Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery

AIMS: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. METHODS AND RESULTS: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-delta PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. CONCLUSION: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.     

Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains

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 community during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inoculation 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 indigenous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided information on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.     

Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non-Saccharomyces yeasts (Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced.     

Molecular profiling of yeasts isolated during spontaneous fermentations of Austrian wines

The aim of the present study was to evaluate the autochthonous yeast population during spontaneous fermentations of grape musts in Austrian wine-producing areas. Investigation of genomic and genetic variations among wine yeasts was a first step towards a long-term goal of selecting strains with valuable enological properties typical for this geographical region. An approach, combining sequences of the D1/D2 domain of the 26S rRNA gene and random amplified polymorphic DNA fingerprinting, was used to characterize yeasts at the species level, whereas the differentiation of Saccharomyces strains was accomplished by amplified fragment length polymorphism fingerprinting. At the beginning of fermentation, representatives of nine genera were identified, with Hanseniaspora and Metschnikowia species characterized most frequently. Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum strains, which were identified throughout the entire fermentation process, showed a high level of genetic diversity. A number of S. cerevisiae strains were common at multiple wineries, but a wide range of strains with characteristic profiles were characterized at individual locations. This biodiversity survey represents a contribution to the investigation and preservation of genetic diversity of biotechnologically relevant yeasts in Austrian wine-making areas.     

The role of indigenous yeasts in traditional Irish cider fermentations

AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.     

Molecular identification of wine yeasts at species or strain level: a case study with strains from two vine-growing areas of Greece

The composition of wine yeast populations, present during spontaneous fermentation of musts from two wine-producing areas of Greece (Amyndeon and Santorini) and followed for two consecutive years, were studied using a range of molecular techniques. Internal Transcribed Spacer (ITS) ribotyping was convincingly applied for yeast species identification, proving its usefulness as a reliable tool for the rapid characterization of species composition in yeast population studies. Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) was shown to be a convenient criterion for the detection of intraspecies genetic diversity of both Saccharomyces and non-Saccharomyces isolate populations. Similarly, polymorphism of amplified delta interspersed element sequences provided an additional criterion for S. cerevisiae strain differentiation. Comparative analysis of S. cerevisiae genetic diversity, using mtDNA restriction patterns and delta-amplification profiles, showed a similar discriminative power of the two techniques. However, by combining these approaches it was possible to distinguish/characterize strains of the same species and draw useful conclusions about yeast diversity during alcoholic fermentation. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were (i) almost complete absence of non-S.cerevisiae species from fermentations of must originating from the island Santorini, (ii) a well recorded strain polymorphism in populations of non-Saccharomyces species originating from Amyndeon and (iii) an unexpected polymorphism concerning S. cerevisiae populations, much greater than ever reported before in similar studies with wine yeasts of other geographical regions.     

Dynamics of the yeast strain population during spontaneous alcoholic fermentation determined by CHEF gel electrophoresis

Wine yeasts were isolated from spontaneous alcoholic fermentations performed with white and red grape musts from vintages 1991 and 1992. Yeast cells were analysed by physiological tests and gel electrophoretic karyotyping. It was shown that there is a succession of different strains in the yeast population during the time course of the fermentation process. Furthermore, the composition of the yeast strain population differs from grape must to grape must and from year to year, and may therefore be considered vineyard (terrain)- and vintage-dependent.     

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.     

The molecular genetic differentiation of cultured Saccharomyces strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that baker's yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae and S. bayanus var. uvarum hybrids. The molecular karyotyping of baker's, brewer's, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)5 can be used to study the populations of cultured S. cerevisiae strains.     

Genetic diversity of Saccharomyces cerevisiae strains during the 24 h fermentative cycle for the production of the artisanal Brazilian cachaça

AIMS: Characterization of yeast populations and genetic polymorphism of Saccharomyces cerevisiae strains collected during the short fermentative cycles from the spontaneous fermentations during the artisanal cacha?a production. METHODS AND RESULTS: The prevalent S. cerevisiae strains were analysed by PFG and RAPD-PCR using primers EI1 and M13. The molecular analysis have showed a high degree of genetic polymorphism among the strains within a 24 h fermentative cycle. CONCLUSION: The genetic diversity observed in the S. cerevisiae strains may be occurring due to the existence of a large number of individual genotypes within the species. The unique characteristics of the cacha?a fermentation process probably allows for a faster detection of molecular polymorphisms of yeast strains than other types of fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Spontaneous fermentations to produce cacha?a, due to their characteristics, are an excellent model for the study of molecular diversity of S. cerevisiae strains during the production of fermented beverages.     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.     

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.     

Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine

Individual yeast strains belonging to the Saccharomyces sensu stricto complex were isolated from Amarone wine produced in four cellars of the Valpolicella area (Italy) and characterized by conventional physiological tests and by RAPD-PCR and mtDNA restriction assays. Thirteen out of 20 strains were classified as Saccharomyces cerevisiae (ex S. cerevisiae p.r. cerevisiae and p.r. bayanus) and the remaining as Saccharomyces bayanus (ex S. cerevisiae p.r. uvarum). RAPD-PCR method proved to be a fast and reliable tool for identification of Saccharomyces sensu stricto strains and also gave intraspecific differentiation. Restriction analysis of mtDNA permitted to distinguish S. cerevisiae and S. bayanus species and to discern polymorphism among S. cerevisiae isolates. The assessment of the phenotypic diversity within the isolates by gas-chromatographic analysis of secondary fermentation products was explored. Small quantities of isobutanol were produced by most of the strains and higher amounts by some S. cerevisiae strains with phenotypes Gal^- and Mel^-; all S. bayanus strains produced low amounts of amilyc alcohols. From this study it appears that each winery owns particular strains, with different genetic and biochemical characteristics, selected by specific environmental pressures during the Amarone winemaking process carried out at low temperature in presence of high sugar content.     

Yeast population dynamics during the fermentation and biological aging of sherry wines

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of "fino" sherry wine making. The four races of "flor" Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-flor S. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations of S. cerevisiae strains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.     

Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.     

Molecular typing demonstrates homogeneity of Saccharomyces uvarum strains and reveals the existence of hybrids between S. uvarum and S. cerevisiae, including the S. bayanus type strain CBS 380

PCR/RFLP of the NTS2 sequence of rDNA was shown to be suitable for differentiating Saccharomyces sensu stricto species. We previously showed that, within the presently accepted S. bayanus taxon, strains formerly classified as S. uvarum represented a distinct subgroup (Nguyen and Gaillardin, 1997). In this study, we reidentified 43 more strains isolated recently from wine, cider and various fermentation habitats, and confirmed by karyotyping, hybridization and mtDNA analysis the homogeneity of strains from the S. uvarum subspecies. Molecular typing of nuclear and mitochondrial genomes of strains preserved in collections, and often originating from beer like S. pastorianusNT, revealed the existence of hybrids between S. uvarum and S. cerevisiae. Surprisingly, S. bayanusT CBS380 appeared itself to be a hybrid between S. uvarum and S. cerevisiae. This strain has a mitochondrial genome identical to that of S. uvarum, and a very similar karyotype with 13 isomorphic chromosomes, six of which at least hybridize strongly with S. uvarum chromosomes or with a S. uvarum specific sequence. However, four of the chromosome bands of S. bayanusT bear Y' sequences indistinguishable from those of S. cerevisiae, a feature that is not observed among presently isolated S. uvarum strains. Because of the hybrid nature of S. bayanus(T) and of the scarcity of similar hybrids among present days isolates, we propose to reinstate S. uvarum as a proper species among the Saccharomyces sensu stricto complex.     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

Physiological properties of some yeast strains

Twenty yeast strains have recently been isolated in pure cultures from natural and industrial sources and identified based mainly on physiological properties. The majority of the strains (15) are alcohologenic belonging to the genus Saccharomyces and comprise two brewer's (beer) yeast strains (S. carlsbergensis= S. uvarum A and B), two baker's yeast strains (S. cerevisiae CA and CP), one spirit yeast strain (S. cerevisiae CF) and ten wine yeast strains (S. cerevisiae var. ellipsoideus = S. ellipsoideus 1, 3, 4, 6, 8 and 9; S. oviformis 2, 5 and 7; and S. uvarum 10). The other 5 yeast strains belong to different species: Kloeckera apiculate, Candida mycoderma (Mycoderma vini), Pichia membranaefaciens, Rhodotorula glutinis and Torulopsis holmii, respectively.     

Transformation of lignins from grape solids during alcoholic fermentation

Conversion of lignins contained in solid parts of Rkatsiteli grapes (crests, seeds, and skin) during alcoholic fermentation by wine yeast in Reader's medium was studied. Various species of wine yeast were used: Saccharomyces oviformis, S. vini Kakhuri 42, S. chodati Teliani 79, and S. uvarum Tsinandali 77. We found that lignins from solid parts of grapes are partially decomposed during alcoholic fermentation, which releases low-molecular-weight aromatic compounds into the medium. A peculiar feature of lignin decomposition during alcoholic fermentation is the formation of reduction products.     

Evidence for domesticated and wild populations of Saccharomyces cerevisiae

Saccharomyces cerevisiae is predominantly found in association with human activities, particularly the production of alcoholic beverages. S. paradoxus, the closest known relative of S. cerevisiae, is commonly found on exudates and bark of deciduous trees and in associated soils. This has lead to the idea that S. cerevisiae is a domesticated species, specialized for the fermentation of alcoholic beverages, and isolates of S. cerevisiae from other sources simply represent migrants from these fermentations. We have surveyed DNA sequence diversity at five loci in 81 strains of S. cerevisiae that were isolated from a variety of human and natural fermentations as well as sources unrelated to alcoholic beverage production, such as tree exudates and immunocompromised patients. Diversity within vineyard strains and within saké strains is low, consistent with their status as domesticated stocks. The oldest lineages and the majority of variation are found in strains from sources unrelated to wine production. We propose a model whereby two specialized breeds of S. cerevisiae have been created, one for the production of grape wine and one for the production of saké wine. We estimate that these two breeds have remained isolated from one another for thousands of years, consistent with the earliest archeological evidence for wine-making. We conclude that although there are clearly strains of S. cerevisiae specialized for the production of alcoholic beverages, these have been derived from natural populations unassociated with alcoholic beverage production, rather than the opposite.