Genetic and phenotypic diversity of autochthonous Saccharomyces spp. strains associated to natural fermentation of 'Malvasia delle Lipari'

AIMS: Characterize from both genetic and phenotypic standpoints the indigenous strains of Saccharomyces spp. associated with natural fermentation of 'Malvasia delle Lipari'. METHODS AND RESULTS: A total of 192 yeast isolates were obtained from completed fermentation of a mix of 'Malvasia delle Lipari' (92%) and 'Corinto nero' (8%) grapes in two wineries in Salina Island (Sicily, Italy). Fifty-one Saccharomyces spp. isolates were characterized using ITS-PCR, random amplified polymorphic DNA-PCR and mitochondrial DNA restriction fragment length polymorphism and 12 biotypes were identified. Representative strains of each biotype, tested for their physiological traits, exhibit different killer activity, fermentation vigour, production of hydrogen sulphide and show similar beta-glucosidase and proteolytic activity. CONCLUSIONS: It is possible to cluster in different groups naturally occurring indigenous biotypes of Saccharomyces cerevisiae from 'Malvasia delle Lipari' on the basis of molecular profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Deeper insight on indigenous wine yeast of a conserved environment. The knowledge gained might offer a contribution to the selection of autochthonous wine yeast as starters for controlled fermentations.     

Differentiation of industrial wine yeast strains using microsatellite markers

AIMS: To differentiate nine industrial wine strains of Saccharomyces cerevisiae using microsatellite (simple sequence repeats, SSR) markers. METHODS AND RESULTS: Six of the strains were indigenous yeasts currently used as high-density starter monocultures by the Uruguayan wine industry. Unequivocal differentiation of these six native strains and three commercial S. cerevisiae wine strains was achieved by PCR amplification and polymorphism analysis of loci containing microsatellite markers. CONCLUSION: We recommend the use of this reproducible and simple molecular method to routinely discriminate wine yeast strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Microsatellites are superior to other methods for typing yeasts because the results can be exchanged as quantitative data. Knowledge of the frequencies of the alleles for different SSR markers will eventually lead to an accurate typing method to identify industrial wine yeast strains.     

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.     

Typing of Saccharomyces cerevisiae and Kloeckera apiculata strains from Aglianico wine

AIMS: Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation. METHODS AND RESULTS: Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae. CONCLUSIONS: The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.     

Microsatellite PCR profiling of Saccharomyces cerevisiae strains during wine fermentation

AIMS: Use of microsatellite PCR to monitor populations of Saccharomyces cerevisiae strains during fermentation of grape juice. METHOD AND RESULTS: Six commercial wine strains of S. cerevisiae were screened for polymorphism at the SC8132X locus using a modified rapid PCR identification technique. The strains formed four distinct polymorphic groups that could be readily distinguished from one another. Fermentations inoculated with mixtures of three strains polymorphic at the SC8132X locus were monitored until sugar utilization was complete, and all exhibited a changing population structure throughout the fermentation. CONCLUSIONS: Rapid population quantification demonstrated that wine fermentations are dynamic and do not necessarily reflect the initial yeast population structure. One or more yeast strains were found to dominate at different stages of the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The population structure of S. cerevisiae during mixed culture wine fermentation is dynamic and could modify the chemical composition and flavour profile of wine.     

Saccharomyces cerevisiae biodiversity in spontaneous commercial fermentations of grape musts with 'adequate' and 'inadequate' assimilable-nitrogen content

AIM: To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content. METHODS AND RESULTS: Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity. CONCLUSIONS: The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.     

Ecological success of a group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrids in the northern european wine-making environment

The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of S. kudriavzevii genetic content in three representative strains. We developed microsatellite markers for S. kudriavzevii and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12 S. cerevisiae and 7 S. kudriavzevii microsatellite loci and found that these strains are the products of multiple hybridization events between several S. cerevisiae wine yeast isolates and various S. kudriavzevii strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (δμ)(2) genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by S. cerevisiae/S. kudriavzevii cryophilic hybrids to winery environments in cool climates.     

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.     

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.     

