluble water-extractable arabinoxylan fraction (WE-AX). The xylanase pretreated (WU-AX) had a stimulatory effect upon colonic bifidobacteria throughout all three vessels. Counts of Bacteroides spp. and Clostridium spp. were also both significantly reduced. Addition of the WU-AX substrates to the first vessel resulted in induction of bacterial synthesis of extracellular hydrolytic enzymes xylanase and ferulic acid esterase which are both required for bacterial metabolism of WU-AX; this induction was significantly greater with the xylanase treated WU-AX.

Isolation of commensal bacteria from umbilical cord blood of healthy neonates born by cesarean section.

In a previous study, lactic acid bacteria were isolated from meconium obtained from healthy neonates born by cesarean section. Such a finding suggested that term fetuses are not completely sterile, and that a mother-to-child efflux of commensal bacteria may exist. Therefore, presence of such bacteria in umbilical cord blood of healthy neonates born by elective cesarean section was investigated. The blood samples were submitted to an enrichment step and then inoculated onto agar plates. All the identified isolates belonged to the genus Enterococcus, Streptococcus, Staphylococcus, or Propionibacterium. Later, a group of pregnant mice were orally inoculated with a genetically labeled E. faecium strain previously isolated from breast milk of a healthy woman. The labeled strain could be isolated and polymerase chain reaction detected from the amniotic fluid of the inoculated animals. In contrast, it could not be detected in the samples obtained from a noninoculated control group.