Detección y caracterización de Brettanomyces bruxellensis y Trigonopsis cantarellii en el contexto enológico stars

Abstract

Brettanomyces/Dekkera is currently the leading cause of microbiological alterations in wines, especially those premium red wines that undergo aging processes in wooden barrels. This yeast is responsible for huge economic losses in the winemaking industry in all producing countries of the world. The first objective of this thesis was to develop a rapid method of quantitative PCR (qPCR) for detection and quantification of Brettanomyces/Dekkera cells in red wine samples taken at different stages of production and conservation, as well as in samples of interest to the winemaker. The first milestone achieved in this work was to eliminate the serious interferences involving macromolecular aggregates of solid particles and polyphenolic compounds present in non-finished wines that are responsible for inhibition of DNA polymerase reactions. Once optimization of the qPCR method was achieved, a comparative study was carried out with the method of classical microbiological culture in selective medium, using a total of 324 Tempranillo red wines from both Rioja and Ribera del Duero appellations, and collected during the period 2009 - 2011. Statistical comparisons showed a good correlation between the results of both methods, emphasizing the greater specificity of qPCR, for which no false-positive results were found and that the detection and quantification limits were between 16 - 25 Brettanomyces cells/mL in those red wines with turbidity and in the presence of up to 7.5 105 CFU/mL of other yeast populations. The method of microbiological culture in selective medium allowed detection and quantification of down to 1 Brettanomyces/Dekkera CFU/mL in wines, and also in cellar air. The next goal was to study the sensitivity of Brettanomyces/Dekkera to different antimicrobial agents used in winemaking and to determine the most effective method against this spoiling yeast. The antimicrobial agents studied were: potassium metabisulphite, enological tannins solution, chitosan and dimethyl dicarbonate. Within the respective ranges of concentrations permitted in enology, the agent that showed the highest specificity against Brettanomyces/Dekkera was chitosan, and the agent that showed the greatest efficacy was potassium metabisulphite, showing growth inhibitory activity and biocide effect against all the strains tested (MIC90 and MBC90 = 48 mg/L, in the presence of 12.5 % ethanol in the medium). This agent was used subsequently to study Brettanomyces/Dekkera naturally contaminated red wines stored in bottles and kept under cellar conditions. A direct correlation was demonstrated between the Brettanomyces/Dekkera populations (CFU/mL) and the increase of concentrations of volatile phenols (4-ethylguaiacol, 4- ethylphenol, 4-vinylphenol and 4-propylguaiacol) in wines, being completely excluded the possibility of lactic acid bacteria or other microorganisms responsible for the production of these compounds. A concentration of 100 mg/L MBP was fully effective and prevented both the growth of Brettanomyces/Dekkera, and the consequent increase of volatile phenols in the studied wines. In this thesis the following species of Saccharomycetales yeasts were also identified in samples of wines undergoing aging in wooden barrels collected in the period 2008 - 2011 and suspected of being contaminated under sensory analysis once they presented some evidence of aromatic deviation: Pichia mandshurica, Pichia holstii, Arthroascus schoenii, Trigonopsis variabilis, and Trigonopsis cantarelli. The most recurrent species was T. cantarellii, and represented 54.5 % of all studied isolates. This yeast species was characterized and proved its ability to grow in dry red wines (< 0.2 g/L reducing sugars) with a high ethanol content (up 13.8 %) and greater resistance to potassium metabisulphite than Brettanomyces/Dekkera. T. cantarellii yeasts showed very slow growth, and the ability to generate potentially spoiling acids was demonstrated in 48 % of the studied strains, and likewise, the potential to generate some deviation aromas was found on 72 % of strains, although with a lower potential than those usually found for Brettanomyces/Dekkera.