Release of macromolecules by Saccharomyces cerevisiae during ageing of French flor sherry wine "Vin jaune".

The French flor sherry wine "Vin jaune" spends 6 years and 3 months in the same barrel under a yeast velum. Because of temperature variations in the cellars, this velum sinks partially into the wine and a deposit of dead yeasts cells accumulates in the bottom of the barrels, favouring the formation of new velum. Growth and autolysis occur simultaneously. This study investigated the evolution of macromolecules released by yeasts during the ageing of "Vin jaune" in a model system closely simulating winemaking. It was observed that the release of macromolecules during the formation of the velums by living yeasts was low but greatly increased when the velums fell and yeast viability decreased. The release of macromolecules was then due to the autolysis of dead cells. Analysis of macromolecules during ageing revealed that they contained 73.3-78.5% neutral sugars and 6-7% proteins according to the ageing stage. Their amino acid composition did not change during ageing. A high content of serine and threonine commonly involved in O-glycosidic linkages present in yeast mannoproteins was observed. Throughout ageing, the mannose and glucose contents of macromolecules increased but the ratio of polymeric mannose to glucose decreased. Size exclusion chromatography showed that mannoproteins released in wine were partially hydrolysed by yeast beta-1,3-glucanases freed in wine.

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.

Aroma compound sorption by oak wood in a model wine.

Oak wood used for wine barrels was immersed into a model wine containing eight aroma compounds (e.g., aromatic and terpene alcohols, ethyl esters, and aldehyde), for which activity coefficients in water and model wine were determined using the mutual solubility measurement. A mass balance of these volatiles considering their reactivity in model wine was established. For most of the studied aroma compounds, and mainly for linalool and ethyl octanoate, a sorption behavior into wood was reported for the first time. This phenomenon was selective and could not be related to the solubilities in model wine and hydrophobicities of the studied aroma compounds, suggesting that acid-base and polar characteristics of wood were more involved in this sorption mechanism. This study has also shown that the level of sorption is a function of the ratio of wood surface area/solution volume.

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 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.

Purification method for the isolation of monophosphate nucleotides from Champagne wine and their identification by mass spectrometry.

Monophosphate nucleotides are difficult to identify in Champagne wine because they are present in small concentrations in a complex mixture. A method for the isolation, separation and identification of reference compounds, which achieved on average 79% recovery (except for cytidine derivatives), was developed and applied to wine. Some monophosphate nucleotides were then isolated from a Champagne wine aged on lees for 8 years, by ultrafiltration followed by a semi-preparative HPLC step using a strong anion-exchange column. The fraction obtained was subjected to HPLC in a reversed-phase column to remove the salt previously introduced, before identification of compounds by HPLC coupled to a mass spectrometer. For the first time in wine, 5'-IMP, 5'-AMP, 5'-CMP, 5'-GMP, 5'-UMP and the 3'- and/or 2'-isomers of the four latter compounds were identified by comparing their HPLC and electrospray ionization mass spectrometry data with those of reference nucleotides.

Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis.

Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm. Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis. Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium. Ten sterols were identified by gas chromatography/mass spectrometry. A second group of six sterols was separated and partially characterized. Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S. cerevisiae. Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol. In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium. At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the medium.

Effect of ethanol on the glucose-induced movements of protons across the plasma membrane of Saccharomyces cerevisiae NCYC 431.

The study of glucose-induced proton fluxes in Saccharomyces cerevisiae NCYC 431 showed a decrease of proton net efflux by ethanol across the plasma membrane of energized cells. Furthermore a negative net proton efflux (an influx) occurred from a given ethanol concentration (between 1.3 and 1.5 M) whatever the experimental conditions used, thus allowing the definition of a nil-net exchange step where no net movement of protons across the plasma membrane could be observed. A new technique of ethanol tolerance determination in yeast based upon a correlation for the same ethanol concentration between both the collapse of the proton gradient and the growth cessation in cultures supplemented with ethanol after 8 h incubation was proposed. The defined method also showed a cumulated effect of temperature and ethanol on Saccharomyces cerevisiae NCYC 431.

[Influence of nitrogen components of wine on the growth of lactic acid bacteria]. / Influence des constituants azotés du vin sur la croissance des bactéries lactiques.

Three different fractions of nitrogen compounds have been extracted from wine: proteins, peptides, amino acids. They have been tested to support the growth of malolactic bacteria in acid medium: only the peptidic fraction supports this growth.