Indigenous yeast population from Georgian aged wines produced by traditional “Kakhetian” method

The yeast microbiota present in wines produced by the ancient “Kakhetian” method in Georgia (EU) was studied. This technique involves the use of terracotta vessels (amphoras), during spontaneous fermentation, maceration phase and wine ageing. The analysed yeasts were collected from wines after maturation for one year in ten amphoras from a Georgian winery. The 260 isolates were all identified as Saccharomyces cerevisiae, and the majority were classified as flor yeasts by restriction analysis of ITS region. A first technological and molecular screening was used to select 70 strains for further characterization. Both genetic and metabolic characterization discriminated flor from non-flor strains. The combined results obtained by analysis of interdelta region and mtDNA-RFLP yielded 23 different biotypes; no biotype was common to flor and non-flor strains. The wines produced by flor yeasts showed a high content in acetaldehyde, acetic acid, acetoin, whereas the level of other compounds was similar to wines obtained by non-flor strains. This study represents the first report on the composition of yeast microbiota involved in the maturation of this traditional wine. These flor strains represent an interesting yeast population, in possession of peculiar characteristics allowing them to survive during wine ageing, becoming the dominant flora in the final wine.     

Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex

Along the fermentation process yeasts are affected by a succession of stress conditions that affect their viability and fermentation efficiency. Among the stress conditions the most relevant are high sugar concentration and low pH in musts, temperature and, as fermentation progresses, ethanol accumulation. Nowadays, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing search for particular wine yeast strains possessing a wide range of optimized, improved or novel enological characteristics. Traditionally, the species S. cerevisiae and S. bayanus within the Saccharomyces sensu stricto species are considered some of the most important yeast species involved in fermentation processes. However, in the last years, hybrid strains between the species S. cerevisiae, S. bayanus and S. kudriavzevii have been described as yeasts conducting the alcoholic fermentations and some of them are commercially available. Our results indicate that yeasts in the Saccharomyces sensu stricto complex were not affected by low pH or high glucose content in the media; however temperature and ethanol concentration variables appreciably affected their growth. The strains pertaining to S. cerevisiae were able to tolerate high temperature stress, whereas strains within S. bayanus and S. kudriavzevii were better adapted to growth at lower temperatures. Regarding to alcohol tolerance, S. cerevisiae is tolerating alcohol better than S. bayanus or S. kudriavzevii. Surprisingly, the natural hybrids between these species have adapted to growth under ethanol and temperature stress by inheriting competitive traits from one or another parental species. These results open new perspectives in the construction of new hybrid strains with biotechnological interest, as the characteristics of the parents may result in interesting combinations in the hybrids.     

啤酒酵母乙醛代谢关键酶相关性研究

乙醛是啤酒中的主要风味物质,其代谢主要来自酵母细胞。酵母中乙醇脱氢酶及乙醛脱氢酶是乙醛代谢的关键酶,对乙醛变化起着重要作用。跟踪啤酒酵母发酵过程中相对酶活力及乙醛变化,发现两种乙醇脱氢酶和乙醛脱氢酶的相对酶活力与发酵过程乙醛含量变化具有一定相关性。同时对低产乙醛啤酒酿酒酵母kb2-4与出发菌株啤酒酵母kb进行发酵试验,跟踪检测相对酶活力及乙醛含量,其乙醇脱氢酶Ⅰ和乙醇脱氢酶Ⅱ及乙醛脱氢酶相对酶活力均高于出发菌株,平均增幅分别为15.5%,11.6%和5%。3种酶活性的变化协同作用可以使乙醛含量降幅最大为33.8%。     

Release of macromolecules by Saccharomyces cerevisiae during ageing of French flor sherry wine "Vin jaune"

