José Manuel Guillamón

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region

Abstract In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.

Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae.

The influence of fermentation temperature (from 15 to 35 degrees C) on a mixed strain population was studied. Mitochondrial DNA analysis was used to differentiate Saccharomyces cerevisiae strains and the frequency of each strain during the alcoholic fermentation was determined. The chemical analyses of resulting wines were carried out. The temperature determined how Saccharomyces strains developed and how effectively they fermented. Some strains performed better at high temperatures and others at low temperatures. The maximal population size was similar at all temperatures. At low temperatures, however, it was reached later though it remained constant throughout the alcoholic fermentation. On the other hand, viable cells decreased at high temperatures, especially at 35 degrees C. Obviously, the composition of the wines changed as the temperature of fermentation changed. At low temperatures, alcohol yield was higher. Secondary metabolites to alcoholic fermentation increased as the temperature increased. Glycerol levels were directly affected by temperature.

Yeast population dynamics in spontaneous fermentations: Comparison between two different wine-producing areas over a period of three years

Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.

Effects of fermentation temperature and Saccharomyces species on the cell fatty acid composition and presence of volatile compounds in wine

Low temperature alcoholic fermentations are becoming more frequent due to the wish to produce wines with more pronounced aromatic profiles. However, their biggest drawback is the high risk of stuck and sluggish fermentations. Changes in the plasma membrane composition may be an adaptive response to low temperature fermentations. The production of volatile compounds and the changes in the membrane fatty acids were determined by GC to show the degree of cell adaptation and performance at low temperatures (13 degrees C) taking 25 degrees C as reference. The tests were done in two strains of Saccharomyces cerevisiae and one strain of Saccharomyces bayanus. Low temperatures restricted yeast growth and lengthened the fermentations. The analysis of plasma membrane fatty acids showed that dry yeasts had similar levels of unsaturation, between 70% and 80%, with no medium-chain fatty acids (MCFA). Long-chain saturated fatty acids (SFA) were the most frequent membrane fatty acids throughout the fermentations. Lipid composition changed with the growth temperature. The optimal membrane fluidity at low temperatures was modulated by changes in the unsaturation degree in S. cerevisiae strains. In S. bayanus, however, this change in the unsaturated fatty acid (UFA) percentage was not observed at different growth temperatures but the concentration of MCFA at low fermentation temperatures was higher. Concentrations of volatile compounds were higher in wines produced at lower temperatures and depended on the strain.

Analysis of yeast populations during alcoholic fermentation: A six year follow-up study

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.

Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine

ABSTRACT Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

Isolation and identification of yeasts associated with vineyard and winery by RFLP analysis of ribosomal genes and mitochondrial DNA.

Yeast colonies isolated from vineyard and cellar substrates were analysed in the present study. Yeast species assessment was carried out by amplification and digestion of a region of the ribosomal RNA gene repeat unit. Saccharomyces strains were also characterised using mitochondrial DNA restriction analysis. Oxidative basidiomycetous yeasts without enological potential were predominant in the vineyard environment. Yeasts associated with grape skin depend on grape variety, vintage and degree of grape maturation. These species from grape surface constituted the predominant microbiota in must and they developed during the first stages of the process. Yeasts colonies were also isolated and identified from the walls of a fermentation vat some days before the harvest. Contray to what was expected, Saccharomyces cerevisiae was not the major species isolated as Candida sorbosa represented 76% of the species isolated. Saccharomyces strains isolated from the fermentation vat had been previously isolated in wine fermentations in this cellar. Therefore, these strains should be considered as constant residents of this winery.

Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer.

DNA corresponding to 16S rDNA and the 165-23S rDNA intergenic spacer (ITS) from 22 reference strains of acetic acid bacteria, representing the diversity of the family Acetobacteraceae, and 24 indigenous acetic acid bacteria isolated from wine fermentations were analysed by PCR-RFLP. Frateuria aurantia LMG 1558T and Escherichia coli ATCC 11775T were included as outgroups. PCR-amplified products of about 1450 bp were obtained from the 16S rDNA of all the strains and products of between 675 and 800 bp were obtained from the 16S-23S rDNA ITS. PCR products were digested with 4-base-cutting restriction enzymes in order to evaluate the degree of polymorphism existing among these strains. Of the enzymes tested, Taql and Rsal were the most discriminatory and showed no intraspecific variations in the restriction patterns. Restriction analysis of the 16S rDNA with these enzymes is proposed as a rapid and reliable method to identify acetic acid bacteria at the level of genus and species (or related species group) and its applicability to identification of indigenous acetic acid bacteria was demonstrated. The same degree of distinction as that for the 16S rDNA analysis was obtained within reference strains of acetic acid bacteria by PCR-RFLP of the 16S-23S rDNA ITS. However, 16S-23S rDNA ITS restriction patterns of strains isolated from wine did not match those of any of the reference strains. Thus, PCR-RFLP of the 16S-23S rDNA ITS is not a useful method to identify isolates of acetic acid bacteria at the species level, although it may be an adequate method to detect intraspecific differentiation.

Rapid Characterization of Four Species of the Saccharomyces Sensu Stricto Complex According to Mitochondrial DNA Patterns

Several strains of the four sibling species of the genus Saccharomyces (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species. S. cerevisiae and S. paradoxus presented a greater number of large fragments than S. pastorianus and S. bayanus with all the assay enzymes. With AluI and DdeI, species-specific bands clearly permitted differentiation between S. pastorianus and S. bayanus. To test the resolution of this method, wild Saccharomyces strains were analyzed. The correct assignment of these strains to a known taxon by this rapid method was confirmed by means of electrophoretic karyotyping.

Effect of oenological practices on microbial populations using culture-independent techniques

Sulphur dioxide (SO(2)) addition and yeast inoculation are well-established practices in winemaking for restricting the growth of indigenous yeasts and bacterial populations. The effect of these oenological practices on wine microbial populations has been evaluated using culture-independent methods. These are quantitative PCR (qPCR) for the enumeration of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), and PCR-DGGE to determine the yeast and bacteria species diversity. The PCR-DGGE method detected a low yeast and bacteria species diversity. On the contrary, the specificity of the primers designed for the qPCR allowed that minor microbial groups such as Hanseniaspora were accurately quantified regardless of a large presence of other microbial groups such as Saccharomyces. From an oenological point of view, inoculation increased the proportion of Saccharomyces vs. non-Saccharomyces in a shorter time. Hanseniaspora increased during the first phase and decreased during the latter phases of the process, especially in the sulphited fermentations. Both yeast inoculation and SO(2) kept the LAB populations at very low level, while the AAB populations were hardly affected by these two practices.