**4. Relevance of proteomic analysis in the winemaking process**

In brief, proteomics can be described as a set of techniques for unravelling complex mixtures of proteins. In spite of it being a relatively recent technique, most of the systems used are widely known by the research community. However, the crucial work for its final "take-off" as a viable technique has been the modifications made to the mass spectrometry system, to allow the analysis of peptides and proteins. The exponential growth in the number of entries for genes and/or proteins in the databases now makes protein analysis and identification much easier, as well. This, combined with the use of powerful methods of fractionation and separation of peptides and proteins, such as 2D-PAGE (two dimensional polyacrylamide electrophoresis) and high resolution liquid chromatography, proteomics has been consolidated since the mid-90's, as the science for massive protein analysis; it is now the main methodology for unravelling biological processes, leading some authors to describe the current period as the "post-genomic era".

Proteomics has been defined as the set of techniques for studying the complex mixture of proteins, named the Proteome, that exists in any specific cell, microorganism, tissue, etc, used in specific experimental conditions, culture, sampling, etc. It is a highly dynamic system, and is more complex than genomics because, while the genome of an organism is more or less constant, the number of proteomes obtained from a specific genome is infinite. It depends on the assayed cell, tissue, culture conditions, etc., because each change produces a modification in the observed proteome. An additional factor of complexity derives from the fact that there are changes that occur in proteome that are not encoded in the genome. These changes mainly originate from two sources: (i) the editing of the mRNA; and (ii) posttranslational modifications (PTMs) that normally serve to modify or modulate the activity, function or location of a protein in different physiological or metabolic contexts. More than

winery, effectively creating their own ecosystem, and can subsequently be predominant in the fermentations (Santamaría et al., 2005). In our study, we think that this commercial yeast was present in equipment which was not properly cleaned. When the wine-producer was more careful in the next vintage, there were no problems of contaminations by commercial yeast, and the dominant yeast in the fermentations was the inoculated autochthonous yeast P5 (data not shown). Therefore, it was confirmed that the commercial yeast had not acquired an ecological niche because it presumably did not adapt well to the ecosystem of a

As stated, the results of the RFLP can be obtained 11 and 23 hours after taking the sample for white and red wine respectively. However, this time can be shortened further, because it depends on the method used to rupture cells, on the number of samples analyzed per day, and on whether the samples contain a greater amount of must residues. In the case of red wine, there was another problem in shortening the test time, because the residues were difficult to clear by centrifugation; we think that some compounds remaining in the digested DNA samples were inhibitory for the endonuclease. Therefore, a step has been added in the protocol of the rapid test (Figure 5) in which the sample of the red must is plated on YPDagar and incubated overnight at 28 ºC. Nevertheless, we think that, for red wine, the time taken to obtain the results could also be shortened further, like that for white wine, if the clean biomass can be separated from the must residues in a few minutes. To achieve this,

In brief, proteomics can be described as a set of techniques for unravelling complex mixtures of proteins. In spite of it being a relatively recent technique, most of the systems used are widely known by the research community. However, the crucial work for its final "take-off" as a viable technique has been the modifications made to the mass spectrometry system, to allow the analysis of peptides and proteins. The exponential growth in the number of entries for genes and/or proteins in the databases now makes protein analysis and identification much easier, as well. This, combined with the use of powerful methods of fractionation and separation of peptides and proteins, such as 2D-PAGE (two dimensional polyacrylamide electrophoresis) and high resolution liquid chromatography, proteomics has been consolidated since the mid-90's, as the science for massive protein analysis; it is now the main methodology for unravelling biological processes, leading some authors to describe

Proteomics has been defined as the set of techniques for studying the complex mixture of proteins, named the Proteome, that exists in any specific cell, microorganism, tissue, etc, used in specific experimental conditions, culture, sampling, etc. It is a highly dynamic system, and is more complex than genomics because, while the genome of an organism is more or less constant, the number of proteomes obtained from a specific genome is infinite. It depends on the assayed cell, tissue, culture conditions, etc., because each change produces a modification in the observed proteome. An additional factor of complexity derives from the fact that there are changes that occur in proteome that are not encoded in the genome. These changes mainly originate from two sources: (i) the editing of the mRNA; and (ii) posttranslational modifications (PTMs) that normally serve to modify or modulate the activity, function or location of a protein in different physiological or metabolic contexts. More than

properly-cleaned winery.

further experiments will need to be carried out.

the current period as the "post-genomic era".

