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.     

Reduction of volatile acidity of wines by selected yeast strains

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose-low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose-high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Evidence for viable but nonculturable yeasts in botrytis-affected wine

AIMS: In Botrytis-affected wine, high concentrations of SO2 are added to stop the alcoholic fermentation and to stabilize the wine. During maturing in barrels or bottle-ageing, an unwanted refermentation can sometimes occur. However, results of the usual plate count of wine samples at the beginning of maturing suggest wine microbial stability. The aim of this study was to investigate the mode of yeasts survival after the addition of SO2 and to identify surviving yeasts. METHODS AND RESULTS: Using direct epifluorescence technique, we observed the behaviour of cells after SO2 addition and compared the cell number determined by this method with the result of plate counts. The persistent yeast species were identified using two methods: polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-denaturing gradient gel electrophoresis. They were identified as Saccharomyces cerevisiae and Candida stellata, and after few months of maturing, other spoiling yeasts appeared, like Rhodotorula mucilaginosa or Zygosaccharomyces bailii. CONCLUSIONS: All characteristics of the cells lead to the conclusion that yeast persisted in wine in a viable but nonculturable-like state (VBNC). Suppression of the effect of free-SO2 did not lead to the resuscitation of the cells; however, another method proved the capacity of the cells to exit from the VBNC-like state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of the presence of VBNC-like yeasts in wine. The 'refermentation' phenomenon is probably due to the exit of the VBNC state.     

Yeast community structures and dynamics in healthy and Botrytis-affected grape must fermentations

Indigenous yeast population dynamics during the fermentation of healthy and Botrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations, Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii and Kazachstania sp. populations that have not been reported before for wine fermentations. Importantly, Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e., Saccharomyces cerevisiae in samples from Arcadia and Z. bailii in samples from Attica, could be recovered at the end of Botrytis-affected fermentations. The governing of wine fermentations by Z. bailii is reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy and Botrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations.     

Isolation of culturable yeasts from market wines and evaluation of the 5.8S-ITS rDNA sequence analysis for identification purposes

AIMS: To test the possibility that wines available in the marketplace may contain culturable yeasts and to evaluate the 5.8S-ITS rDNA sequence analysis as adequate means for the identification of isolates. METHODS AND RESULTS: As a case study, typical Greek wines were surveyed. Sequence analysis of the 5.8S-ITS rDNA was tested for its robustness in species or strain identification. Sixteen isolates could be assigned into the species Brettanomyces bruxellensis, Saccharomyces cerevisiae and Rhodotorula pinicola, whereas four isolates could not be safely identified. B. bruxellensis was the dominant species present in house wines, while non-Saccharomyces sp. were viable in aged wines of high alcohol content. CONCLUSIONS: Yeast population depends on postfermentation procedures or storage conditions. Although 5.8S-ITS rDNA sequence analysis is generally a rapid method to identify wine yeast isolates at the species level, or even below that, it may not be sufficient for some genera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to show that commercial wines may possess diverse and potentially harmful yeast populations. The knowledge of yeasts able to reside in this niche environment is essential towards integrated quality assurance programmes. For selected species, the 5.8S-ITS rDNA sequence analysis is a rapid and accurate means.     

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.     

A genetic study of natural flor strains of Saccharomyces cerevisiae isolated during biological ageing from Sardinian wines

In this study, three flor strains of Saccharomyces cerevisiae were genetically characterized. They were isolated from biofilms on Sardinian sherry-like wines produced at family-run wineries where pure cultures of yeasts were not used. The study aimed to investigate the life cycle of these naturally-occurring flor strains, using a genetic procedure supplemented by analysis of subsequent meiotic generations. A semi-homothallic life cycle was found in three strains that could be helpful in a genetic improvement programme.     

