MOLECULAR CHARACTERIZATION, STRESS RESPONSES AND SPECIFIC ENZYMATIC ACTIVITIES OF DEKKERA/BRETTANOMYCES BRUXELLENSIS WINE STRAINS: STRATEGIES OF ANALYSIS AND CONTROL IN THE OENOLOGICAL FIELD.

Among yeasts responsible for wine spoilage, Dekkera/Brettanomyces bruxellensis is the species on which the scientific community has the highest interest. This fact is documented by an increasing in the international publications and by the beginning of the genome sequencing. Recently, it has been traced the evolutional D. bruxellensis lineage by the analysis of promoter sequences, which separated from the Saccharomyces yeasts more than 200 mya. Spoilage caused by D./B. bruxellensis is mainly due to the following issues:  this species remains viable and active in beverages preserved by extreme abiotic stress (anaerobiosis, up to 12-13% ethanol (v/v), minimal amounts of fermentable sugars);  the adopted treatments (sulphiting, membrane filtration, transfer of wine to sanitized barrels) are not always effective.  the off-flavours produced by Brettanomyces include volatile phenols characterised by disagreeable odours. The aim of this thesis was to develop strategies in order to analyse and control the wine spoilage linked to D./B. bruxellensis species. As concern the first issue, spoilage microbial analysis, the present PhD thesis describes: i) the development of new methods for D./B. bruxellensis molecular typing; ii) the phenotypic biodiversity of D./B. bruxellensis species. The topics on the microbial spoilage control were: iii) D./B. bruxellensis response to stress conditions. i) Studies on the natural distribution of D./B. bruxellensis have shown an existing high intraspecies polymorphism degree which is probably due to a fusion event among genomes or to the lacking of a sexual state. Moreover, since D./B. bruxellensis has been mainly associated to fermented beverages that represent mutagenic environments determining the frequent genome rearrangement of D./B. bruxellensis. Genetic variations are usually accumulated with a higher frequency in DNA regions that are not linked to any gene function respect to the coding regions such as introns. Thus, they are considered good indicators in evolutional studies; in S. cerevisiae, the lariat branch point TACTAAC and the 5’ splice site GTATGT (5’ss) are conserved sequences that were used to build primers for the Intron Splice Site amplification analysis (ISS-PCR) described for inter- and intraspecific characterisation of S. cerevisiae. The main goals of this first topic was to develop new methods for D./B. bruxellensis molecular typing. The setting up of a multiplex PCR protocol throughout the use of modified oligonucleotides that targeted 5’ss -GTAAGT- has confirmed a high polymorphism among D. bruxellensis genomes. Thus, a further optimisation of the primers, a simple capillary electrophoresis protocol that can accurately separates the amplified fragments and clear rules for the ISS profiles elaboration were applied. The results points out that the genetic signatures obtained exploiting the ISS as molecular targets are able to show genetic differences that, up to now, only other laborious technique can put in evidence (Karyotyping, PFGE-RFLP, AFLP). The proposed protocol has proved to be reliable and robust. Moreover, considering that a positive correlation between the extent of non-protein-coding DNA and the eukaryotic complexity degree has been observed, the ISS fingerprinting can represents a useful tool to analyse the evolution rate of a yeast species. ii) D./B. bruxellensis yeasts have evolved numerous developmental options in order to adapt and survive the changing status of the environment. Independent studies showed that distinct genetic groups of D./B. bruxellensis can have different physiological characteristics and strong differences in their ability to produce 4-ethylphenols. The main goal of second topic was to characterise D./B. bruxellensis from a phenotipic point of view. In particular, the physiological diversity within D./B. bruxellensis strains was investigated studying the growth and the production of volatile phenols and biogenic amines under a wine model condition. Moreover, the carbon compounds assimilation, H2S production, and vinylphenol reductase (VPR) activity were also analysed. The potential hazard of spoilage when D./B. bruxellensis grows in oenological conditions was confirmed since most of the analysed strains were able to produce volatile phenols or showed a detectable VPR specific activity. Actually metabolic traits, as growth rate and off-flavour production, proved to be related and strain-dependent suggesting that an early detection and identification of “fast-growing yeasts” and “fast volatile phenols producers” could be essential to introduce adequate corrective measures. The experiments on carbon assimilation revealed that about 30% of the analysed yeasts has a own pattern in the utilization of carbonious sources. Three different ranges of VPR activity and H2S production were used to assign the physiological profiles. Combining the results from the three phenotypic experiments, about 70% of the CBS collection can be discriminated at strain level. The present work suggested that the chromosomal rearrangement occurred in this species has generated strains with a high physiological polymorphic state. iii) A goal of the wine industry is to reduce the risk of wine being spoiled by microbial activity. The main aim of the third topic of this research was to study the response to stress conditions in D./B. bruxellensis due to the yeast exposition to an electric current treatment and exogenous SO2. Results indicated that a similar effect occurred on cells after the current treatment in comparison to the SO2 exposition; both treatments resulted in a reduced microbial cell survival in the studied red wine. The kinetics of volatile phenol accumulation confirmed that, the use of an electric field could be adequate to hinder the yeast spoilage. As concern the latter issue, the SO2 resistance, a metabolomic study on the effect of the SO2 addiction to D./B. bruxellensis cultures was carried out too. Results displayed that among the metabolic pathways resulted to be affected by exogenous sulphite concentration, aminoacid metabolisms seem to be involved in the SO2 tolerance. Unlike what was observed in S. cerevisiae, neither adenine nor methionine modified the toxicity level of SO2 under laboratory conditions. The ethanol concentration seems to increase the sensitivity to sulphite suggesting that a membrane system, such as the sulphite efflux pump of S. cerevisiae, could be present in this species. In conclusion, the main research products of this PhD thesis were:  a new PCR protocol to typing D./B. bruxellensis that uses specific primers for this yeast species, and a precise and reliable fragment separation protocol by capillary electrophoresis. Actually, this method shows a high reproducibility (94%), it is rapid in comparison to other techniques that in the past allowed a discrimination at strain level of D./B. bruxellensis isolates (Karyotyping, RFLP-PFGE, AFLP, etc.), and it represents a useful tool to monitor the yeast evolution rate;  the collection of D./B. bruxellensis phenotypic features that never have been used to evaluate the biodiversity degree in this species, such as the VPR specific activity, H2S production, and the assimilation of carbon compounds different from ones found in wine. The compilation of a database collecting both genetic and phenotypic traits of different D./B. bruxellensis strains is the future perspective to offer an efficient way to counteract this spoilage yeast;  a new technology to reduce the survival of D. bruxellensis in wine using a low electric current (LEC) treatment.  the understanding of some metabolic mechanisms involved in the SO2 response in D./B. bruxellensis. This step will allow the following upgrade toward the study of molecular mechanisms, and metabolic pathways that this yeast can activate to protect itself against the exposure to high concentration of exogenous sulphur dioxide.