Effect of selenium on growth and antioxidant enzyme activities of wine related yeasts.

The use of supplements in the diet is a common practice to address nutritional deficiencies. Selenium is an essential micronutrient with an antioxidant and anti-carcinogenic role in human and animal health. There is increasing interest in developing nutritional supplements such as yeast cells enriched with selenium. The possibility of producing beverages, namely wine, with selenium-enriched yeasts, led us to investigate the selenium tolerance of six wine related yeasts. The production of such cells may hamper selenium toxicity problems. Above certain concentrations selenium can be toxic inducing oxidative stress and yeast species can show different tolerance. This work aimed at studying selenium tolerance of a diversity of wine related yeasts, thus antioxidant response mechanisms with different concentrations of sodium selenite were evaluated. Viability assays demonstrated that the yeast Torulaspora delbrueckii showed the highest tolerance for the tested levels of 100 µg mL(-1) of sodium selenite. The evaluation of antioxidative enzyme activities showed the best performance for concentrations of 250 and 100 µg mL(-1), respectively for the yeast species Saccharomyces cerevisiae and Hanseniaspora guilliermondii. These results encourage future studies on the possibility to use pre-enriched yeast cells as selenium supplement in wine production.

Partial 26S rDNA restriction analysis as a tool to characterise non-Saccharomyces yeasts present during red wine fermentations.

Restriction patterns of amplified regions of ribosomal large subunit RNA encoding genes (26S rDNA) were evaluated as a routine methodology to examine yeast species diversity during red wine fermentation. The results were confirmed by sequencing of D1/D2 region of 26S rDNA. Red wine production was carried out using a yeast starter culture together with different commercial products, namely enzymes, fermentation activators and tannins and their influence on the non-Saccharomyces yeast population was studied. Yeast strains were isolated using lysine agar as a selective medium for non-Saccharomyces yeasts, after morphological characterisation of colonies. Amplification of 26S rDNA followed by digestion with three restriction enzymes applied to the 121 isolates, generated 19 profiles and a very high correlation with sequencing results was achieved. Although a starter yeast culture was added, results showed that several yeast species were present during all stages of fermentation, independent of the conditions tested, emphasizing the diversity of microorganisms associated with winemaking. On the other hand commercial additives did not significantly influence the diversity of yeast population during the fermentation process. For non-Saccharomyces strains, restriction patterns of a PCR amplified 26S rDNA region proved to be an adequate tool for clustering strains at species level and enabled the monitoring of yeast population dynamics during red wine fermentation.

Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains.

Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts.

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.

Influence of ethanol and temperature on the cellular fatty acid composition of Zygosaccharomyces bailii spoilage yeasts.

Changes in the fatty acid profile of Zygosaccharomyces bailii strains, isolated from different sources, after growth at increasing concentrations of ethanol and/or decreasing temperatures were determined. Differences in fatty acid composition between Zygosaccharomyces bailii strains at standard conditions (25 degrees C, 0% initial ethanol) were observed and could be related to ethanol tolerance. Zygosaccharomyces bailii strain isolated from wine showed the highest ethanol tolerance in relation to growth rate. Surprisingly, an increase in ethanol concentration or a decrease in growth temperature caused a decrease in the degree of unsaturation of total cellular fatty acids. On the other hand, the mean chain length increased (high ethanol concentration) or decreased (low temperature) depending on the stress factor. When both stress situations (high ethanol concentration and low temperature) were present at the same time, the degree of unsaturation remained approximately constant. With decreasing temperatures, the C16/C18 ratio increased in studies of initial ethanol content below 5%, and above 5% ethanol, decreased.

RAPD analysis: a rapid technique for differentiation of spoilage yeasts.

Techniques for the identification of the spoilage yeasts Saccharomyces cerevisiae and members of the Zygosaccharomyces genus from food and beverages sources were evaluated. The use of identification systems based on physiological characteristics resulted often in incomplete identification or misidentification. Also the cellular fatty acid analysis failed on differentiating species within the Zygosaccharomyces genus. However, the Random Amplified Polymorphic DNA (RAPD) assay, using selected 10-mer oligonucleotides, allowed discrimination between all species tested. For this RAPD assay, a simple and reproducible method of DNA isolation from spoilage yeast cells is described.