Application of Multi Locus Sequence Typing to the analysis of the biodiversity of indigenous Saccharomyces cerevisiae wine yeasts from Lebanon

AIMS: To assess suitability of Multi Locus Sequence Typing (MLST) for investigating the biodiversity of wine yeast strains. This method was compared with established ones like microsatellite analysis or amplification of genomic regions flanked by repeated (delta) elements. METHODS AND RESULTS: DNA fragments were amplified and sequenced for 26 loci representing housekeeping genes, open reading frames (ORFs) of unknown functions or intergenic regions. A set of seven loci was tested on 84 Saccharomyces cerevisiae strains, including 65 strains isolated from traditional wineries in Lebanon, commercial wine strains and Asian isolates. An overall sequence diversity of 2.05% was observed, consisting of single nucleotide polymorphisms, 60% of them occurring in a heterozygous state. The number of polymorphic sites per locus varied between 4 and 14. The same set of strains was analysed by microsatellite typing on six polymorphic loci and by interdelta amplification. CONCLUSIONS: Clustering of MLST profiles clearly differentiated the Asian group of strains from Lebanese and European commercial strains that appear closely related. The current MLST scheme appears less discriminatory (92.27%) on closely related wine yeasts than microsatellite or interdelta typing (>99%). SIGNIFICANCE AND IMPACT OF THE STUDY: MLST is a highly reliable method for relatedness inference and promising for wine yeast typing.     

Molecular typing of wine yeast strains Saccharomyces bayanus var. uvarum using microsatellite markers

The Saccharomyces bayanus var. uvarum yeasts are associated with spontaneous fermentation of must. Some strains were shown to be enological yeasts of interest in different winemaking processes. The molecular typing of S. bayanus var. uvarum at the strain level has become significant for wine microbiologists. Four microsatellite loci were defined from the exploration of genomic DNA sequence of S. bayanus var. uvarum. The 40 strains studied were homozygote for the locus considered. The discriminating capacity of the microsatellite method was found to be equal to that of karyotypes analysis. Links between 37 indigenous strains with the same geographic origin could be established through the analysis of microsatellite patterns. The analysis of microsatellite polymorphism is a reliable method for wine S. bayanus var. uvarum strains and their hybrids with Saccharomyces cerevisiae identification in taxonomic, ecological studies and winemaking applications.     

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.     

Saccharomyces cerevisiae wine yeast populations in a cold region in Argentinean Patagonia. A study at different fermentation scales

AIMS: To study the diversity and dynamics of indigenous Saccharomyces wine populations during Malbec spontaneous fermentation, a representative Patagonian red wine, at both industrial and laboratory scale. METHODS AND RESULTS: Two molecular techniques, including restriction fragment length polymorphism of mitochondrial (mt) DNA and polymorphism of amplified delta interspersed element sequences, were used for characterization of indigenous yeasts at strain level. The mtDNA restriction patterns showed the major discriminative power; however, by combining the two molecular approaches it was possible to distinguish a larger number of strains and, therefore, draw more representative conclusions about yeast diversity. Although a great diversity of wild Saccharomyces cerevisiae strains was observed, only nine represented more than half of the total Saccharomyces yeast biota analysed; five of these were common and took over the Malbec must fermentation in both vinifications. CONCLUSIONS: Many different indigenous S. cerevisiae strains were identified; nevertheless, the dominant strains in both industrial and laboratory vinification processes were just a few and the same. SIGNIFICANCE AND IMPACT OF THE STUDY: Small-scale fermentation appears to be a valuable tool in winemaking, one especially helpful in evaluating microbiological aspects of as well as possible interactions between inoculated selected strains and native strains.     

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.     

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.     

Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level

AIMS: In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. METHODS AND RESULTS: Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. CONCLUSIONS: Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.     

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.     

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.     

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...     

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.     

Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history

Fermented beverages and foods have played a significant role in most societies worldwide for millennia. To better understand how the yeast species Saccharomyces cerevisiae, the main fermenting agent, evolved along this historical and expansion process, we analysed the genetic diversity among 651 strains from 56 different geographical origins, worldwide. Their genotyping at 12 microsatellite loci revealed 575 distinct genotypes organized in subgroups of yeast types, i.e. bread, beer, wine, sake. Some of these groups presented unexpected relatedness: Bread strains displayed a combination of alleles intermediate between beer and wine strains, and strains used for rice wine and sake were most closely related to beer and bread strains. However, up to 28% of genetic diversity between these technological groups was associated with geographical differences which suggests local domestications. Focusing on wine yeasts, a group of Lebanese strains were basal in an F(ST) tree, suggesting a Mesopotamia-based origin of most wine strains. In Europe, migration of wine strains occurred through the Danube Valley, and around the Mediterranean Sea. An approximate Bayesian computation approach suggested a postglacial divergence (most probable period 10,000-12,000 bp). As our results suggest intimate association between man and wine yeast across centuries, we hypothesize that yeast followed man and vine migrations as a commensal member of grapevine flora.     

