Genetic biodiversity of enological Saccharomyces isolated from grapes must natural fermentations.

Spontaneous grapes must fermentation, induced by the indigenous micro flora, is believed to be associated with a specific vineyard and to give a distinctive style and quality to that wine. The alcohol-tolerant Saccharomyces cerevisiae strains invariably dominate the latter stage of natural wine fermentation. The S. cerevisiae population and other specific yeasts present in the vineyard niche habitats are considered autochthonous and their involvement in natural fermentation allows the production of wines with particular features in each microclimatic area. The present study was aimed to the individuation of autochthonous yeast strains useful in the improvement of oenological production of Salento, which is a very important wine-producing area of Southern Italy. Grapes were sampled from the most representative areas of Salento region (Ugento e Guagnano for "Negroamaro" and Gioia del Colle for "Primitivo") and separately subjected to natural fermentation in an experimental scale. The identification of micro biota present during the last step of wine fermentation (>1 °Bé) of grapes, was carried out to select autochthonous yeast strains for industrial wine production. Aliquots of must samples of several dilutions were isolated after spreading on YPD agar medium supplemented. As preliminary screening, averages of 1500 colonies for each of the three fermentations were assayed for H2S production on BIGGY agar. The colonies appearing white or light brown (about the 10% of initial 1500 individual clones) were selected and recognized as S. cerevisiae by a microbiological screening based on the implementation of the taxonomical keys for identification of yeasts belonging to Saccharomyces genus. Identification at strain level of S. cerevisiae isolates is a fundamental step to investigate the biodiversity of this yeast and to examine population dynamics during the fermentative process. The criterion for strain differentiation was provided by the amplification of genomic sequence blocks flanked by delta elements of retrotrasposon origin. Interdelta (ID) typing has proved to be a very convenient method that can advantageously replace the other methods for molecular characterization of S. cerevisiae strains. For the first time, a recently developed method has been applied for the ID by the use of fluorescent primers and automatic sequencers. When compared with gel-based analytical methods, the use of capillarity system and automated analysis increase data throughput, scoring and reliability, decreasing the overall experimental error. The results of the genetic characterization of S. cerevisiae strains associated to natural fermentations of must from "Primitivo" and "Negroamaro" grapes and their implications for the selection of autochthonous industrial starters will be discussed.