Claudine Charpentier

Production and Tolerance of Ethanol in Relation to Phospholipid Fatty-acyl Composition in Saccharomyces cerevisiae NCYC 431

Summary: Accumulation of ethanol in supernatants from anaerobic cultures of Saccharomyces cerevisiae NCYC 431 closely paralleled growth during the early exponential phase of batch growth, and continued after growth had ceased. During the 8–64 h period of the fermentation, the intracellular ethanol concentration was greater than the extracellular concentration. Ethanol was very rapidly extracted from organisms by washing with water. During growth up to 32 h, there was a progressive decrease in fatty-acyl unsaturation in phospholipids, and a corresponding proportional increase in saturation. Thereafter, the trend was very slightly reversed. Supplementing cultures with ethanol (0.5 or 1.0m) after 8h incubation retarded growth rate, while supplementation with 1.5 m-ethanol immediately stopped growth. In cultures supplemented with 0.5 or 1.0 m-ethanol, viability was not lowered, but supplementation with 1.5 m-ethanol caused a rapid decline in viability. Supplementation of cultures with ethanol at any of the three concentrations led to an increase in the proportion of mono-unsaturated fatty-acyl residues in cellular phospholipids, especially in C18 residues, which was accompanied by a decrease in the proportion of saturated residues.

Alteration in membrane fluidity and lipid composition, and modulation of H+-ATPase activity in Saccharomyces cerevisiae caused by decanoic acid

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.

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25°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 & Biotechnology (2001) 26, 235–240.

Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae.

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.

Effect of ethanol on membrane fluidity of protoplasts fromSaccharomyces cerevisiae andKloeckera apiculata grown with or without ethanol, measured by fluorescence anisotropy

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.

Yeast adapted to wine: Nitrogen compounds released during induced autolysis in a model wine

As important as the blend of base wines before bottling, one of the most important steps in the champagne-making process is the long ageing on lees. Two yeast strains of Saccharomyces cerevisiae MC001 and MC002, used in champagne wine production, were allowed to autolyse. After 8 days of autolysis, active dry yeasts adapted to wine released 1.7- to 1.8-fold more nitrogen compounds than nonadapted active dry yeast. The nitrogen content (total, proteins, peptides and amino) present in autolysates was measured for yeasts adapted to wine. The composition of free amino acids and amino acids constituting peptides showed no difference between the two strains of yeast used. Studies of intracellular proteolytic activity and release of peptides showed no correlation between these two phenomena. These results indicate that yeasts adapted to wine give results similar to those that occur in wine during ageing. Journal of Industrial Microbiology & Biotechnology (2002) 29, 134–139 doi: 10.1038/sj.jim.7000291

Effects of physico-chemical parameters of a model wine on the binding of γ-decalactone on bovine serum albumin

Abstract To understand the effect of temperature, pH and the composition of alcoholic beverages in flavour-protein interactions, the binding of γ-decalactone to bovine serum albumin (BSA) was investigated using the equilibrium dialysis method. Thermodynamic analysis revealed that the affinity of aroma compound for BSA is higher at 10 °C than at 20 and 30 °C, while the number of binding sites (n = 6–7) is not modified at the three temperatures. pH did not have any appreciable effect on flavour binding in the presence of ethanol, but it was observed that a decrease of 1.8 pH unit reduces binding by 40% in its absence. The presence of ethanol has no effect on the number of binding sites and on the standard free energy (ΔG °) of the interactions. On the other hand, the binding constant (k) was 4.8-fold higher in water than in model wine (pH 3.5, ionized compounds, 10% w/w ethanol); so, the affinity of volatile compound was clearly lower in the model wine than in water.

Population Structure and Comparative Genome Hybridization of European Flor Yeast Reveal a Unique Group of Saccharomyces cerevisiae Strains with Few Gene Duplications in Their Genome

Wine biological aging is a wine making process used to produce specific beverages in several countries in Europe, including Spain, Italy, France, and Hungary. This process involves the formation of a velum at the surface of the wine. Here, we present the first large scale comparison of all European flor strains involved in this process. We inferred the population structure of these European flor strains from their microsatellite genotype diversity and analyzed their ploidy. We show that almost all of these flor strains belong to the same cluster and are diploid, except for a few Spanish strains. Comparison of the array hybridization profile of six flor strains originating from these four countries, with that of three wine strains did not reveal any large segmental amplification. Nonetheless, some genes, including YKL221W/MCH2 and YKL222C, were amplified in the genome of four out of six flor strains. Finally, we correlated ICR1 ncRNA and FLO11 polymorphisms with flor yeast population structure, and associate the presence of wild type ICR1 and a long Flo11p with thin velum formation in a cluster of Jura strains. These results provide new insight into the diversity of flor yeast and show that combinations of different adaptive changes can lead to an increase of hydrophobicity and affect velum formation.

Modelling the inhibitory effect of copper sulfate on the growth of Penicillium expansum and Botrytis cinerea

Aims:  This study aimed to investigate the effect of copper sulfate (from 0 to 8 mmol kg−1) on radial growth rate and lag time of two moulds responsible for vine grapes spoilage: Penicillium expansum strain 25·03 and Botrytis cinerea, strains BC1 and BC2.

Research report: Interactions between toxic fatty acids for yeasts and colloids, cellulose and yeast ghost using the equilibrium dialysis method in a modelwine system

Abstract The capacity of different materials (yeast walls, colloids and cellulose) to bind octanoic acid and decanoic acid was investigated in a model wine. The interactions between these toxic fatty acids and the soluble or insoluble material was shown using the equilibrium dialysis method. Yeast walls adsorb decanoic acid and to a lesser extent octanoic acid which confirms previous results. In comparison, colloids from both grape or yeast weakly bind decanoic acid and do not significantly bind octanoic acid. No interactions between cellulose and fatty acid were observed. According to the yeast wall composition, lipids seem to play a key role for binding. With regards to our results, the effect of colloids on fermentation rate and malolactic activity is discussed.