The French flor sherry wine "Vin jaune" spends 6 years and 3 months in the same barrel under a yeast velum. Because of temperature variations in the cellars, this velum sinks partially into the wine and a deposit of dead yeasts cells accumulates in the bottom of the barrels, favouring the formation of new velum. Growth and autolysis occur simultaneously. This study investigated the evolution of macromolecules released by yeasts during the ageing of "Vin jaune" in a model system closely simulating winemaking. It was observed that the release of macromolecules during the formation of the velums by living yeasts was low but greatly increased when the velums fell and yeast viability decreased. The release of macromolecules was then due to the autolysis of dead cells. Analysis of macromolecules during ageing revealed that they contained 73.3-78.5% neutral sugars and 6-7% proteins according to the ageing stage. Their amino acid composition did not change during ageing. A high content of serine and threonine commonly involved in O-glycosidic linkages present in yeast mannoproteins was observed. Throughout ageing, the mannose and glucose contents of macromolecules increased but the ratio of polymeric mannose to glucose decreased. Size exclusion chromatography showed that mannoproteins released in wine were partially hydrolysed by yeast beta-1,3-glucanases freed in wine.     

Tolerance mechanism of the ethanol-tolerant mutant of sake yeast

Several ethanol-tolerant mutants have been bred from industrial sake yeasts, but the mechanism of ethanol tolerance in these mutants has not been elucidated. After the determination of the entire genome sequence of Saccharomyces cerevisiae, various methods to monitor the whole-gene expression of the yeast have been developed. In this study, we used a commercially available nylon membrane on which virtually every gene of S. cerevisiae was spotted to compare expression profiles between the ethanol-tolerant mutant and its parent sake yeast to investigate the mechanism of ethanol tolerance in this mutant. As a result, we found that several genes were highly expressed only in the ethanol-tolerant mutant but not in the parent strain. These genes were known to be induced in cells that were exposed to various stresses, such as ethanol, heat, and high osmolarity, or at the stationary-phase but not at the log-phase. In the ethanol-tolerant mutant, the expression level of these stress-responsive genes was further increased after exposure to ethanol. We also found that substances such as catalase, glycerol and trehalose that may have protective roles under stressful conditions were accumulated in high amounts in the ethanol-tolerant mutant. The ethanol-tolerant mutant also exhibited resistance to other stresses including heat, high osmolarity and oxidative stress in addition to ethanol tolerance. These results indicate that the mutant exhibits multiple stress tolerance because of elevated expression of stress-responsive genes, resulting in accumulation of stress protective substances.     

Functional genomics of commercial baker's yeasts that have different abilities for sugar utilization and high-sucrose tolerance under different sugar conditions

In the modern baking industry, high-sucrose-tolerant (HS) and maltose-utilizing (LS) yeast were developed using breeding techniques and are now used commercially. Sugar utilization and high-sucrose tolerance differ significantly between HS and LS yeasts. We analysed the gene expression profiles of HS and LS yeasts under different sucrose conditions in order to determine their basic physiology. Two-way hierarchical clustering was performed to obtain the overall patterns of gene expression. The clustering clearly showed that the gene expression patterns of LS yeast differed from those of HS yeast. Quality threshold clustering was used to identify the gene clusters containing upregulated genes (cluster 1) and downregulated genes (cluster 2) under high-sucrose conditions. Clusters 1 and 2 contained numerous genes involved in carbon and nitrogen metabolism, respectively. The expression level of the genes involved in the metabolism of glycerol and trehalose, which are known to be osmoprotectants, in LS yeast was higher than that in HS yeast under sucrose concentrations of 5-40%. No clear correlation was found between the expression level of the genes involved in the biosynthesis of the osmoprotectants and the intracellular contents of the osmoprotectants. The present gene expression data were compared with data previously reported in a comprehensive analysis of a gene deletion strain collection. Welch's t-test for this comparison showed that the relative growth rates of the deletion strains whose deletion occurred in genes belonging to cluster 1 were significantly higher than the average growth rates of all deletion strains.     