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.     

Comparison of two alternative dominant selectable markers for wine yeast transformation

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10(3) transformants per microg of DNA for the three wine yeast strains tested.     

New PCR-based methods for yeast identification

AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.     

Toward a global database for the molecular typing of Saccharomyces cerevisiae strains

Most of the yeast strains used in fermented beverages and foods are classified as Saccharomyces cerevisiae. However, different strains are suitable for different fermentation processes. The purpose of this work is the proposal of a standardized methodology for the molecular genotyping of S. cerevisiae strains based on polymorphisms at microsatellite loci and/or single nucleotide polymorphisms (SNPs). Single nucleotide variants in the coding region of FLO8, a key regulator of flocculation and pseudohyphae formation, were analyzed in a subset of Uruguayan wine strains. Polymorphism analysis at nine microsatellite loci (selected from 33 loci tested) was performed in a collection of 120 strains, mostly wine strains, from different origins. From a total of 184 different alleles scored, 50 were exclusive alleles that could identify 29 strains. Four selected microsatellite loci are located within or near genes of putative enological interest. The Uruguayan strains are highly diverse and evenly distributed in the phylogenetic reconstructions, suggesting an evolutionary history previous to human use. The Saccharomyces cerevisiae Microsatellites and SNPs Genotyping Database is presented (www.pasteur.edu.uy/yeast). Comparison of standardized results from strains coming from different settings (industrial, clinical, environmental) will provide a reliable and growing source of information on the molecular biodiversity of S. cerevisiae strains.     

Phenotypic landscape of Saccharomyces cerevisiae during wine fermentation: evidence for origin-dependent metabolic traits

The species Saccharomyces cerevisiae includes natural strains, clinical isolates, and a large number of strains used in human activities. The aim of this work was to investigate how the adaptation to a broad range of ecological niches may have selectively shaped the yeast metabolic network to generate specific phenotypes. Using 72 S. cerevisiae strains collected from various sources, we provide, for the first time, a population-scale picture of the fermentative metabolic traits found in the S. cerevisiae species under wine making conditions. Considerable phenotypic variation was found suggesting that this yeast employs diverse metabolic strategies to face environmental constraints. Several groups of strains can be distinguished from the entire population on the basis of specific traits. Strains accustomed to growing in the presence of high sugar concentrations, such as wine yeasts and strains obtained from fruits, were able to achieve fermentation, whereas natural yeasts isolated from "poor-sugar" environments, such as oak trees or plants, were not. Commercial wine yeasts clearly appeared as a subset of vineyard isolates, and were mainly differentiated by their fermentative performances as well as their low acetate production. Overall, the emergence of the origin-dependent properties of the strains provides evidence for a phenotypic evolution driven by environmental constraints and/or human selection within S. cerevisiae.     

A breeding strategy to harness flavor diversity of Saccharomyces interspecific hybrids and minimize hydrogen sulfide production

Industrial food-grade yeast strains are selected for traits that enhance their application in quality production processes. Wine yeasts are required to survive in the harsh environment of fermenting grape must, while at the same time contributing to wine quality by producing desirable aromas and flavors. For this reason, there are hundreds of wine yeasts available, exhibiting characteristics that make them suitable for different fermentation conditions and winemaking practices. As wine styles evolve and technical winemaking requirements change, however, it becomes necessary to improve existing strains. This becomes a laborious and costly process when the targets for improvement involve flavor compound production. Here, we demonstrate a new approach harnessing preexisting industrial yeast strains that carry desirable flavor phenotypes - low hydrogen sulfide (H(2) S) production and high ester production. A low-H(2) S Saccharomyces cerevisiae strain previously generated by chemical mutagenesis was hybridized independently with two ester-producing natural interspecies hybrids of S.?cerevisiae and Saccharomyces kudriavzevii. Deficiencies in sporulation frequency and spore viability were overcome through use of complementary selectable traits, allowing successful isolation of several novel hybrids exhibiting both desired traits in a single round of selection.     

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.     

Field-flow fractionation as analytical technique for the characterization of dry yeast: correlation with wine fermentation activity

Important oenological properties of wine depend on the winemaking yeast used in the fermentation process. There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed. Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use. Laboratory-scale fermentations were performed using two different S. cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation. The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye. In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells. Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L. During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed. Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF. These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast-based bioprocesses such as wine production.