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America

AIMS: The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. METHODS AND RESULTS: Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. CONCLUSIONS: Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

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.     

Molecular identification and characterization of wine yeasts isolated from Tenerife (Canary Island, Spain)

AIMS: The present study was aimed at the identification, differentiation and characterization of indigenous yeasts isolated from Tenerife vineyards (viticulture region that has never been characterized before). Microbiota were studied from 14 samples taken during fermentations carried out in the 2002 vintage, from 11 wineries belonging to five wine regions on Tenerife Island. METHODS AND RESULTS: Yeasts' strains were identified and characterized through restriction analysis of the 5.8S-internal transcribed spacer region and the mitochondrial DNA. At the beginning of alcoholic fermentation, 26 yeast species were found, where 14 species were present in significant frequencies in only one sample. Likewise, the Saccharomyces cerevisiae strains isolated are very specific, as they were only present in one wine region. CONCLUSIONS: There were isolated specific yeasts from each region on Tenerife Island. The founded yeasts may be responsible for distinctive and interesting properties of the studied wines. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms part of an extensive taxonomic survey within the ecological framework of vineyards in Tenerife. This investigation is an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the grape growing regions of this island. The results obtained demonstrate the value of using molecular genetic methods in taxonomic and ecological surveys. The results also shed some light on the ecology and oenological potential of S. cerevisiae strains isolated from this unique environment.     

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.     

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.     

Sequence diversity of yeast 2 microns RAF gene and its co-evolution with STB and REP1.

Despite the extensive study of yeast 2 microns plasmid, the exact function of plasmid-encoded RAF gene is not clear. Variants of 2 microns plasmids from industrial Saccharomyces cerevisiae yeasts were isolated and characterized. Sequencing of RAF alleles revealed about 8% nucleotide and 10% amino acid diversities between 2 microns variants of closely related strains, RAF sequence variations were correlated with STB-REP1 sequence diversity. We also used restriction fragment length polymorphism linkage to screen a large number of yeast strains from different fermentation industries. The results clearly show a tight linkage of STB-REP1-RAF variations. Thus, our observations suggest that plasmid-borne cis- and trans-acting elements co-evolved to form an optimal molecular parasite and that RAF may play a role in active plasmid partitioning.     

Classical and molecular analyses to characterize commercial dry yeasts used in wine fermentations

AIMS: The aim of the work was to apply PCR-temperature gradient gel electrophoresis (PCR-TGGE) and restriction enzyme analysis (RE) assays to identify commercially available starters of Saccharomyces cerevisiae sensu stricto complex. METHODS AND RESULTS: To characterize an analysed pool of 62 active dry yeasts of different brands used in wine fermentation practices, classical microbiological tests were also performed as well as evaluation of contamination with lactic acid bacteria and non-Saccharomyces yeasts. PCR-TGGE and RE were used in order to provide fast and reliable methods to identify and differentiate enological yeasts. Proposed molecular methods enabled to identify particular strains within 36 h after colony isolation and directly from dry yeast suspension. CONCLUSIONS: The methods are highly recommended to obtain reliable results on yeast strain differentiation in a significantly shorter time if compared to classical fermentation tests. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtaining of yeast strain differentiation in a short time and without plating is a good tool for a rapid discrimination among enological strains used as starters in enological practices.     

Combination of 10% EDTA, Photosan, and a blue light hand-held photopolymerizer to inactivate leading oral bacteria in dentistry in vitro

Aims:  To study the yeast diversity of Nigerian palm wines by comparison with other African strains.
Methods and Results:  Twenty-three Saccharomyces cerevisiae strains were obtained from palm wine samples collected at four locations in eastern Nigeria, and characterized using different molecular techniques: internal transcribed spacer restriction fragment length polymorphism and sequence analysis, pulsed field gel electrophoresis, inter delta typing and microsatellite multilocus analysis. These techniques revealed that palm wine yeasts represent a group of closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote strains and an allelic richness similar to wine suggestive of local domestication. Several other African yeast strains were not connected to this group. Ghana sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin.
Conclusions:  Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates.
Significance and Impact of the Study:  This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration.     

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.     

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.