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ABSTRACT: In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in β-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R(2) = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l(-1)) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l(-1)) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.
MIRCEN Journal of Applied Microbiology and Biotechnology 01/2013; · 1.08 Impact Factor
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ABSTRACT: Air-liquid biofilm formation is largely dependent on Flo11p and seems related to cell lipid content and composition. Here, it is shown that in the presence of cerulenin, a known inhibitor of the fatty acid synthase complex, biofilm formation is inhibited together with FLO11 transcription in a flor strain of Saccharomyces cerevisiae, while the administration of saturated fatty acids to cerulenin-containing medium restores biofilm formation and FLO11 transcription. It is also shown that, in biofilm cells, the FLO11 transcription is accompanied by the transcription of ACC1, ACS1 and INO1 key genes in lipid biosynthesis and that biofilm formation is affected by the lack of inositol in flor medium. These results are compatible with the hypothesis that the air-liquid biofilm formation depends on FLO11 transcription levels as well as on fatty acids biosynthesis.
FEMS Yeast Research 07/2012; 12(7):864-6. · 2.40 Impact Factor
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ABSTRACT: Pichia fermentans DiSAABA 726 is a biofilm-forming yeast that undergoes dimorphic transition. Under yeast-like morphology it controls brown rot caused by Monilia spp. on apple fruit, while under pseudohyphal form, it shows pathogenic behaviour itself on peach fruit. The present study investigates the nutritional factors that induce and separate yeast-like and pseudohyphal morphologies under laboratory conditions. We show that P. fermentans DiSAABA 726 produces mainly yeast-like cells on media containing millimolar concentrations of urea and diammonium phosphate, and forms pseudohyphae at micromolar concentrations of these two salts. With ammonium sulphate, yeast-like or pseudohyphal morphology depends on the N concentration and the pH of the culture media. Amino acids such as methionine, valine, and phenylalanine invariably induce pseudohyphal morphology irrespective of the N concentration and the pH of the culture media. Methionol, 1-butanol, isobutanol, and isopropanol induce pseudohyphal growth, while phenylethanol and isoamyl alcohol fail to induce the formation of filaments. Thus, the morphogenesis of P. fermentans DiSAABA 726 depends more on the nitrogen source than on the N concentration, and is regulated by the quorum-sensing molecules that are generally produced from amino-acid assimilation under nitrogen starvation.
Fungal Biology 07/2012; 116(7):769-77. · 1.43 Impact Factor
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ABSTRACT: Bacillus thuringiensis is a Gram positive bacterium that is used for the production of biopesticides. The toxic action of different strains and
serovars ofB. thuringiensis can be extremely selective towards specific pests, or, in contrast, it can affect a wide variety of non-target organisms
such as insects, vertebrates or humans. A reliable characterization of the cultivated strains is of primary importance for
the biopesticide industry, in order to assess the contamination of the final product with strains with different pesticide
actions or that might be dangerous for human health. The aim of this study was to develop useful methods for the typing of
differentB. thuringiensis strains using two PCR-based methods, RAPD and Rep-PCR with BOXA1R and ERIC2 primers. The molecular fingerprints obtained
using ERIC2-PCR showed a reliable ability to discriminateBacillus thuringiensis strains.
Annals of Microbiology 04/2012; 56(1):71-76. · 0.69 Impact Factor
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ABSTRACT: Air-liquid biofilm formation appears to be an adaptive mechanism that promotes foraging of Saccharomyces cerevisiae flor strains in response to nutrient starvation. The FLO11 gene plays a central role in this phenotype as its expression allows yeast cells to rise to the liquid surface. Here, we investigated the role of ammonium depletion in air-liquid biofilm formation and FLO11 expression in a S. cerevisiae flor strain. The data obtained show that increasing ammonium concentrations from 0 to 450 m m reduce air-liquid biofilm in terms of biomass and velum formation and correlate with a reduction of FLO11 expression. Rapamycin inhibition of the TOR pathway and deletion of RAS2 gene significantly reduced biofilm formation and FLO11 expression. Taken together, these data suggest that ammonium depletion is a key factor in the induction of air-liquid biofilm formation and FLO11 expression in S. cerevisiae flor strains.
