SPARKLING WINE PRODUCTION BY CHAMPENOISE METHOD IN LOMBARDY REGION: YEAST POPULATION BIODIVERSITY AND TECHNOLOGICAL ASPECTS OF INDIGENOUS SACCHAROMYCES CEREVISIAE AS POTENTIAL STARTERS

Sparkling wine obtained through the Champenoise method represents a relevant cultural and outstanding economical fact in Italy. There are two Lombardy sparkling wines belonging to DOCG: (Denominazione di Origine Controllata e Garantita) Franciacorta DOCG and Oltrepò Pavese Metodo Classico which high quality production is remarkable. Commercial starters belonging to Saccharomyces species currently used in Italy for sparkling wine production have been isolated from French territories on the basis of the quality characteristics of Champagne wine since they possess very good fermentative and oenological capabilities. Winemaking is a process on which interactions between Saccharomyces and non-Saccharomyces yeasts take place, influencing wine quality at both levels, sensory and chemical; so assessment of yeast biodiversity is relevant for understanding their evolution in winemaking and consequently increasing the control capability on such processes. Despite the Lombardy region is a very important region for sparkling wines production, a biodiversity analysis of yeast involved in the winemaking process in this region, has still not been carried out. This PhD was focused on the study of yeasts population biodiversity involved in winemaking process in Franciacorta and Oltrepò pavese areas as well as the evaluation of technological aspects of indigenous S. cerevisiae to be used as potential new starters in the sparkling wine production made by Champenois method. In particular, the main research activities and corresponding results were: Genetic identification of yeast population involved in winemaking process in Franciacorta and Oltrepò Pavese areas Samples of vineyard air, must before SO2 addition and base wine were collected during 2009, 2010 and 2011 vintages in Franciacorta and Oltrepò pavese areas. For genetic identification of yeast isolates, genomic DNA was extracted according to Querol et al.(1992). Analysis of RFLP-ITS (Fernandez-Espinar et al., 2000) and D1/D2 of 26S rDNA sequence (Kurtzman and Robnett, 1998) were performed. For the identification at strain level, interdelta analysis (δ-PCR) (Legras and Karst, 2003) was carried out for isolates identified as S. cerevisiae during the 2009 and 2010 vintages. The amplified fragments from δ-PCR were run in capillary electrophoresis (Tristezza et al., 2009). A data elaboration was performed calculating the genetic similarity according to Dice´s coefficient (Dice, 1945). Dendrograms were constructed by the unweighted pair group method using arithmetic averages (UPGMA). During this study it was possible to create a collection of 492 yeast isolates from 24 vineyards belonging to 19 wineries among Oltrepò Pavese and Franciacorta areas. A total of 13 genus and 25 yeast species were isolated and identified during vintages for 3 years. 186 S. cerevisiae isolates were obtained during 2009 and 2010 vintages and from fingerprinting, a great genetic biodiversity was obtained among the analyzed patterns which presented a genetic similarity in a range of 0.2% to 95% by which the 98% of the isolates were S. cerevisiae strain-specific. In conclusion, through yeast biodiversity in Franciacorta and Oltrepò Pavese areas, it was possible to observe that there is a great opportunity to select autochthonous S. cerevisiae strains with typical oenological characters which could be representative of these oenological areas. Yeast population evolution during controlled and spontaneous fermentations A monitoring of yeast populations evolution during controlled and spontaneous alcoholic fermentations was carried out in three Franciacorta wineries during 2009 vintages. Genetic identification of the isolates was performed as described above except for capillary electrophoresis. The amplified fragments from δ-PCR were directly elaborated through software Gel compare II (Bionumerics Applied Maths, Belgium) and Dendrograms were constructed by the unweighted pair group method using arithmetic averages (UPGMA). A total of 323 isolates were collected from both controlled and spontaneous fermentation of which ninety-eight percent (98%) were presumptively classified as S. cerevisiae according to the results of RFLP-ITS of rDNA analysis. Through δ-PCR a total of 319 genetic profiles were obtained and analyzed. From the elaboration of the results, 164 different strains among controlled and spontaneous alcoholic fermentation was distinguished (corresponding to 51% of the population investigated). The highest S. cerevisiae biodiversity was found in a winery from controlled fermentation with 53 different genetic patterns corresponding to 82% of the yeast population. In conclusion, this study represented a first approach to population dynamics of oenological yeasts in an important viticulture region as Franciacorta. The obtained results have an important significance for the local industry, especially since the controlled fermentations are not always lead by commercial starter strains only, but even by a high percentage of indigenous strains that took place in the process. Selection and evaluation of indigenous Saccharomyces cerevisiae strains for Sparkling wine production From the two previous phases, sixteen indigenous S. cerevisiae strains were selected and their technological and qualitative characterizations were performed. By the microfermentations in laboratory, ethanol, glycerol and acetic acid production were tested by enzymatic protocols. Sniffing tests of wines were realized with a tasters panel and statistical analysis of data was carried out through ANOVA (Statgraphics Centurion Plus 5.1). On the basis of microfermentation and sniffing results, two strains of each Oltrepò Pavese and Franciacorta area were chosen to be used as indigenous starters in tirage proves at pilot scale in different Franciacorta and Oltrepò pavese wineries. The test was monitored at different time intervals thorough viable counts determination, cellular vitality evaluation and yeast genetic identification in order to determine the indigenous strain dominance and its permanence during fermentation. In addition, samples of wine were subjected to tasting six months after tirage proves to evaluate the intensity and pleasantness. Statistical analysis of data was carried out through ANOVA (Statgraphics Centurion Plus 5.1). In conclusion, four indigenous starters representative of each area demonstrated a high potential to implant and carry out the fermentation successfully. In addition, this potential was supported through tasting-wine proves on which wines produced by these indigenous starters were sensorially accepted at 6 months of aging. This result represents an opportunity of developing indigenous starters formulation to improve the Franciacorta DOCG and Oltrepò Pavese Metodo Classico DOCG production. From these results, future prospects are promising. Development of a protocol for recovery of yeast DNA from sparkling wines Simultaneously to the previous research, a study to set up a protocol for extraction and amplification of yeast DNA from sparkling wine was carried out, aimed to a development of a molecular test that allows the verification of the sparkling wine authenticity.. Sparkling wine bottles from both Oltrepò Pavese and Franciacorta territories obtained during tirage test in wineries were used. Wine samples were filtered at different time intervals and four different DNA extraction protocols were tested. In conclusion, through this study a protocol based on the combination of multiple methods Magnetic Beads+Organic Solvents+Real-time PCR was developed. This molecular approach could be used to improve the traceability for the DOCG sparkling wines in Lombardy, allowing the use of S. cerevisiae as “trace element” during the process of sparkling wine authentication and as a helpful criterion to investigate DOCG status. Future developments of the protocol will be focused on applying the same DNA extraction protocol to commercial bottles ready for the consumer market.