"Selection and characterization of microorganisms for the production of distillates" 

Grappa is an alcoholic beverage obtained ​​by distilling grape marcs from grapes grown exclusively in Italy. The production process begins in the cellar, where the wine making takes place: after the harvesting and pressing of grapes, the must is separated from the grape marcs, made up mainly of skins and seeds, with or without stalks. Grape marcs obtained from white wine-making contain sugars not yet fermented. For this reason the vegetable material brought to the distillery is stored for a certain period of time, to allow alcoholic fermentation to take place. Grape marcs can be stored from a few days to a few weeks in different ways, in silos or in plastic tunnels. During this period, the main chemical process performed by yeasts is sugar degradation and production of alcohol, although a large number of other molecules with positive (aromatic substances) and negative (toxic compounds) effects, can originate from the indigenous microflora. In order to avoid the deterioration of the vegetable material due to inappropriate storage conditions, modern distilleries have introduced some technological treatments (pH improvement, temperature control, absence of oxygen); the use of selected yeasts to control microbial populations has also received some interest recently. In this regard we must say that international studies in the field of Grappa are almost non-existent, since its production is limited by law to the national territory. This study is part of a larger project, financed by the Veneto region, on the selection of yeasts to be used as fermentation starters, inoculated into grape marcs destined to Grappa production. The first objective on schedule was to study the yeasts microflora composition and population dynamics, using conventional and molecular methods found in the literature or developed by the Microbiology Group in the Department of Agricultural Biotechnology. Two Grappa varieties were chosen, Moscato and Prosecco, from two distinct areas of the Veneto region. The yeast population counts were carried out at the beginning and after 4 and 15 days of storage in anaerobic conditions. About seventy isolates for each sample were identified to species level by RAPD -PCR analysis followed by sequencing of the ITS region. At the start of storage a good biodiversity was observed in both grape marc groups, with a high percentage of Hanseniaspora opuntiae, but the absence of Saccharomyces strains, which become however dominant after only 4 days of fermentation, settling at a certain level until the end of fermentation. The effects of the refrigeration and acidification treatments on yeast population dynamics were subsequently examined during storage.  A total of 200 isolates were collected from acidified and non-acidified Prosecco grape marcs, after 4 and 15 days of storage. The identification of oenological strains belonging to the Saccharomyces sensu stricto group was performed by multiplex PCR, and their characterization at strain level was obtained by analysis of mitochondrial DNA. Genetic analyses highlighted the development of different strains in the acidified group and the non-acidified control group. The acidification treatment, applied in order to inhibit bacterial microflora, proved to be capable of influencing the yeast population from a qualitative rather than quantitative point of view. The decrease in pH produced a big selection effect, as only 18 of the 121 identified strains were found in both conditions. A total of 104 isolates, identified as belonging to the Saccharomyces cerevisiae group, were chosen from a pool of about 1000 yeasts, collected during various experiments from grape marcs of different origin or subjected to technological treatments during the ensilage period. The isolates were grown on synthetic must to assess their fermentation performance. The yeasts isolated from Moscato grape marcs showed the best characteristics in terms of fermentation vigor, expressed as sugar consumption after 2 and 7 days of fermentation. The foam production level was also investigated, and a sensory evaluation was carried out at the end of fermentation to identify possible unpleasant smells. Other technological characteristics were examined, such as resistance to the antioxidant and antiseptic agent sulphur dioxide and the production of hydrogen sulphide. As for resistance to SO₂, all isolates can tolerate a concentration of 50 mg/l, commonly used in cellars. At the intermediate dose of 100 mg/l, the most resistant strains are from non acidified Prosecco grape marcs.

It is also known that microbial glycosidase activity contributes to the aroma of wines and spirits through the hydrolysis of non volatile and odourless glycoconjugate precursors, which release terpenols and other aromatic compounds. Glycosides made up of a terpene hydrocarbon (aglycone) and a carbohydrate are concentrated in the skins, and the contribution of glycosidase activity is crucial for the expression of Grappa's aroma. Glycosidase activity was therefore quickly tested in all isolates with an especially developed fluorimetric test and by growing the cells in a minimal medium containing the disaccharide cellobiose as the sole carbon source. Statistical analysis was applied to the technological and quality characteristics monitored in all isolates in order to identify the yeasts best suited to be used as starters in distillates production. Recently it was suggested that glucosidase activity is related to the higher exo-1,3-glucanase EXG1 activity of Saccharomyces cerevisiae, which is involved in processes such as growth, budding and mating. Therefore the chosen approach was based on the development of mutants for the exoglucanase genes. In particular, two deletion mutants were constructed for the EXG1 and EXG2 genes, following a protocol based chiefly on homologous recombination. The effect of deletion on glucosidase activity was tested with a fluorimetric assay, showing a slight decrease in the null EXG1 mutant. In addition, the mutated strains were grown in natural Prosecco must placed in 1L fermenters, but fermentation curves revealed no significant differences between the strains. We can therefore conclude that the deletion of exoglucanase genes  probably does not influence the growth of yeasts. The overall results indicate that grape marcs of different origin or subjected to technological treatments provide a large source of yeasts that can be selected as starters in the production of Grappa.