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.     

Use of high-ethanol-resistant yeast isolates from Nigerian palm wine in lager beer brewing

High-ethanol-resistant yeasts, characterized as Saccharomyces sp., were isolated from Nigerian palm wine with added sucrose for high gravity brewing. The yeast isolates that survived the highest ethanol production were used to ferment brewery wort and produced 8.2 to 8.5% (v/v) ethanol; values almost double that of the control yeast from a local brewery.     

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.     

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.     

High degree of correlation between molecular polymorphism and geographic origin of wine yeast strains

AIMS: To guarantee the endemic genetic background of the isolates obtained in yeast isolation programs, it is necessary to differentiate between endemic and commercial strains because the progressive use of commercial yeast in wine areas around the world would affect the autochthonous yeast populations. METHODS AND RESULTS: Mitochondrial DNA restriction analysis, electrophoretic karyotyping and random amplification of polymorphic DNA (RAPD) were evaluated as experimental approaches to correlate genomic polymorphism and geographic origin of native wine yeast strains. The three molecular methods were capable of detecting a European commercial strain among native Chilean strains; however, RAPD proved to have the best performance. CONCLUSIONS: The molecular polymorphism analysis is useful to evaluate the geographical origin of native yeast isolates and confirms or refutes the genetic background of currently marketed strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits a genetic characterization of native yeast populations and confirms its utility as a tool for evaluating if a native isolate derives from the region where it was collected, permitting, furthermore, to develop studies on the evolution of native yeast populations and to evaluate the effect of introduced yeasts on these populations.     

Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis

Aims:  To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location.
Methods and Results:  Random amplification of polymorphic DNA (RAPD) was used to discriminate between 58 Tunisian strains and 21 strains from various other countries of P. savastanoi pv. savastanoi , the causal agent of olive knot disease. Isolates were separated into three groups by cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14). Group 1 contained isolates from the southeast of Tunisia and European strains. Group 2 comprised strains isolated from the north of Tunisia exclusively while group 3 encompassed the majority of isolates obtained from five orchards located in the centre of Tunisia.
Conclusions:  The results indicated that isolates of P. savastanoi pv. savastanoi were genetically distinct according to geographic regions. RAPD grouped isolates derived from the same orchard as identical.
Significance and Impact of the Study:  This is the first application of RAPD in the delineation of P. savastanoi pv. savastanoi strains.     

A note on the leavening activity of yeasts isolated from Nigerian palm wine

The role of the yeast flora of Nigerian palm wine in the leavening activity of the beverage was investigated by subjecting organisms from the wine to dough-raising tests. Those with appreciable leavening activity were identified as Saccharomyces cerevisiae and Candida spp. They produced maximum dough volumes in 3–4 h at 37°C. The study has provided experimental evidence that yeasts contribute to the leavening activity of palm wine and has identified strains which have potential utility in commercial bread baking.     

Characterization of vanA-type Enterococcus faecium isolates from urban and hospital wastewater and pigs

Aims:  In this study we analysed urban, hospital wastewater and pig faeces samples to investigate the presence of vancomycin-resistant Enterococcus faecium strains (VREF) and to determine potential links among the strains originating from the above sources and VREF strains causing clinical infections.
Methods and Results:  Urban, hospital wastewater and pig faeces exhibited high VREF prevalence of 52%, 87% and 85%, respectively. Pulsed field gel electrophoresis (PFGE) clustering of VREF genotypes as well as discriminant analysis of antibiotic resistance patterns of VREF strains revealed their source specificity while strains isolated from hospitalized humans were genetically distinct.
Conclusions:  PFGE genotypes and antimicrobial resistance patterns in VREF isolates are distinguishable by each sample origin. The observed high genetic diversity of VREF suggests horizontal transfer of genetic elements among VREF. Phenotypic and genotypic data indicate that VREF isolates of hospital-treated wastewater might pass to the urban wastewater system.
Significance and Impact of the Study:  This study provides information to understand the origin and the mechanism of circulation of vancomycin resistance in food animals and wastewater treatment plants for minimizing the risk of transmission of VRE in human population.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Induction of production and secretion β(1→4) glucanase with Saccharomyces cerevesiae by replacing the MET10 gene with egl1 gene from Trichoderma reesei

