Molecular Approaches for the Individuation and Characterization of Technological and Quality Traits in Microorganisms of enological interest

The genus Saccharomyces includes microorganisms important for many technological applications, particularly in the industry of fermented foods. In the enological field, yeast strains currently used in wine fermentations (belonging to Saccharomyces sensu stricto group) were selected from natural spontaneous fermentations and are specifically adapted to the winery’s environment. Some of their essential properties that are at the basis of strain selection are the capability of these yeasts to transform carbohydrates into alcohol by fermentation, with high transformation efficiency and remarkable tolerance to high alcohol levels. Yeasts belonging to the S. sensu stricto group are also adapted to grow in grape musts with high sugar content, low pH, limited nitrogen, lipids and vitamins concentrations and presence of added sulphites. During the selection programs of wine yeasts, the first stages of the experimental work regard the collection of several hundred isolates that are screened with the aim to find those that possess important enological properties. In most of the cases, this equivalent to establish that these strains belong to the Saccharomyces sensu stricto complex. For that reason, the availability of a molecular test that could rapidly, ditely and easily identifies these enological yeast would be extremely useful. In the first part of this work the analysis of yeast ITS region on rDNA was proposed as screening method. Using a tailor-made profile database, more then 350 natural isolates collected from marks of Prosecco and Moscato grape were subjected to molecular identification. As the protocol needs several time consuming experimental steps (amplification and digestion) a new method was proposed. For this purpose an original pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, was constructed. These generate an amplification fragment specific for the Saccharomyces sensu stricto species, while no signal was obtained from Saccharomyces sensu lato strains or from another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a common band for all strains. This was used as a positive experimental control in multiplex PCR analysis. The new method, and also ITS analysis, allow a “genotypic characterization” of enological strains that is required to start a “technological characterization” for the definition of the enological traits. The innovative DNA-microarray technology that has become a standard tool for the analysis of genome-wide expression profiles, can be used to investigate, from a molecular point of view, the differences in the expression of technological and quality characters of enological yeast in laboratory and also winery conditions. For this purpose in the second part of the work, using microarray technology, an investigation of yeast metabolic shifts at transcriptional level in both laboratory and industrial conditions was faced up. Two commercial yeast strains widely used in wineries, Lallemand 71B and Lallemand EC1118, have been compared during fermentation of both 1 l and 100 l natural white must. These strains have different behaviours and attitudes: 71B is known to be a strong producer of fermentative aromas, EC1118 is an efficient fermenter, quite neutral from an aromatic point of view. Comparing the two strains, the metabolic pathway of sulphured amino acids production displayed an higher expression level in 71B, together with the sulphite efflux responsible gene SSU1. Moreover, genes involved in the production of fermentative aromas, such as esters and higher alcohols, showed a slightly higher expression in 71B: all these evidences have been confirmed by Real-time PCR, another high throughput tool for expression analysis. The up-regulated genes during the scale-up experiment, on the other hand, seem to be linked to anaerobiosis stress response, probably due to small differences in fermentation conditions which have been sensed by yeast. As final remark, this study tries to give a contribution for understanding the genetic basis of the differences that are found in fermentation performances of wine yeasts in winery conditions. Furthermore it may help in assessing the eproducibility of yeast behaviours during alcoholic fermentation, when a laboratory scale is used.