酿酒酵母中亚硫酸盐转运基因SSU1的分子进化分析

SSU1基因是涉及亚硫酸外排及SO₂耐受性的重要因素之一。为了研究酿酒酵母(Saccharomyce cerevisiae)中SSU1对SO₂耐受性及其分化机制, 文章探讨了SSU1基因在酿酒酵母中的遗传特征及进化规律。基于SSU1基因序列的聚类分析表明, 酿酒酵母群体可通过该基因分为3个亚群, 且与其分离的地理位置无关; 基于群体数据的McDonald-Kreitman 检验表明, SSU1基因在酿酒酵母中受到适应性选择的作用; Ka/Ks检验表明, 在酿酒酵母中, 不同的亚群间有Ka/Ks显著大于1 的值, 且PAML的支系模型检验到正选择作用在群体中特定的支系上; PAML的支系-位点模型检验获得9个潜在正选择作用位点, 其中有4个发生在受正选择作用的特定支系中; 基于ssu1p蛋白结构的分析表明, 在特定支系存在的正选择作用位点中, 除345(R/K)位点上两氨基酸替换均为碱性氨基酸外, 其他3个位点均是极性氨基酸/疏水性氨基酸之间替换, 考虑不同区域的氨基酸pKa值对其维持正常的功能有着重要的作用, 在该类位点的替换可能影响到ssu1p蛋白对SO₂的转运作用。     

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.     

一株分离于工业污水池的耐碱酵母

目的:从新疆温泉县一个碱性工业污水处理池中分离并鉴定耐碱酵母菌。方法:用稀释平板法分离菌种,通过形态学观察、生理生化特征及26S rDNA D1/D2区基因序列分析鉴定菌种。结果:从水样中分离得到一株耐碱酵母菌,它们能在pH3.5~11.0,12%NaCl,4~45℃生长,经形态观察及生理生化特征鉴定为酵母属,对其26S rDNA 5’端D1/D2区基因序列进行了PCR扩增并测序,GenBank注册号为DQ132884,同源序列分析结果表明该序列与酿酒酵母(Saccharomyces cerevisiae)Sb4有99.8%的同源性,因此将其命名为酿酒酵母(Saccharomyces cerevisiae)XJU-2,该菌种已保藏于中国微生物菌种保藏委员会普通微生物中心(CGM-CC),保藏号为CGMCC No.2.3095。结论:XJU-2的最高耐碱值可达pH 11.0,而且酸碱耐受范围很大,性能明显优于国内外已报道的酿酒酵母菌种。     

Peculiarities of flor strains adapted to Sardinian sherry-like wine ageing conditions

Saccharomyces cerevisiae flor yeasts, which are subjected to stressful conditions during wine ageing, exhibit a number of characteristics which distinguish them from non-flor S. cerevisiae wine strains. In the present work, 22 flor and 14 non-flor S. cerevisiae wine strains are compared, in order to elucidate other possible peculiarities of these yeasts. The results obtained demonstrate that in contrast to the homothallic nature of the non-flor strains, 77% of the flor strains exhibit two variants of a semi-homothallic life cycle. Moreover, the flor-forming ability is shown to be inversely correlated to spore viability and the utilisation of maltose and galactose.     

基于核内核糖体小亚基序列的蝗总科系统发育关系分析

用核糖体SSURdna全序列对蝗总科（Acridoidea）进行了分子系统学研究。依据测定的8种蝗虫的SSU Rdna全序列 (平均 1.844 bp),并从GenBank中选取了6种内群种类和2种外群种类的SSU Rdna同源序列,进行序列分析。利用Clustal、MEGA 和 PHYLIP 软件构建分子系统树（距离邻接法Neighbor-Joining,NJ;最小进化法 Minimum Evolution）。结果显示： (1) 蝗总科是一个单系类群;(2) 锥头蝗科（Chrotogonidae）和瘤锥蝗科（Pyrgomorphidea）亲缘关系较近,为蝗总科最原始的类群;(3) 网翅蝗科（Arcypteridae）和槌角蝗科（Gomphoceridae）有较近的亲缘关系; (4) 斑翅蝗科 （Oedipodidae）为最进化的类群; (5) SSU Rdna序列保守性强,转换transition）取代的速率大于或接近颠换（transversion）取代的速率;(6) 在系统树中,总科首先分离,大多数同科不同属的类群以高置信度聚合在一起,说明SSU Rdna序列适合用于蝗总科的系统发育关系分析。     

Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts

Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.     

FLO11 is essential for flor formation caused by the C-terminal deletion of NRG1 in Saccharomyces cerevisiae

The flor strains of Saccharomyces cerevisiae form a flor on the surface of wine after alcoholic fermentation. High hydrophobicity of the cell surface is suggested to be important for flor formation by the flor wine yeasts. However, the molecular mechanism of flor formation is not clear. We found that expression of C-terminal deleted NRG1 lacking its two C2H2 zinc finger motifs (NRG1(1-470)) on the multicopy plasmid conferred the ability to form a flor to a non-flor laboratory strain. The cell surface hydrophobicity of NRG1(1-470) was higher than of the non-flor strain. Disruption of the Nrg1p-repressed gene FLO11, which encodes a cell surface glycoprotein that functions as a flocculin or an adhesin, abolished flor formation. Moreover, expression of FLO11 on a multicopy plasmid could also cause flor formation. These results indicate that FLO11 is essential for flor formation by NRG1(1-470). In addition, the results suggest that the C-terminal truncated form of Nrg1p exerts a dominant negative effect on FLO11 repression, resulting in FLO11 expression and, thus, flor formation.     

Characterization of Ccw7p cell wall proteins and the encoding genes of Saccharomyces cerevisiae wine yeast strains: relevance for flor formation

The specific flavour of Sherry-type wines requires aromatic compounds produced as by-products of the oxidative metabolism of yeasts that are able to form a biofilm (flor) at the wine surface. A similar yeast pellicle develops on the surface of 'Tokaji Szamorodni', one of the traditional Hungarian botrytized wines, during maturation. In this work, patterns of biotinylated cell wall proteins extracted from film-forming and nonfilm-forming Saccharomyces cerevisiae strains were compared. It was found that all the tested 23 film-forming 'Szamorodni' yeast strains had a decreased size of the Ccw7/Hsp150 protein, one of the members of the Pir-protein family. Sequencing of the encoding genes revealed that the strains were lacking three out of the 11 repeating sequences characteristic to this protein family. One of the film-forming strains contained CCW7 alleles of different length, which was generated by intragenic tandem duplication of a sequence containing two repetitive domains. Unlike the film-forming strains, 16 nonfilm-forming wine yeasts isolated from a different botrytized wine, 'Tokaji Aszu', showed pronounced polymorphism of the CCW7 locus. It is highly probable that the modified Ccw7 protein does not contribute to the increased hydrophobicity of film-forming strains but it may influence molecular reorganization of the cell wall during stress adaptation.     

Phylogenetic utility of protein (RPB2, β-tubulin) and ribosomal (LSU,SSU) gene sequences in the systematics of Sordariomycetes (Ascomycota,Fungi)

The Sordariomycetes is an important group of fungi whose taxonomic relationships and classification is obscure. There is presently no multi-gene molecular phylogeny that addresses evolutionary relationships among different classes and orders. In this study, phylogenetic analyses with a broad taxon sampling of the Sordariomycetes were conducted to evaluate the utility of four gene regions (LSU rDNA, SSU rDNA, beta-tubulin and RPB2) for inferring evolutionary relationships at different taxonomic ranks. Single and multi-gene genealogies inferred from Bayesian and Maximum Parsimony analyses were compared in individual and combined datasets. At the subclass level, SSU rDNA phylogenies demonstrate their utility as a marker to infer phylogenetic relationships at higher levels. All analyses with SSU rDNA alone, combined LSU rDNA and SSU rDNA, and the combined 28 S rDNA, SSU rDNA and RPB2 datasets resulted in three subclasses: Hypocreomycetidae, Sordariomycetidae and Xylariomycetidae, which correspond well to established morphological classification schemes. At the ordinal level, the best resolved phylogeny was obtained from the combined LSU rDNA and SSU rDNA datasets. Individually, the RPB2 gene dataset resulted in significantly higher number of parsimony informative characters. Our results supported the recent separation of Boliniaceae, Chaetosphaeriaceae and Coniochaetaceae from Sordariales and placement of Coronophorales in Hypocreomycetidae. Microascales was found to be paraphyletic and Ceratocystis is phylogenetically associated to Faurelina, while Microascus and Petriella formed another clade and basal to other members of Halosphaeriales. In addition, the order Lulworthiales does not appear to fit in any of the three subclasses. Congruence between morphological and molecular classification schemes is discussed.     

Inhibition of malolactic fermentation by cryotolerant yeasts

White wines produced by some cryotolerant strains of Saccharomyces cerevisiae are more resistant to malolactic fermentation than those produced by normal strains: e.g. for two months of storage, the wines, inoculated with Leuconostoc oenos or Lactobacillus plantarum, were fully stabilized with levels of 51-65 mg total SO 2 /l and 5.70-5.75 g titratable acidity/l. The use of these yeasts in wine-making can decrease the quantities of sulfites added to stabilize wines.     

Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by real-time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 x 10(5) to 3.8 CFU/ml in sweet wine and from 5 x 10(6) to 50 CFU/ml in red wine.