Yeast 12/2011; 28(12):809-14. · 1.89 Impact Factor
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ABSTRACT: The influence of fenamidone ((S)-1-anilino-4-methyl-2-methylthio-4-phenylimidazolin-5-one), pyraclostrobin (methyl 2-[1-(4-chlorophenyl)pyrazol-3-yloxymethyl]-N-methoxycarbanilate), indoxacarb (methyl 7-Chloro-2,5-dihydro-2-[[(methoxycarbonyl) [4- (trifluoromethoxy) phenyl] amino] carbonyl] indeno[1,2-e][1,3,4] oxadiazine-4a(3H)-carboxylate), and deltamethrin ([cyano-[3-(phenoxy)phenyl]methyl] 3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropane-1-carboxylate) on spontaneous fermentation carried out by natural yeast grapes microflora, was studied during the wine-making process. Aliquots of pesticide standard solutions were added to the grapes before crushing, to reach a concentration equal or half the maximum residue limit (MRL). Vinifications were performed, with maceration (R), or without maceration (W). During the wine-making process, samples were taken at the beginning (one hour after grapes crushing), at the middle and at the end of the spontaneous fermentation process. At half the MRL concentration, deltamethrin affected Pichia sp. population with a decrease of almost 50 %, while fenamidone decreased Candida sp., Candida stellata at 83, and 36%, respectively. Metschnikowia pulcherrima population decreased in all samples when compared to the control. Experiments at MRL levels showed a strong reduction for all non-Saccharomyces yeast species, when grapes had been treated with pyraclostrobin, fenamidone, and deltamethrine, except for Candida sp. which was found to have been affected only by fenamidone residues. Growth zone inhibition test showed only an in vitro activity of pyraclostrobin over Kloeckera spp., C. stellata, and M. pulcherrima. Microvinification experiments produced wines with no differences concerning S. cerevisiae population as well as production of ethanol and residual sugars. Experiments showed that at the end of the fermentation process pesticides were adsorbed by the lees and grape skins, and no pesticides residue was detectable in wine.
Journal of Environmental Science and Health Part B Pesticides Food Contaminants and Agricultural Wastes 08/2011; 46(6):491-7. · 1.10 Impact Factor
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ABSTRACT: Flor strains of Saccharomyces cerevisiae form a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.
Applied and environmental microbiology 06/2010; 76(12):4089-91. · 3.69 Impact Factor
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ABSTRACT: During fermentation of high-sugar-containing medium lacking lipid nutrients, wine yeasts undergo oxidative stress and oxidative damage to cell membranes and proteins. Considering that cell membranes are important stress sensors, and that under hypoxic conditions wine yeasts modulate cell membranes composition by incorporating lipids available in the growth medium, in the present work, the effects of lipid nutrition on wine yeast oxidative stress response were evaluated on two strains of Saccharomyces cerevisiae. Biomarkers of oxidative stress, oxidative damage and antioxidant response were evaluated together with viability and acetic acid production during fermentation of a synthetic must lacking lipid nutrients as compared to added oleic acid and ergosterol. The results show that the availability of lipid nutrients causes a significant reduction in the intracellular content of reactive oxygen species and in the oxidative damage to membranes and proteins, as indicated by flow cytometry of cells stained with dihydroethidum (DHE) and propidium iodide (PI) and by Western blot of protein carbonyls. Accordingly, lipid nutrients feeding results in the increase in cell viability and superoxide activity, and the reduction in trehalose accumulation, proteinase A activity and production of acetic acid. In summary, these results are compatible with the hypothesis that the supplementation of lipid nutrients mitigates oxidative stress and oxidative damage in wine strains of S. cerevisiae during growth under unfavourable conditions.
International journal of food microbiology 06/2010; · 3.01 Impact Factor
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ABSTRACT: In Saccharomyces cerevisiae, FLO11 encodes an adhesin that is associated with different phenotypes, such as adherence to solid surfaces, hydrophobicity, mat and air-liquid biofilm formation. In the present study, we analysed FLO11 allelic polymorphisms and FLO11-associated phenotypes of 20 flor strains. We identified 13 alleles of different lengths, varying from 3.0 to 6.1 kb, thus demonstrating that FLO11 is highly polymorphic. Two alleles of 3.1 and 5.0 kb were cloned into strain BY4742 to compare the FLO11-associated phenotypes in the same genetic background. We show that there is a significant correlation between biofilm-forming ability and FLO11 length both in different and in the same genetic backgrounds. Moreover, we propose a multiple regression model that allows prediction of air-liquid biofilm-forming ability on the basis of transcription levels and lengths of FLO11 alleles in a population of S. cerevisiae flor strains. Considering that transcriptional differences are only partially explained by the differences in the promoter sequences, our results are consistent with the hypothesis that FLO11 transcription levels are strongly influenced by genetic background and affect biofilm-forming ability.
