Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches.

The process of cocoa fermentation is a very important step for the generation or aromatic compounds, which are attributable to the metabolism of the microorganisms involved. There are some reports about this process and the identification of microorganisms; however, there are no reports identifying the yeasts involved in a Mexican cocoa fermentation process using molecular biology techniques, including restricted fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The aim of this study was to identify the main yeast species associated with Mexican cocoa fermentations employing culture-dependent and -independent techniques achieving two samplings with a 1 year time difference at the same site. Isolation of the microorganisms was performed in situ. Molecular identification of yeast isolates was achieved by RFLP analysis and rDNA sequencing. Total DNA from the microorganisms on the cocoa beans was utilized for the DGGE analysis. Bands from the DGGE gels were excised and sequenced. Nineteen isolated yeasts were identified (al specie level), three of which had never before been associated with cocoa fermentations worldwide. The detected predominant yeast varied from one technique to another. Hanseniaspora sp. resulted dominant in DGGE however Saccharomyces cerevisiae was the principal isolated species. In conclusion, the culture-dependent and -independent techniques complement each other showing differences in the main yeasts involved in spontaneous cocoa fermentation, probably due to the physiological states of the viable but non culturable yeasts. Furthermore important differences between the species detected in the two samplings were detected.

Application of ISSR-PCR for rapid strain typing of Debaryomyces hansenii isolated from dry-cured Iberian ham.

Yeast populations of dry-cured Iberian ham isolated from seven industries in the province of Badajoz were characterized by ISSR-PCR using the (CAG)4 primer and PCR-RFLP of the ITS1-5.8S rRNA-ITS2 fragment, and identified by DNA sequencing. A total of 242 isolates were analyzed, indicating the primary species present was Debaryomyces hansenii at 80.9% of the isolates followed by Candida zeylanoides at 10.3% of the isolates. The remainders of isolates were identified as Yamadazyma triangularis, Sporobolomyces roseus, Meyerozyma guilliermondii, Rhodotorula slooffiae, and Cryptococcus victoriae. The ISSR-PCR method was a fast and reliable method which was able to discriminate species at a level comparable to restriction analyses of the ITS1-5.8S rRNA-ITS2 region. This method allowed for strain typing of D. hansenii, yielding 29 different PCR patterns within 196 isolates. Moreover, ISSR-PCR using the (CAG)4 primer indicated that this technique could be a promising tool for rapid discrimination of yeast starter cultures and spoilage species in dry-cured Iberian ham.

Optimization of experimental and modelling parameters for the differentiation of beverage spoiling yeasts by Matrix-Assisted-Laser-Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in response to varying growth conditions.

The growth of spoiling yeasts in beverages results in reduced quality, economic and image losses. Therefore, biochemical and DNA-based identification methods have been developed but are mostly time-consuming and laborious. Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) could deliver discriminative peptide mass fingerprints within minutes and could thus be a rapid and reliable tool for identification and differentiation. However, routine analysis of yeasts by MALDI-TOF MS is yet impaired by low reproducibility and effects of different physiological states of organisms on the reliability of the identification method are still controversial. The aim of this study was to optimize sample preparation and measurement parameterization using three spoilage yeasts (Saccharomyces cerevisiae var. diastaticus, Wickerhamomyces anomalus and Debaryomyces hansenii). The influence of environmental or physiological parameters including oxygen availability, different nutrients, cell density and growth phase were analysed and revealed small differences in mass fingerprints. Yeasts grown in the presence or absence of oxygen were precisely differentiated along these differences in mass fingerprints and a crude classification of growth phase was possible. Cell concentration did not affect the spectra distinctly, neither qualitatively nor quantitatively, and an influence of available nutrients could not be measured in each case. However, core mass peaks remained constant under all tested conditions enabling reliable identification.

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces subspecies.

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.

Characterization of the yeast ecosystem in grape must and wine using real-time PCR.

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.