**4. Relevance of proteomic analysis in the winemaking process** 

200 different PTMs have been described (including phosphorylation, methylation, acetylation, etc.) that transform each single gene into tens or hundreds of different biological functions. Before the advances made in proteomics, the differential analysis of the genes that were expressed in different cell types and tissues in different physiological contexts was done mainly through analysis of mRNA. However, for wine yeast, it has been proved that there is no direct correlation between mRNA transcripts and protein content (Rossignol et al., 2009). It is known that mRNA is not always translated into protein, and the amount of protein produced by a given amount of mRNA depends on the physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of its abundance and diversity.

In terms of methodology, proteomics approaches are classified in two groups: (i) gel-free systems based on the use of various chromatography methods; and (ii) gel-based methods that use mainly two-dimensional polyacrylamide gel electrophoresis (2DE). This latter approach will form the focus of our discussion here, given the subject matter of this book. As a succinct summary, the typical workflow of a proteomic experiment begins with the experimental design. This must be studied in depth, and it will delimit the conclusion obtained, even more so when comparisons are made between two strains, cultures or physiological stages, among others. As an optimum, only one factor among the various different assayed conditions must change (Fernandez-Acero et al., 2007). Several biological replicates, usually from 3 to 5, will be required depending on the strategy adopted. The next key step is to obtain a protein extract of high enough quality to separate complex mixtures of proteins. Usually, the protein extraction is done in sequential steps. First the tissue, cells, etc. are ruptured using mechanical or chemical techniques. Then, proteins are precipitated and cleaned. Most existing protocols use acetone and trichloroacetic acid. In the next step the proteome is defined and visualized using electrophoretic techniques. 2DE has been widely used for this purpose. Using this technique, proteins are separated using two different parameters. In the first dimension, proteins from the purified extract are separated by their iso-electric point using an iso-electrofocusing (IEF) device. Then, the focused strips are loaded in a polyacrylamide gel where the proteins are separated by their molecular weight. This system allows the separation of hundreds of proteins from one complex mixture. The gels are visualized with unspecific (Comassie, Sypro, etc.) or specific (e.g. Phospho ProQ diamond) protein stains. The gels are digitalized and analyzed with specific software to reveal the significant spots. These spots are identified using mass spectrometry; commonly, for 2DE approaches, MALDI TOF/TOF is used. The huge list of identified proteins obtained is studied to discover the biological relevance of each identification.

In spite of the many achievements of proteomics, only a few proteomic studies have been carried out on wine yeast, whereas mRNA expression has been widely used to study a broad range of industrial conditions. However, Rossignol et al. (2009) show that substantial changes in protein levels during alcoholic fermentation are not directly associated with changes in the transcriptome; this suggests that the mRNA is selectively processed, degraded and/or translated. This conclusion is important: it is the proteome, not the genome nor the transcriptome, that is the relevant level of analysis for understanding the adaptations of wine yeasts during alcoholic fermentation, since these are responsible for the phenotype.

The usual strategy for wine production is the inoculation of selected yeast strains into the must, decreasing the lag phase, a quick and complete fermentation of the must, and a high

Application of Gel Electrophoresis Techniques to

future as a new commercial starter (Fleet, 2008).

conditions of the fermentation.

unwanted situation.

winemaking process.

processes in the near future.

**6. Acknowledgements** 

the Study of Wine Yeast and to Improve Winemaking 17

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

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

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

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

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

This work was supported by grants PETRI 95-0855 OP from the DGICYT of the Ministry of Science and Innovation, and OT 054/174/015/020/114/136/104 from Bodegas Barbadillo

S.L. of Sanlúcar de Barrameda, Spain, and CDTI-IDI-20101408.

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 nutrient, glucose.

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 adaptation to a new medium.

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 relevance of this achievement for winery processes could be significant.
