Quantificació, Identificació i Tipificació de llevats mitjançant lús de diferents tècniques moleculars

• Autores: Núria Hierro Crivillé
• Directores de la Tesis: Albert Mas Barón (dir. tes.), José Manuel Guillamón Navarro (dir. tes.)
• Lectura: En la Universitat Rovira i Virgili ( España ) en 2007
• Idioma: catalán
• Tribunal Calificador de la Tesis: Amparo Querol Simón (presid.), MªJesús Torija (secret.), Antonio J. Palacios García (voc.), Josep Cano (voc.), Sergi Ferrer (voc.)
• Materias:
  • Ciencias tecnológicas
    • Tecnología bioquímica
      • Microbiología industrial
• Enlaces
  • Tesis en acceso abierto en: TDX
• Resumen

  • Alcoholic fermentation is a complex microbial process that is characterized by a succession of different species of yeasts. Yeasts with low fermentation activity, such as Candida spp., Hanseniaspora spp., Pichia spp., Rhodotorula spp. and Kluyveromyces spp., are predominant in grape musts and during the early stages of fermentation. Subsequently, Saccharomyces cerevisiae proliferates, dominates and completes the wine fermentation. Non-Saccharomyces spp. can contribute to the aroma properties and chemical composition of the resulting wine. However, they can be both beneficial and harmful. For a good control of the wine making process, cellars need fast, simple and reliable methods for identifying and typing yeasts as well as for detecting and enumerating yeasts.The objectives of this thesis were: (i) to develop molecular techniques for characterization, identification and quantification of wine yeasts, (ii) to evaluate the usefulness of these techniques to identify and quantify yeasts isolated from natural samples of wine fermentation. For this purpose, we used the oligonucleotides based on the repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) elements described in bacteria to characterize reference yeast strains. We also evaluated the usefulness of the complementary primers to ISS (intron splice site) for the same objective. We also developed real-time, or quantitative, PCR (QPCR) to rapidly detect and quantify the total number of yeasts, Saccharomyces spp. and Hanseniaspora spp. in wine without culturing and we also performed a reverse transcriptase QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. We later validated all these techniques with natural samples of wine.We characterized 41 reference strains belonging to 15 species by three PCR methods: PCR-ISS, ERIC-PCR and REP-PCR. We demonstrated the reproducibility of the techniques, but some problems of reproducibility were sometimes detected with the REP-PCR. The REP-PCR amplification patterns from strains of the same species were identical and some polymorphism was detected by PCR-ISS and ERIC-PCR in strains of the same species. In spite of this intraspecific variability, the strains of the same species showed enough species-specific bands to enable yeast identification. Therefore, all three techniques are useful for rapid, simple and relatively cheap yeast identification. Afterwards, we proved the usefulness of these PCR techniques to monitor yeast diversity during industrial wine fermentation. We evaluated how the growth dynamics of non-Saccharomyces yeasts is affected by new winemaking practice of cold maceration, and by grape maturity. Half of the isolates were ascribed to Candida stellata, Hanseniaspora uvarum and H. osmophila. The other isolates we identified were identified as minor species. As general conclusions we could indicate that the higher the degree of ripeness, the higher the proportion of C. stellata in the grape must. Also, it seemed that cold maceration favored the presence of H. osmophila, C. tropicalis and Z. bisporus. We also developed a QPCR assay for detecting and quantifying the total yeast population, Saccharomyces spp. and Hanseniaspora spp. in a sample of wine. The QPCR assays were first conducted on reference yeast strains from pure cultures to detect the specificity of the primers. We later validated this technique with artificially contaminated and natural samples of wine and compared the results with those obtained from plating to determine effectiveness of QPCR. We obtained a very good correlation between the predicted number of cells determined by QPCR and by plating. The detection of RNA by reverse transcriptase PCR (RT-PCR) is considered to be a better indicator of cell viability than the detection of DNA. We compared total yeast quantification by plating and by QPCR using DNA as template with Saccharomyces cerevisiae quantification using RNA. The counts obtained by the three methods were similar.