Proteome analysis of grape skins during ripening.

The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation.

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P(50))]. P(50) was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.

Impact of oxygen addition during enological fermentation on sterol contents in yeast lees and their reactivity towards oxygen.

During enological fermentations, superfluous oxygen consumption by yeast cells is observed. The superfluous oxygen consumed by the yeast cells is mainly related to the operation of non-respiratory oxygen consumption pathways resulting in an overall decrease in the total sterol fraction in yeast. On the other hand, yeast lees remaining at the end of alcoholic fermentations exhibit specific oxygen utilization rates ranging from 1 to 4 micromol O2 h- 10(-10) cells from the second to the thirteenth month of wine aging. This oxygen consumption capacity of yeast lees was independent of residual cell viability. In this study, we investigated the potential relationship between the oxygen added to commercial yeast strains during enological fermentation and the capacity of the corresponding yeast lees to interact with oxygen. Additions of low (7 mg l(-)) and excess (37 mg l(-1)) amounts of oxygen at the end of the cell growth phase were compared in terms of repercussions on the oxygen consumption activity of the corresponding yeast lees. As expected, the superfluous oxygen consumption by yeast cells during fermentation had a positive influence on the fermentation kinetics and increased cell biomass formation. Oxygen consumption rates and the total capacity of oxygen consumption by the corresponding yeast lees clearly decreased when oxygen was added during fermentation. This marked decrease in yeast lees reactivity towards oxygen was concomitantly related to an increase in ergosterol synthesis and to oxygen-dependent sterol degradation. Such degradation occurred when oxygen was added in excess. Therefore, oxygenation control during fermentation appears to be a potential way to optimize both the fermentation kinetics and control yeast lees reactivity towards oxygen. For practical applications, oxygenation control during alcoholic fermentation may be considered as a general tool for decreasing the highly reductive effect of yeast lees during wine aging.