**1. Introduction**

Yeasts are unicellular fungi that are frequently used as a model and tools in basic science studies. This is the case of the laboratory yeasts *Saccharomyces cerevisiae*, which were introduced in the laboratory for genetics and molecular studies in about 1935. There is, however, a second type of yeast comprising those used in industrial processes, for example, in brewing, baking and winemaking. Wine yeast and its properties have been known to humans for as long as civilizations have existed, and the earliest evidence of this yeast has been dated to Neolithic times (Mortimer, 2000).

Most wine yeast strains are diploid and have a low frequency of sporulation. Another important characteristic of wine yeasts, and those used in other industries, is their highly polymorphic chromosomes: their genetic constitution is affected by the frequent and extensive mutation they undergo. These effects include (i) aneuploidy, (ii) polyploidy, (iii) amplification and deletion of chromosomal region or single gene, and (iv) the presence of hybrid chromosomes. The chromosomal polymorphism obtained by applying the technique known as pulsed field gel electrophoresis (PFGE) has been used to characterize and to classify strains that belong to the same species.

In the wine industry, knowledge of the yeast species responsible for the alcoholic fermentation is important because these yeasts with their metabolism contribute significantly to the organoleptic characteristics of the finished wine (Fleet, 2008). The diverse range of yeasts associated with the vinification process can be classified in two groups. The first group is formed principally by the genera *Hanseniaspora*, *Torulaspora*, *Metschnikowia*, *Candida*, *Zygosaccharomyces*, etc. These yeasts initiate spontaneous alcoholic fermentation of the must, but they are soon replaced by the second group, formed by *Saccharomyces* yeasts, which are present during the subsequent phases of the fermentation until it is completed. Within the genus *Saccharomyces* the species most relevant for the fermentation process are *S. cerevisiae* and *S. bayanus* var. *uvarum*; this is because they have become of interest for their biotechnological properties. However, there is currently increasing interest in the non-*Saccharomyces* yeasts for the development of innovative new styles of wine (Viana et al.,

Application of Gel Electrophoresis Techniques to

of an electric field.

temperature (14 ºC).

species (Naumov et al., 2000, 2002).

Plugs (intact cells)

Suspension cells

obtain the karyotype.

blending stages.

the Study of Wine Yeast and to Improve Winemaking 5

manipulation of the genetic material to prevent possible rupture of the chromosomes. The cells are then embedded in blocks of agarose which are subsequently treated with a reducing agent and K proteinase to destabilize the wall and cytoplasmatic membranes, respectively (Figure 1), thus facilitating the release of the DNA when submitted to the action

This methodology for correctly obtaining the karyotype of *S. cerevisiae* is based on the procedure described by Carle & Olson (1985) and optimized by Rodríguez et al. (2010). It also depends on the concentration of the agarose gel (1%), buffer (0.5 x TBE), initial and final switch (60-120 seconds respectively), run time (24 hours), voltage (6 V/cm) and buffer

The analytical results given by this technique are the number and size of the yeast chromosomes, and it allows specific strains of *Saccharomyces* to be differentiated because their karyotypes show distinct bands running below the 500-kb marker. It also allows the differentiation between *S. cerevisiae* and *Saccharomyces bayanus* var. *uvarum* (*S. uvarum*)

> Plugs (broken cells)

Fig. 1. Methodology for characterizing yeast strains, using pulsed field gel electrophoresis to

In previous research the PFGE technique has been used to analyse the dynamics of the yeast population during the spontaneous fermentations of wine (Demuyter et al., 2004; Martínez et al., 2004; Naumov et al., 2002; Raspor et al., 2002; Rodríguez et al., 2010), and it has also been used to characterize other industrial yeasts including baker's and brewer's yeast (Codón et al., 1998). Another relevant application of PFGE has been to characterize the yeast population which is present in the *flor velum* that grows on the surface of fino-type sherry wines in the barrel, during their biological ageing process (Mesa et al., 1999, 2000). The results revealed an interesting correlation between the yeast genotypes and the different

One disadvantage of the technique is that it is laborious, expensive and requires specialized personnel; increasingly, therefore, analysts are resorting to other simpler and faster techniques to discriminate between yeast clones, like, for example, interdelta analysis of sequences or microsatellite analysis (Cordero-Bueso et al., 2011; Le Jeune et al., 2007; Schuller et al., 2007). However, the methodology proposed in Figure 1 enables a large

PFGE (CHEF system)

+ + + +


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kb

Reduction solution (β-mercaptoethanol) 24 h

Lysis solution (proteinasa k and N-lauroylsarcosine) 24 h

Washing

2009). In the industry, knowledge of specific strains of these microorganism species is important for (i) their selection; (ii) their use as starter cultures; and (iii) improving the fermentation process.

During the 1990's the development of molecular techniques has enabled the identification and characterization of different strains belonging to the same species of yeast, and it has been possible to establish the ecology of spontaneous fermentations in many of the world's winemaking regions (Fleet, 2008). These techniques also constitute a powerful tool not only for the selection of the most suitable yeast, since they tell us which yeasts are the most representative in the fermentation process, but also for obtaining information on the addition to the must of particular strains of yeast in the case of inoculated fermentations (Rodríguez et al., 2010).

Two of the approaches most often used for the molecular characterization of industrial yeast are analysis of the electrophoretic karyotypes by pulsed-field gel electrophoresis (PFGE) and analysis of the restriction fragment length polymorphism of the mitochondrial DNA (mtDNA-RFLP). We have used PFGE in winemaking to analyse the diversity of wild yeasts in spontaneous fermentation of a white wine produced in a winery in SW Spain with the object of selecting the most suitable autochthonous starter yeast; and from the results of the inoculation, we were able to make decisions for improving the efficiency of the process and to establish procedures for the proper performance of the inoculation (Rodríguez et al., 2010). We have also applied the analysis of the karyotypes to characterize natural yeasts in biodynamic red wines in another region of Spain. In this chapter we also evaluate the use of the mtDNA-RFLP technique for quick monitoring of the dominance of inoculated strains in industrial fermentation, without any need for the prior isolation of yeast colonies (Rodríguez et al., 2011).

Another electrophoretic technique has been used to show substantial changes in protein levels in selected wine yeasts under specific growth conditions. It has recently been stated that the proteome is "the relevant level of analysis to understand the adaptations of wine yeasts for fermentation" (Rossignol et al., 2009). Following this, in-depth studies are now being made of the proteome of wine yeast strains and the relationship between the proteome and wine quality and winery processes. We are now exploring more generally the relevance of proteomics to wine improvement. In this chapter, we will summarize the efforts being made by the proteomics research community to obtain the knowledge needed on proteins in the post-genomics era
