Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang, China
ABSTRACT The evolution of yeast species and Saccharomyces cerevisiae genotypes during spontaneous fermentations of Muscat blanc planted in 1957 in Jingyang region of China was followed in this study. Using a combination of colony morphology on Wallerstein Nutrient (WLN) medium, sequence analysis of the 26S rDNA D1/D2 domain and 5.8S-ITS-RFLP analysis, a total of 686 isolates were identified at the species level. The six species identified were S. cerevisiae, Hanseniaspora uvarum, Hanseniaspora opuntiae, Issatchenkia terricola, Pichia kudriavzevii (Issatchenkia orientalis) and Trichosporon coremiiforme. This is the first report of T. coremiiforme as an inhabitant of grape must. Three new colony morphologies on WLN medium and one new 5.8S-ITS-RFLP profile are described. Species of non-Saccharomyces, predominantly H. opuntiae, were found in early stages of fermentation. Subsequently, S. cerevisiae prevailed followed by large numbers of P. kudriavzevii that dominated at the end of fermentations. Six native genotypes of S. cerevisiae were determined by interdelta sequence analysis. Genotypes III and IV were predominant. As a first step in exploring untapped yeast resources of the region, this study is important for monitoring the yeast ecology in native fermentations and screening indigenous yeasts that will produce wines with regional characteristics.
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ABSTRACT: Various molecular approaches have been applied as culture-independent techniques to monitor wine fermentations over the last decade. Among them, those based on RNA detection have been widely used for yeast cell detection, assuming that RNA only exists in live cells. Fluorescence in situ hybridization (FISH) targeting intracellular rRNA is considered a promising technique for the investigation of wine ecology. For the present study, we applied the FISH technique in combination with epifluorescence microscopy and flow cytometry to directly quantify populations of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris during alcoholic fermentations. A new specific probe that hybridizes with eight species of Hanseniaspora genus and a second probe specific for Starm. bacillaris were designed, and the conditions for their application to pure cultures, mixed cultures, and wine samples were optimized. Single and mixed fermentations were performed with natural, concentrated must at two different temperatures, 15°C and 25°C. The population dynamics revealed that the Sacch. cerevisiae population increased to 10(7)-10(8)cells/ml during all fermentations, whereas H. uvarum and Starm. bacillaris tended to increase in single fermentations but remained at levels similar to their inoculations at 10(6)cells/ml in mixed fermentations. Temperature mainly affected the fermentation duration (slower at the lower temperature) but did not affect the population sizes of the different species. The use of these probes in natural wine fermentations has been validated.International Journal of Food Microbiology 08/2014; 191C:1-9. DOI:10.1016/j.ijfoodmicro.2014.08.014 · 3.16 Impact Factor
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ABSTRACT: Mixed inoculation of Saccharomyces cerevisiae strains is used in winemaking for achieving high sensory quality of the wine. However, information on the diversity and population of yeasts during inoculated fermentation is very limited. In this study, we evaluated the effect of mixed inocula with different inoculation timing on the yeast commu-nity during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inocula-tion of both strains. Wallersterin Laboratory Nutrient (WLN) medium and sequence of the 26S rDNA D1/D2 domain were used to compare the diversity of yeast species. Five species, including Candida diversa, Hanseniaspora opuntiae, H. uvarum, Issatchenkia orientalis and I. terricola, were identified in the grape must, with Issatchenkia sp. being predominant (67.5 %). Three to four species were involved in each fermentation treatment. The fermentations by mixed inocula presented more yeast species than by pure inocula. Interdelta sequence typing was used to identify S. cerevisiae strains. Ten genotypes were identified among 322 isolated S. cerevisiae strains. Their distribution varied among different stages of fermentations and different inoculation treatments. The inoculated strains were not predominant, while indige-nous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided informa-tion on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.Annals of Microbiology 08/2014; 65(2). DOI:10.1007/s13213-014-0934-8 · 1.04 Impact Factor
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ABSTRACT: Two molecular methods, interdelta polymorphism fingerprinting of genomic DNA and COX1 intron polymorphism fingerprinting of mitochondrial DNA, were used to characterize 52 strains of Saccharomyces identified to species by 26S rDNA sequencing only in order to assess the potential of these techniques for strain typing and genetic analysis individually and in combination. Two laboratory isolates derived from the same parentage served as a control. Forty-seven S. cerevisiae strains representing a mix of commercial strains and vineyard or winery isolates were used. Five of these strains have been given the commercial designation S. cerevisiae race bayanus. Three strains were identified as S. bayanus, S. kudriavzevii, and S. servazzii from their 26S rDNA sequence and thus may be hybrids. Forty-four genetic patterns were found among the strains by interdelta polymorphism fingerprinting and 47 genetic patterns were found by COX1 intron polymorphism fingerprinting. Each method allowed differentiation of the majority of strains but grouped strains into different clusters. Strains were clustered into eight groups using both techniques in combination. The two laboratory strains clustered together but were in a larger grouping of wine strains. Saccharomyces bayanus, S. kudriavzevii, and S. servazzii were separated from each other but each clustered with S. cerevisiae strains and could not be differentiated from S. cerevisiae by either method. Three of the S. cerevisiae race bayanus strains clustered together but the other two were in different groupings. Of the 10 commercial strains, 904 (French Red) was grouped alone, while 522 (Montrachet) and 905 (Premier Cuvee) were grouped with wild isolates from different areas. Thus both methods reveal patterns of strain similarity but neither method differentiated strains by species or by origin.American Journal of Enology and Viticulture 02/2013; 65(1):109-116. DOI:10.5344/ajev.2013.13056 · 1.63 Impact Factor