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ABSTRACT: Restriction digestion analysis of the ITS products was tested as an easy method to identify isolates of filamentous fungi on grapes. Endonucleases SduI, HinfI, MseI, HaeIII were used. Endonucleases BfmI, Cfr9I, Hpy188I, MaeII or PspGI were used as necessary to complete discrimination. The 43 species studied generated 42 different composite profiles. Only the species P. thomii and P. glabrum gave the same composite profile. 96.3% strains tested could be differentiated to the species level with only four enzymes. Hundred ninety nine strains of filamentous fungi were isolated from various vineyards in Burgundy and identified by this method. Penicillium (58.5%) was the genus the most frequently isolated and no strains of the genus Aspergillus was isolated. P. spinolusum was the most isolated species of Penicillium (22.70%). The species C. cladiosporioides, B. cinerea, E. nigrum, A. alternata, T. koningiopsis, P. diplodiella, C. herbarum, A. alternatum, T. cucumeris and F. oxysporum were also isolated. This technique is a rapid and reliable method appropriate for routine identification of filamentous fungi. This can be used to screen large numbers of isolates from various environments in a short time. This is the first exhaustive study of fungal diversity at species level in vineyard.
Food Microbiology 09/2011; 28(6):1145-54. · 3.28 Impact Factor
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G Spano,
P Russo,
A Lonvaud-Funel,
P Lucas, H Alexandre,
C Grandvalet,
E Coton,
M Coton,
L Barnavon,
B Bach, [......],
A Bunte,
C Magni,
V Ladero,
M Alvarez,
M Fernández,
P Lopez,
P F de Palencia,
A Corbi,
H Trip,
J S Lolkema
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ABSTRACT: Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods.
European journal of clinical nutrition 11/2010; 64 Suppl 3:S95-100. · 3.07 Impact Factor
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ABSTRACT: Volatile phenols, such as 4-ethylphenol, are responsible for a "horsey" smell in wine. Thus, the study of volatile phenol sorption in yeasts, and their subsequent elimination from wine, helps to optimize eco-friendly wine curative processes. Here, we compared the influences of spray drying, lyophilization and evaporative drying at low water activity on yeast, for improving the 4-ethylphenol sorption capacity in a synthetic model wine. The changes that occur in the physico-chemical characteristics of the yeast surface (surface hydrophobicity, electron-donor character and zeta potential) during these drying processes were determined to assess if any correlation exists between these factors and the 4-ethylphenol sorption capacities of the cells. Evaporative drying at low water activity, spray drying and lyophilization induced, respectively, 61.5%, 169% and 192% greater 4-ethylphenol sorption than biomass without drying treatment. Surface hydrophobicity of yeasts was also significantly greater, but the zeta potential of yeast cells was significantly lower after the drying processes. This is the first report investigating changes to the physico-chemical variables affected during yeast drying. These cell surface modifications were correlated with the 4-ethyphenol sorption value measured.
International journal of food microbiology 08/2009; 135(2):152-7. · 3.01 Impact Factor
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ABSTRACT: Growth of the lactic acid bacterium (LAB) Oenococcus oeni, which is involved in malolactic fermentation during the winemaking process, is stimulated by peptides originating from yeast. In this study, we investigated the impact of peptides on O. oeni growth, peptidase activity and the expression of genes encoding the studied peptidases.
Low levels of PepN activity and very high levels of PepI activity were observed in O. oeni, whereas levels of PepX activity were intermediate. The level of biosynthesis of these O. oeni peptidases was shown to depend on peptides present in the culture medium. These results were confirmed by transcriptional analyses of putative pep genes. The mechanism of repression by peptides did not involve a CodY-like regulator.
Peptides from yeast decrease the levels of enzymatic activity and relative gene expression of O. oeni peptidases. Peptidases specific for proline-containing peptides are important for O. oeni nitrogen metabolism.
We report here for the first time that the enzymes involved in the assimilation of proline-containing peptides by O. oeni differ from the well-described proteolytic system of milk LAB. This may reflect a specific adaptation to the wine environment.
Journal of Applied Microbiology 04/2009; 106(3):801-13. · 2.34 Impact Factor
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ABSTRACT: Because the yeast Brettanomyces produces volatile phenols and acetic acid, it is responsible for wine spoilage. The uncontrolled accumulation of these molecules in wine leads to sensorial defects that compromise wine quality. The need for a rapid, specific, sensitive and reliable method to detect this spoilage yeast has increased over the last decade. All these requirements are met by real-time PCR. We here propose improvements of existing methods to enhance the robustness of the assay. Six different protocols to isolate DNA from a wine and three PCR mix compositions were tested, and the best method was selected. Insoluble PVPP addition during DNA extraction by a classical phenol:chloroform protocol succeeded in the relief of PCR inhibitors from wine. We developed an internal control which was efficient to avoid false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The method was evaluated by an intra-laboratory study for its specificity, linearity, repeatability and reproducibility. A standard curve was established from 14 different wines artificially inoculated. The quantification limit was 31 cfu/mL.