Aims:  To construct novel brewer's yeast strains with the ability to degrade β-glucan and increase sulfite levels in beer brewing by genetic manipulation.
Methods and Results:  The recombinant plasmid pA15ME containing P_met10-egl1-T_met10 expression cassette was constructed. Bam HI-linearized target plasmid pA15ME was transformed into the industrial brewer's yeast strain Z0103 to replace the MET10 locus through one-step gene replacement. The recombinants Z8, Z7 and Z3 with the ability to secrete active endo-β-1,4-glucanase I into the culture medium were isolated by Congo red dyeing. The enzymatic activities of EG I of Z8, Z7 and Z3 were 3·3, 1·5, 1·3 U l⁻¹, and the hydrolysing degrees of β-glucans in wort were increased 11·9%, 8·6% and 6·9%, respectively, than that of original strain Z0103. The MET10 gene deletions were confirmed by real-time PCR, and the sulfite levels of the culture mediums inoculated with Z8, Z7 and Z3 were increased 26%, 16% and 17%, respectively, compared to that of Z0103.
Conclusions:  The novel endoglucanase-producing brewer's yeast strains with inserted endoglucanase gene and deficient MET10 gene led to reduced content of barley β-glucans, enhanced filterability and increased sulfur dioxide in fermenting wort. Thus, the cost for addition of microbial β-glucanase enzyme and sulfite preparations in normal beer brewing processes could be reduced.
Significance and Impact of the Study:  These results suggested that genetic engineering approach is a powerful tool to construct the novel recombinant brewer's yeast strains with different properties to reduce the cost of beer brewing and improve the flavour of a beer, and the strains obtained have potential application value in beer brewing.     

Comparative genomics among Saccharomyces cerevisiae x Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins

ABSTRACT: BACKGROUND: Interspecific hybrids between S. cerevisiae x S. kudriavzevii have frequently been detected in wine and beer fermentations. Significant physiological differences among parental and hybrid strains under different stress conditions have been evidenced. In this study, we used comparative genome hybridization analysis to evaluate the genome composition of different S. cerevisiae x S. kudriavzevii natural hybrids isolated from wine and beer fermentations to infer their evolutionary origins and to figure out the potential role of common S. kudriavzevii gene fraction present in these hybrids. RESULTS: Comparative genomic hybridization (CGH) and ploidy analyses carried out in this study confirmed the presence of individual and differential chromosomal composition patterns for most S. cerevisiae x S. kudriavzevii hybrids from beer and wine. All hybrids share a common set of depleted S. cerevisiae genes, which also are depleted or absent in the wine strains studied so far, and the presence a common set of S. kudriavzevii genes, which may be associated with their capability to grow at low temperatures. Finally, a maximum parsimony analysis of chromosomal rearrangement events, occurred in the hybrid genomes, indicated the presence of two main groups of wine hybrids and different divergent lineages of brewing strains. CONCLUSION: Our data suggest that wine and beer S. cerevisiae x S. kudriavzevii hybrids have been originated by different rare-mating events involving a diploid wine S. cerevisiae and a haploid or diploid European S. kudriavzevii strains. Hybrids maintain several S. kudriavzevii genes involved in cold adaptation as well as those related to S. kudriavzevii mitochondrial functions.     

Fluoroquinolone resistance in clinical and environmental isolates of Escherichia coli in Mexico City

Aims:  To assess the different phenotypes and mechanisms of fluoroquinolone (FQ) resistance in clinical and environmental isolates of Escherichia coli .
Methods and Results:  We compared FQ-resistant E. coli isolates, measuring minimal inhibitory concentrations (MIC) of ciprofloxacin, along with susceptibility to other antibiotics. We also searched for the presence of efflux pumps, using efflux inhibitors, and for plasmid-borne FQ-resistance by PCR. We found that, aside from the higher FQ-resistance prevalence among clinical strains, environmental ones resist much lower concentrations of ciprofloxacin. Efflux pumps mediate fluoroquinolone resistance as frequently among environmental isolates than in clinical strains. Plasmid-borne qnrA genes were not detected in any resistant strain.
Conclusions:  Environmental FQ-resistant strains may have a nonclinical origin and/or a selective pressure different from the clinical use of FQs.
Significance and Impact of the Study:  The identification of the source of low-level FQ-resistant strains (ciprofloxacin MIC c . 8 μg ml⁻¹) in the environment could be important to curb the rapid emergence and spread of FQ-resistance in clinical settings, as these strains can easily become fully resistant to FQ concentrations achievable in fluids and tissues during therapy.     

Genetic and physiological variants of yeast selected from palm wine

Genetic screening of 1200-palm wine yeasts lead to the selection of fourteen isolates with various genetic and physiological properties. Nine of the isolates were identified as Saccharamyces species, three as Candida species, one as Schizosaccharomyces species and one as Kluyveromyces species. Five of the isolates were wild type parents, two were respiratory deficient mutants (rho) and nine were auxotrophic mutants. Four isolates were heterozygous diploid (αa) and two were homozygous diploid (aaα α) for the mating a mating types were further identified on mating with type loci. Four Mat α and four Mat a types were further identified on mating with standard haploid yeast strains. Forty-five percent sporulated on starvation medium producing tetrads. Fifty-two percent of the four-spored asci contained four viable spores. Maximum specific growth rate [μ_max] of the fourteen isolates range from0.13–0.26, five isolates were able to utilize exogenous nitrate for growth. Percentage alcohol production range between 5.8–8.8% for palm wine yeast, 8.5% for bakers’ yeast and 10.4% for brewers yeast. The palm wine yeast were more tolerant to exogenous alcohol but had a low alcohol productivity. Hybridization enhanced alcohol productivity and tolerance in the palm wine yeasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine

Aims:  To explain the role of Saccharomyces cerevisiae and Saccharomyces uvarum strains (formerly Saccharomyces bayanus var. uvarum ) in wine fermentation.
Methods and Results:  Indigenous Saccharomyces spp. yeasts were isolated from Amarone wine (Italy) and analysed. Genotypes were correlated to phenotypes: Melibiose⁻ and Melibiose⁺ strains displayed a karyotype characterized by three and two bands between 225 and 365 kb, respectively. Two strains were identified by karyotype analysis (one as S. cerevisiae and the other as S. uvarum ). The technological characterization of these two strains was conducted by microvinifications of Amarone wine. Wines differed by the contents of ethanol, residual sugars, acetic acid, glycerol, total polysaccharides, ethyl acetate, 2-phenylethanol and anthocyanins. Esterase and β-glucosidase activities were assayed on whole cells during fermentation at 13° and 20°C. Saccharomyces uvarum displayed higher esterase activity at 13°C, while S. cerevisiae displayed higher β-glucosidase activity at both temperatures.
Conclusions:  The strains differed by important technological and qualitative traits affecting the fermentation kinetics and important aroma components of the wine.
Significance and Impact of the Study:  The contribution of indigenous strains of S. cerevisiae and S. uvarum to wine fermentation was ascertained under specific winemaking conditions. The use of these strains as starters in a winemaking process could differently modulate the wine sensory characteristics.     

Comparison of Campylobacter fla-SVR genotypes isolated from humans and poultry in three European regions

Aims:  The genetic diversity of Campylobacter isolated from human infection and from poultry was assessed in strains originating in three different European regions in order to compare these two hosts and to investigate European regional differences.
Methods and Results:  Randomly chosen isolates originated from Norway, Iceland and Basque Country in Spain were genotyped by sequencing of the short variable region (SVR) of flaA . A total of 293 strains were investigated, c . 100 per country with half originated from either host. The results indicate extensive diversity in both hosts and identified differences in the nature and distribution of genotypes between the countries. These differences could in part be related to geographical location, in that Campylobacter genotypes from Iceland and Norway were more similar to each other than either was to Basque Country.
Conclusions:  Differences between the countries exceeded the observed differences between human and poultry isolates within a country.
Significance and Impact of the Study:  Regional differences are extensive and should not be ignored when comparing genotyping data originating from different international studies.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Petite mutation in aged and oxidatively stressed ale and lager brewing yeast

Aims:  To determine the role of oxidative stress and chronological ageing on the propensity of brewing yeast strains to form respiratory deficient 'petites'.
Methods and Results:  Four industrial yeast strains (two ale and two lager strains) were exposed to oxidative stress in the form of H₂O₂ (5 mmol l⁻¹) for two hours. Cell viability and occurrence of petites were determined by the slide culture and TTC-overlay techniques, respectively. Increases in petite frequency were observed but only in those strains sensitive to oxidative stress. Chronological ageing under aerobic conditions led to an increase in petites in strains sensitive to oxidative stress. No such increase was observed under anaerobic conditions.
Conclusion:  Ageing may contribute to mitochondrial DNA damage and increase the propensity of brewing yeast cells to become respiratory deficient. Tolerant strains may be less likely to generate petites as a result of serial re-pitching.
Significance and Impact of the Study:  Continuous re-use of brewing yeast is associated with an increase in the frequency of petites within brewery yeast slurries, a phenomenon resulting in reduced fermentative capacity. The cause of petite generation during brewery handling is unknown. We show that endogenous oxidative stress has the potential to generate petites within brewing yeast populations.     

Characterization of Aeromonas encheleia strains isolated from aquatic environments in the Czech Republic

Aims:  Characterization and identification of Aeromonas strains isolated from surface and underground waters using phenotypic and genotyping methods.
Methods and Results:  Biotyping using the ENTEROtest 24 kit and conventional biochemical and physiological tests assigned four strains to Aeromonas encheleia , whereas three isolates were identified as ambiguous Aeromonas bestiarum/Aeromonas caviae and one strain as Aeromonas eucrenophila/Aeromonas encheleia . Further characterization grouped the analysed strains together with Aer. encheleia CCM 4582^T and assigned the analysed group as members of Aer. encheleia species using ribotyping, whole-cell protein analysis and ERIC-PCR fingerprinting. The results obtained were verified by DNA gyrase A subunit gene sequencing. All analysed isolates showed unique molecular patterns, except for isolates P 1769 and CCM 7407, which revealed the same Eco RI ribotype profile and proved to be identical strains.
Conclusions:  Our results imply that Aer. encheleia strains occur in unpolluted surface as well as in underground waters and demonstrate applied methods as suitable for their identification.
Significance and Impact of the Study:  To our best knowledge, this is the first report of the isolation and identification of Aer. encheleia in the Czech Republic.     

The genome sequence of the wine yeast VIN7 reveals an allotriploid hybrid genome with Saccharomyces cerevisiae and Saccharomyces kudriavzevii origins

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S.?cerevisiae and non-S.?cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S.?cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S.?cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S.?cerevisiae and S.?kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation.