**5. Conclusions**

Application of the PFGE technique allows the yeast population in the wine fermentation process to be characterized. The technique has been reported to be the most efficient for

degree of reproducibility of the final product. The development of global analysis methodology has allowed a detailed analysis to be made of changes in gene expression and protein levels at various time-points during vinification. Zuzuarregui et al. (2006) presented a comparison between the mRNA and protein profiles of two yeast strains with different fermentation behaviours, which correlates with divergence in the fermentation profiles. The results indicate changes in the mRNA and protein levels and, probably, post-translational modifications of several proteins, some of them involved in stress response and metabolism. Another proteomic approach was aimed at studying the adaptation of a wild-type wine yeast strain, isolated from a natural grape must, to physiological stresses during spontaneous fermentation (Trabalzini et al., 2003). Using 2DE, changes in the yeast proteome were monitored during glucose exhaustion, before the cells begin their stationary phase. The proteome adaptation of *S. cerevisiae* seems to be directed or caused by the effects of ethanol, leading to both hyperosmolarity and oxidative responses. Through the use of a wild-type *S. cerevisiae* strain and PMSF, which is a specific inhibitor of vacuolar proteinase B, it was also possible to distinguish the specific contributions of the vacuole and the proteasome autoproteolytic process. This is the first study that follows the adaptation of a physiologically wild wine yeast strain progressively to the exhaustion of an essential

To monitor yeast stress Salvadó et al. (2008), using ADWY (active dried wine yeast) inoculated into the must, have observed its behaviour in different stress situations, i.e. high sugar concentration or low pH. The main responses after inoculation in a fermentable medium were the activation of several genes of the fermentation pathway and the monoxidative branch of the pentose pathway, and the induction of a huge cluster of genes related to ribosomal biogenesis and protein synthesis. The changes that occur during the lag phase are characterized by an overall change in the protein synthesis and reflect the physiological conditions of the yeast, which affects the fermentative capacity and fermentation performance. Certain enological practices increase these stressful conditions for ADWY. This is the case of low-temperature fermentation, which improves taste by restructuring flavour profiles, with potential enological applications. This study focuses on changes that occur in ADWY after inoculation in a synthetic wine. These changes reflect

Previous reports have shown that proteomic analysis of wine yeast is the most relevant tool for understanding the physiological changes involved in winery processes. The information obtained may improve the quality of the final product. Our group has been a pioneer in fungal proteomic approaches (Fernandez-Acero et al., 2007, 2011; Garrido et al., 2010), and in line with this, we are now exploring the relevance of proteomics in wine improvement (Muñoz-Bernal et al., 2011). Our group has developed new protocols for obtaining the proteome and subproteomes of yeast, and the results to date suggest that there is a lot of biological information to be studied and analyzed from the proteomic perspective. The

Application of the PFGE technique allows the yeast population in the wine fermentation process to be characterized. The technique has been reported to be the most efficient for

relevance of this achievement for winery processes could be significant.

nutrient, glucose.

adaptation to a new medium.

**5. Conclusions** 

discriminating between *S. cerevisiae* yeast clones (Schuller et al., 2004), and it differentiates these from the specie *S. bayanus* var. *uvarum* (Naumov et al., 2002). It is also able to reveal the occurrence of gross chromosomal rearrangements, which account for the rapid evolution shown by yeast in industrial environments (Infante et al., 2003). Using PFGE, we have detected a high degree of polymorphism in the population of spontaneous fermentations of different types of wine produced in different regions of Spain, and it was observed that there were yeast strains that were specific to a particular phase of the fermentation process. This suggests that yeast strains with different karyotypes also differ in their adaptation to the evolving environment at different phases of the fermentation process. Studies for the molecular characterization of wine yeast represent a first step for selecting autochthonous yeast strains which are better adapted to specific conditions of a particular wine-making region. Moreover, such knowledge in respect of yeast populations may lead to the identification of a new natural source of wine yeast that could be used by the industry in the future as a new commercial starter (Fleet, 2008).

Studies by PFGE of the yeast population in inoculated fermentations also allow producers to understand and make informed decisions for improving their processes. Our results suggest that the success of the inoculation protocol is highly dependent on adequate preparation of the inoculums, which must facilitate the adaptation of the inoculated strains to the final conditions of the fermentation.

The RFLP test designed to monitor and confirm that the population of the inoculated yeast has reached and maintained predominance, in white or red wines, is proposed as a response to one of the major challenges for microbiological control in the wine industry. In our results real situations are shown taking place during actual wine fermentations; for example spontaneous fermentations sometimes occur before the inoculation. We offer a test which the winemaker can use to obtain a reliable indication of whether or not wild yeasts are displacing the inoculated strains. If the strategy presented is followed, the wine producer would be able to identify and correct in time the unwanted evolution of the yeast population - usually by re-inoculating the selected strains and/or correcting a deviation in temperature or change in some other parameter of the vessel that might have caused the unwanted situation.

Our studies are among the first examples carried out at the industrial scale showing how molecular techniques can be successfully applied to improve quality and efficiency in the winemaking process.

Despite the achievements already made, we are also exploring the potential use of the latest molecular proteomics techniques to unravel the biological component of the complex winemaking processes. Proteomics data collected to date strongly suggest that these techniques are potentially very useful for controlling the fermentation process and for assuring the quality of the finished wine; they offer excellent prospects for improving these processes in the near future.