International journal of food microbiology 01/2009; 129(3):237-43. · 3.01 Impact Factor
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ABSTRACT: To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA-1163 strain.
The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5.8 x 10(3) per microg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 125 kV cm(-1), under a resistance of 200 omega and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB).
An effective protocol to transform O. oeni ATCC BAA-1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA-1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein.
This is the first report of a successful electroporation of O. oeni ATCC BAA-1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.
Letters in Applied Microbiology 11/2008; 47(4):333-8. · 1.62 Impact Factor
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edited by Tec & Doc, Lavoisier Ed., Paris., 07/2008;
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ABSTRACT: Oenococcus oeni is a slow-growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino-acid medium. We aimed to characterize the oligopeptide use of this micro-organism.
Several peptides of two to eight amino-acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium.
Oenococcus oeni was able to transport the oligopeptides with two to five amino-acid residues tested and to hydrolyse them further.
This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.
Journal of Applied Microbiology 03/2008; 104(2):573-80. · 2.34 Impact Factor
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7ième Symposium International d'Œnologie., Bordeaux, France; 06/2003
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ABSTRACT: Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25 degrees C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l(-1), respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties.
Journal of Industrial Microbiology and Biotechnology 05/2001; 26(4):235-40. · 2.73 Impact Factor
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ABSTRACT: Analysis of velum-forming yeast cell wall components released by beta-1,3-glucanase treatment were compared with those of a non velum-forming yeast. SDS-PAGE electrophoresis and Western blotting with ConA-peroxidase staining of mannoproteins allowed us to identify a 49-kDa mannoprotein present in the cell wall of the velum-forming yeast and hardly visible in the control. The cell wall nature of this protein was confirmed by labelling with the non-permeable sulfosuccinimydiyl-6-(biotinamido)hexanoate reagent. A partial purification of this mannoprotein by anion exchange HPLC followed by surface hydrophobicity determination revealed that the fraction containing the 49-kDa mannoprotein was the most hydrophobic. Since cell surface hydrophobicity plays an important role in aggregate formation, it is likely that this mannoprotein is involved in velum formation.
FEMS Microbiology Letters 05/2000; 185(2):147-50. · 2.04 Impact Factor
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ABSTRACT: A pma1-1 mutant of Saccharomyces cerevisiae with reduced H(+)-ATPase activity and the isogenic wild-type strain accumulated high levels of trehalose in response to a temperature upshift to 40 degrees C and after addition of 10% ethanol, but only modest levels in response to a rapid drop in external pH and after addition of decanoic acid. There was, however, no correlation between the absolute levels of trehalose in the stressed cells and their viability. All these treatments induced a significant decrease in intracellular pH, and surprisingly, this decrease was very similar in both strains, indicating that intracellular acidification could not be the triggering mechanism for trehalose accumulation in response to stress. A careful investigation of metabolic parameters was carried out to explain how trehalose accumulated under the four different stress conditions tested. No single and common mechanism for trehalose accumulation could be put forward and the transcriptional activation of TPS1 was not unequivocally related to trehalose accumulation. Another finding was that a pma1-1 mutant exhibited a two- to threefold greater capacity to accumulate trehalose than the isogenic wild-type. This enhanced disaccharide synthesis could be attributed to a twofold higher trehalose-6-phosphate synthase activity, together with a fourfold higher content of intracellular UDP-Glc. In addition, this mutant showed 1.5-fold higher levels of ATP compared to the wild-type. The various stress treatments studied showed that a drop in intracellular pH does not correlate with trehalose accumulation. It is suggested that plasma membrane alteration could be the physiological trigger inducing trehalose accumulation in yeast.
Microbiology 05/1998; 144 ( Pt 4):1103-11. · 3.06 Impact Factor
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ABSTRACT: Recently a number of studies have focused on the factors responsible for the occurrence of stuck and sluggish fermentations.
Results from these studies indicate that together with nutritional deficiencies and inhibitory substances, technological practices
could lead to such situations. This review explains, from a biochemical point of view, the influence of nutritional deficiencies,
inhibitory substances and technological practices on yeast cell development and physiology and the fermentation process.
