La biodiversità come risorsa: studio della variabilità intraspecifica nella specie Oenococcus oeni, per la valorizzazione di tratti fenotipici peculiari

The strains belonging to the species Oenococcus oeni are the main microbial agents responsible for malolactic fermentation (MLF), as they are perfectly adapted to survive in a harsh environment such as wine. Oenococcus oeni is indeed able to grow in starving conditions, at low pH values, and it tolerates very high concentrations of ethanol. Depending on the strain, O. oeni gives to wine also typical aromatic notes. Thanks to these characteristics, strains of O. oeni are the most suitable microorganisms to be used as MLF starter. This species is very interesting not only for economic and technological reasons, but also for basic studies. Oenococcus oeni has indeed been suggested by some authors as a model species both to study the behaviour of lactic acid bacteria in acidic environments, and to analyse the evolutionary mechanisms of microbial genome. In fact, O. oeni is prone to accumulate mutations as it lacks two components of the MMR (MisMatch Repairing) system, necessary to correct DNA replication errors. This leads O. oeni to increase variability by accumulating both point mutations in the genome, and the acquisition of exogenous DNA by horizontal gene transfer. Nonetheless, the analysis of the microbial biodiversity has often focused on few topics with the purpose to ensure certain outcomes for starter producing companies and winemakers. Therefore the framework of metabolic and physiological potential of the O. oeni species is not yet complete. The present research was preceded by numerous ecological studies to analyse the diffusion and the biodiversity of O. oeni strains in Verona wines, especially the Amarone wine. During those researches several strains with different features has been isolated: some strains showed interesting technological properties, while many of them were unsuitable in winemaking trials. Considering the variability showed by the species O. oeni and the different aptitudes of the strains present in the collection of the Department of Biotechnology to carry out MLF in winemaking processes, in this PhD thesis the attention was focused on a small number of strains with unique characteristics. The final aims were to deepen the knowledge about the strains; to develop procedures for their effective differentiation and to exploit the distinctive features of some strains re-evaluating their potential in view of possible applications in winemaking processes. The study was divided into two parts. In the first part, a model collection of ten O. oeni strains, representing the variability observed in previous research was phenotypically and genotypically characterized. Then a correlation between the response to high concentrations of ethanol and the isolation source was demonstrated, through the analysis of membrane fluidity changes in response to different concentrations of alcohol. Finally, the possibility to differentiate strains by the expression patterns of ubiquitous marker genes involved in stress response was evaluated. Moreover, the correlation between the presence in the genome of specific loci associated with an increased resistance and the real properties of the strains was assessed. In the second part, the research focused on the characterization of the strain Br10, which develops in aggregates (pellicles or biofilms), a property never described before for the O. oeni species, and also characterized by a good resistance to various environmental stresses. More in detail, the presence of polysaccharides in the extracellular matrix was determined by colorimetric and enzymatic assays. Moreover, the influence of the culture medium on the capacity to form the aggregates and to carry out MLF in wine was analysed. The possibility to exploit the phenotypic peculiarity showed by the strain Br10 was successfully evaluated, first inoculating the pellicles in difficult wine (after appropriate preadaptation), then performing co-inoculation of yeasts and aggregative bacteria in grape juice. In conclusion, biodiversity represents an important resource, not only from a purely scientific point of view, but also for technological aspects. The constant isolation of new strains and the analysis of their phenotypic and genotypic characteristics are, indeed, the prerequisites to innovate the fermentation processes and to improve the quality of the final products.