Microbiology 10/2009; 155(Pt 12):3838-46. · 3.06 Impact Factor
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ABSTRACT: A gas chromatographic ion-trap mass spectrometry (GC-ITMS) method was developed for the determination of the fungicide zoxamide in grape, must, wine, and spirits. Samples were extracted with hexane and analyzed without any clean up. The gas chromatograph was fitted with a carbofrit inserted into the glass liner to allow large volume injections. Analyses were carried out both in EI and CI mode. Recoveries from fortified samples ranged between 86 and 114% at four different fortification levels (n=6 each), ranging between 0.05 and 2.00 mg/kg. The relative standard deviation was below 19%. Both in EI and CI mode the calculated limit of detection (LOD) and quantification (LOQ) were 0.01 and 0.05 mg/kg (0.08 mg/kg in CI), respectively. Moreover the influence of yeasts and bacteria fermentation was evaluated.
Journal of Chromatography 01/2006; 1097(1-2):165-70. · 4.53 Impact Factor
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ABSTRACT: Saccharomyces cerevisiae flor yeasts, which are subjected to stressful conditions during wine ageing, exhibit a number of characteristics which distinguish them from non-flor S. cerevisiae wine strains. In the present work, 22 flor and 14 non-flor S. cerevisiae wine strains are compared, in order to elucidate other possible peculiarities of these yeasts. The results obtained demonstrate that in contrast to the homothallic nature of the non-flor strains, 77% of the flor strains exhibit two variants of a semi-homothallic life cycle. Moreover, the flor-forming ability is shown to be inversely correlated to spore viability and the utilisation of maltose and galactose.
FEMS Yeast Research 08/2005; 5(10):951-8. · 2.40 Impact Factor
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ABSTRACT: Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface.
Applied and Environmental Microbiology 07/2005; 71(6):2934-9. · 3.83 Impact Factor
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ABSTRACT: Several mutations in genes involved in Saccharomyces mating type switching may affect the homothallic behaviour in wine yeasts. In this study the semi-homothallic (Hq) segregation of a flor wine yeast strain was analysed. We aimed to understand the molecular basis of this behaviour in a flor autochthonous strain, verifying the MAT locus status by a PCR-based HO gene disruption and sequencing of the Y region of the HML, HMR and MAT loci, after nested PCR. Presence of ORFs a1 and a2 in the Y region of the HML locus was found. At the ORF a2 at HML locus, a mutation in the stop codon was found, so the a2 ORF contains 33 more bases.
Antonie van Leeuwenhoek 02/2004; 85(1):29-36. · 2.09 Impact Factor
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ABSTRACT: Several mutations in genes involved in Saccharomyces mating type switching may affect the homothallic behaviour in wine yeasts. In this study the semi-homothallic (Hq) segregation of a flor wine yeast strain was analysed. We aimed to understand the molecular basis of this behaviour in a flor autochthonous strain, verifying the MAT locus status by a PCR-based HO gene disruption and sequencing of the Y region of the HML, HMR and MAT loci, after nested PCR. Presence of ORFs a1 and a2 in the Y region of the HML locus was found. At the ORF a2 at HML locus, a mutation in the stop codon was found, so the a2 ORF contains 33 more bases.
Antonie van Leeuwenhoek 12/2003; 85(1):29-36. · 2.09 Impact Factor
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ABSTRACT: Sardinian sherry strains of S. cerevisiae form a biofilm on the surface of wine at the end of the ethanolic fermentation, when grape sugar is depleted and when further growth becomes dependent on access to oxygen. A point mutation in HSP12 or deletion of the entire gene results in inability to form this film. HSP12 encodes a heat-shock protein previously foundby others to be active during stationary phase, in cells depleted for glucose, and in cells metabolizing ethanol and fatty acids, all conditions associated with sherry biofilms.
Yeast 03/2002; 19(3):269-76. · 1.89 Impact Factor
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ABSTRACT: Fermentation by autochthonous yeasts may produce wines with enological properties that are unique of a specific area or variety of grape must. In order to identify yeast starter strains for the production of the Sardinian wine Cannonau DOC, 66 Saccharomyces cerevisiae strains, isolated from musts and wines Cannonau of six vitivinicole areas in Sardinia, were subjected to enological characterization and molecular identification. The RFLP-PCR fingerprinting of the ITS region of rRNA (ITS1-5,8S- ITS2) as well as ethanol, foam, and H<sub>2</sub>S production were analysed.