Journal of Industrial Microbiology and Biotechnology 12/1997; 20(1):20-27. · 2.73 Impact Factor
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ABSTRACT: Decanoic acid, a lipophilic agent, inhibited in vitro the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae grown in YPD medium. Conversely, when decanoic acid (35 microM) was present in the growth medium, the measured H(+)-ATPase activity was four times higher than that of control cells. Km, and pH and orthovanadate sensitivity were the same for the two growth conditions, which indicated that H(+)-ATPase activation was not due to conformational changes in the enzyme. The activation process was not entirely reversible which showed that plasma membrane H(+)-ATPase activation is due to several mechanisms. 1,6-diphenyl-1,3,5-hexatriene anisotropy performed on protoplasts from cells grown in YPD revealed that as decanoic acid concentration was increased, anisotropy significantly decreased, i.e. membrance fluidity increased. Cells grown in media containing decanoic acid exhibited greater membrane fluidity compared with control cells. Furthermore, these cells did not show any fluidifying effect when increased concentrations of decanoic acid were added. Chemical analysis of cell membrane lipid composition revealed a modification in the distribution of the phospholipid fatty acids and sterols in cells grown in the presence of 35 microM decanoic acid compared with control cells. Our results support the view that the plasma membrane H(+)-ATPase activation induced by decanoic acid is correlated with an alteration in membrane lipid constituents.
Microbiology 04/1996; 142 ( Pt 3):469-75. · 3.06 Impact Factor
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ABSTRACT: The lipid composition of a strain of each of two yeasts, Saccharomyces cerevisiae and Kloeckera apiculata, with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol:phospholipid and sterol:protein ratios and an increase in unsaturation index.
FEMS Microbiology Letters 12/1994; 124(1):17-22. · 2.04 Impact Factor
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ABSTRACT: Determination of the membrane lipid composition of Saccharomyces cerevisiae revealed an increase in the unsaturation index, qualitative and quantitative changes in sterol content and an alteration of the activity of the plasma membrane ATPase when cells were pre-adapted to ethanol. All these changes may constitute different adaptation mechanisms which allow the cell to cope with ethanol stress. The importance of the lipid environment on the plasma membrane ATPase activity is also discussed.
Biotechnology and Applied Biochemistry 11/1994; 20 ( Pt 2):173-83. · 1.53 Impact Factor
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ABSTRACT: Partially (6-fold) purified plasma membrane ATPase from an ethanol-sensitive yeast, Kloeckera apiculata, had an optimum pH of 6.0, an optimum temperature of 35C, a K
m of 3.6 mm ATP and a V
max of 11 mol Pi/min.mg protein. SDS-PAGE of the semi-purified plasma membrane showed a major band of 106 kDa. No in vivo activation of the ATPase by glucose was observed. Although 4% (v/v) ethanol decreased the growth rate by 50% it did not affect the ATPase. Concentrations of ethanol 2% (v/v) did, however, inhibit the enzyme in vitro. The characteristics of the enzyme did not change during growth in the presence of ethanol.
World Journal of Microbiology and Biotechnology 10/1994; 10(6):704-708. · 1.53 Impact Factor
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ABSTRACT: Direct measurements of membrane fluidity by fluorescence anisotropy of protoplasts fromKloeckera apiculata andSaccharomyces cerevisiae, a low and a high ethanol tolerant strain respectively, are presented. The comparison of the behaviour of the two strains
grown with or without ethanol enabled us to demonstrate the existing relationship between ethanol tolerance and membrane fluidity.
Biotechnology Techniques 04/1994; 8(5):295-300.
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ABSTRACT: For the first time, we report that enological yeasts or yeast cell walls can sorb (−)geosmin, an undesirable molecule that causes critical organoleptic defects in wine at low concentrations (around 50 ng l−1). The wine is described as “earthy” or “mouldy”. The influence of various post-harvesting processes on yeast (−)geosmin sorption capacity was studied. The dried yeast biomass obtained by the different processes was analysed by FTIR spectroscopy in ATR mode: structural differences were detected between the samples depending on the strain and the treatment used. Surface proteins and mainly phospholipids from the plasma membrane appeared to induce significantly different signals which may explain the different sorption capacities. The possible involvement of phospholipids in (−)geosmin sorption by yeasts highlights the complexity of this phenomenon.The post-harvesting processes were: spray-drying, mechanical cell disruption, autolysis and two methods of rehydration of dried yeast samples. The results show that, using the optimal combination of processes, the initial (−)geosmin concentration in model wine can be decreased by more than 50%.
Food Chemistry.
