MOLECULAR CHARACTERIZATION, STRESS RESPONSES AND SPECIFIC ENZYMATIC ACTIVITIES OF DEKKERA/BRETTANOMYCES BRUXELLENSIS WINE STRAINS: STRATEGIES OF ANALYSIS AND CONTROL IN THE OENOLOGICAL FIELD.
Tesi di Dottorato
Data di Pubblicazione:
2012
Citazione:
MOLECULAR CHARACTERIZATION, STRESS RESPONSES AND SPECIFIC ENZYMATIC ACTIVITIES OF DEKKERA/BRETTANOMYCES BRUXELLENSIS WINE STRAINS: STRATEGIES OF ANALYSIS AND CONTROL IN THE OENOLOGICAL FIELD / I. Vigentini ; tutor: R.C. Foschino ; co-tutor: C. Picozzi ; coordinatore: M. G. Fortina. Universita' degli Studi di Milano, 2012 Jan 27. 24. ciclo, Anno Accademico 2011. [10.13130/vigentini-ileana_phd2012-01-27].
Abstract:
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. brux
Tipologia IRIS:
Tesi di dottorato
Keywords:
Dekkera/Brettanomyces bruxellensis ; wine spoilage ; molecular typing ; off-flavour production ; metabolomic analysis ; SO2 resistance
Elenco autori:
I. Vigentini
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