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ABSTRACT: In Saccharomyces cerevisiae RAS2 gene switches on cAMP-PKA and MAPK pathways (Sengupta et al .,
2006) that, in turn, regulate FLO11 gene. This gene is involved in many cell-cell adhesion phenotypes like
pseudohyphae formation, adhesive growth and biofilm formation (Reynolds and Fink, 2001; Zara et al .,
2005; Fidalgo et al ., 2006). The aim of this work was to assess the involvement of RAS2 gene in the airliquid
biofilm formation by Saccharomyces cerevisiae flor strains. To do that RAS2 gene was deleted in
the Saccharomyces cerevisiae flor strain 3238-32 and both the wild type and the ras2D mutant were
tested for biofilm formation and FLO11 transcription levels. ras2D maintained the ability to form biofilm,
while significant differences were observed in FLO11 transcriptional levels. Finally, the absence of glucose
and the presence of nitrogen enhanced air-liquid biofilm formation both in the parental and ras2D flor
strains. Thus, RAS2 deletion does not affect air-liquid biofilm formation.
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ABSTRACT: The importance of lipid nutrition and lipid metabolism during must fermentation has been recently
recognized by some authors (Belviso et al. , 2004; Mannazzu et al. , 2008; Zara et al. , 2008 and references therein). In particular it has been shown that during the first hours of fermentation in the absence of exogenous lipids, the induction of key genes in lipid metabolism is strongly correlated with the fermentative performances of Saccharomyces cerevisiae wine strains.
In this work we have analyzed the proteome of two Saccharomyces cerevisiae wine strains, with different susceptibility to stuck fermentation, during growth in the presence and absence of lipid supplements (ergosterol and oleic acid). The aim of the work was to further characterize at the proteome level, the molecular changes that industrial strains of Saccharomyces cerevisiae experiment during fermentation of lipid depleted musts. Total proteins extracted from yeast cells, collected 60h after the inoculum, were subjected to 2D Electrophoresis and the gels obtained were analyzed by means of the PDQuest software (Biorad). Results obtained showed that 50 proteins are differentially expressed in the two strains and that 2, in particular, are highly expressed in the strain resistant to stuck fermentation. Comparison of the 2DE maps obtained with those of Saccharomyces cerevisiae available on line (http://www.ibgc.ubordeaux2.fr/YPM/carte.htm) allowed us to identify the two proteins as two members of the HSP70 family, heat shock proteins that are involved in the general stress response of Saccharomyces cerevisiae. The attribution of the differentially expressed proteins to Gene Onthology categories will be carried out to better understand the molecular basis of yeast adaptation to the lack of lipid nutrients.
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ABSTRACT: Aim: To verify a possible correlation between cell lipid composition, expression of key genes in lipid metabolism and fermentative behaviour of Saccharomyces cerevisiae wine strains.
Methods and Results: The fermentative abilities of two commercial wine strains of S. cerevisiae were tested under stressful conditions. Cell number, glucose and fructose concentrations, expression of ACS1 , ACS2 , ACC1 , OLE1 , ERG9 , ERG10 , ARE1 and ARE2 and lipid content were evaluated. The strain that failed to complete the fermentation had lower amounts of C16:1 and C16:0 fatty acids at the beginning of fermentation (0 h) and late logarithmic phase (72 h). While the amount of C18:1 in this strain was lower than that in the strain that completed the fermentation at 0 h, same levels were observed for both strains at 72 h. The sterol levels were generally higher in the strain that failed to complete the fermentation. Gene expression generally increased from the beginning of the fermentation to the late logarithmic phase in both strains.
Conclusion: A positive correlation between good fermentative ability, elevated fatty acid content and ACC1 gene expression has been identified.
Significance and Impact of the Study: The cell lipid content at the time of inoculum and expression of ACC1 gene of starter strains should be carefully considered in order to identify the possible stuck/sluggish fermentations.
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ABSTRACT: Saccharomyces cerevisiae flor yeasts, which are subjected to stressful conditions during wine ageing, exhibit a number of characteristics which distinguish them from non- flor S. cerevisiae wine strains. In the present work, 22 flor and 14 non- flor S. cerevisiae wine strains are compared, in order to elucidate other possible peculiarities of these yeasts. The results obtained demonstrate that in contrast to the homothallic nature of the non- flor strains, 77% of the flor strains exhibit two variants of a semi-homothallic life cycle. Moreover, the flor -forming ability is shown to be inversely correlated to spore viability and the utilisation of maltose and galactose.