Acetaldehyde tolerance in Saccharomyces cerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis

To identify genes responsible for acetaldehyde tolerance, genome‐wide screening was performed using a collection of haploid Saccharomyces cerevisiae strains deleted in single genes. The screen identified 49 genes whose deletion conferred acetaldehyde sensitivity, and these were termed the genes required for acetaldehyde tolerance. We focused on six of these genes required for acetaldehyde tolerance, ZWF1, GND1, RPE1, TKL1 and TAL1, which encode enzymes in the pentose phosphate pathway (PPP), and OAR1, which encodes for NADPH‐dependent 3‐oxoacyl‐(acyl‐carrier‐protein) reductase. These genes were not only responsible for acetaldehyde tolerance but also turned out to be induced by acetaldehyde. Moreover, the content of oleic acid was remarkably increased in yeast cells under acetaldehyde stress, and supplementation of oleic acid into the media partially alleviated acetaldehyde stress‐induced growth inhibition of strains disrupted in the genes required for acetaldehyde tolerance and OLE1. Taken together, our data suggest that the supply of NADPH and the process of fatty acid biosynthesis are the key factors in acetaldehyde tolerance in the yeast, and that oleic acid plays an important role in acetaldehyde tolerance. Copyright © 2008 John Wiley & Sons, Ltd.     

Relationship between ethanol tolerance, H+ -ATPase activity and the lipid composition of the plasma membrane in different wine yeast strains

Ethanol tolerance, ATPase activity and the lipid composition of the plasma membrane to study potential relationship among them were examined in five different wine yeast strains. Yeast cells were subjected to ethanol stress (4% v/v). Principal component analysis of the results revealed that the wine yeasts studied can be distinguished in terms of ATPase activity and oleic acid (C18:1), and palmitoleic acid (C16:1), in plasma membrane. Multiple regression analysis was used to identify a potential influence of some components of the plasma membrane on ethanol tolerance and ATPase activity. Based on the results, the ergosterol, oleic acid and palmitoleic acid are highly correlated with ATPase activity and ethanol tolerance. Ethanol tolerance and the ATPase activity of the plasma membrane were correlated at the 96.64% level with the oleic acid and ergosterol in plasma membrane. The Saccharomyces cerevisiae var. capensis flor yeast strain, which exhibited the highest ergosterol concentration in plasma membrane when grown in the presence of 4% v/v ethanol, was found to be the most ethanol-tolerant.     

Aroma series as fingerprints for biological ageing in fino sherry‐type wines

BACKGROUND: The contribution of flor yeasts and wood to the ageing of fino sherry type wines was studied by using capillary column gas chromatography to analyse 72 aroma compounds in wines subjected to biological ageing. The odour activity values (OAVs) for the different compounds were classified into eight odorant series that describe the aroma profile of these wines (fruity, chemical, balsamic, vegetable, fatty, empyreumatic, floral and spicy). RESULTS: The fruity samples made up the major series followed by the fatty and spicy at the end of the ageing period. The application of a linear regression model to the OAVs for the different series revealed that the fruity series, which is related to the flor yeasts activity, and the spicy series, which includes the compounds released by wood, are those most closely representing the changes in the aroma of these wines during their ageing. CONCLUSIONS: The comparison of the OAVs for fruity and spicy series along the ageing period can be used to establish a balance between the aroma compounds contributed by the flor yeasts and those extracted from the wood. This balance should be particularly considered in works aimed at shortening the ageing time for these wines. Copyright © 2007 Society of Chemical Industry     

酵母及其管理对乙醛的影响

研究了酵母及其管理对发酵末期乙醛含量的影响。酵母菌株对发酵后期乙醛的含量影响很大。上面酵母比下面酵母的乙醛含量低。不同代数的酵母产乙醛能力不一样,零代酵母的乙醛含量最高,一代酵母的乙醛含量最低。不当的酵母处理方式会导致种酵母的肝糖含量下降和乙醛含量的上升。     

酿酒酵母工业菌株胁迫条件耐受性分析

对酿酒酵母（Saccharomyces cerevisiae）工业菌株胁迫条件，包括高浓度酒精、高渗透压、高温、营养饥饿、氧化胁迫、糠醛毒性的耐受性进行了分析，同时测定了抗生素G418对这些菌株的最低抑菌浓度。结果表明，所测定的酵母菌株对这些逆境条件的耐受性有明显差别，表现出良好耐受性的是6508和安琪酵母菌株，同时多倍性的酿酒酵母工业菌株的耐受性均比单倍性实验室菌株高。     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of <Emphasis Type="Italic">Zygosaccharomyces mellis</Emphasis> strains in stored honey and their stress tolerance

To screen yeast with high sugar tolerance and evaluate their stress tolerance, six yeast strains were selected from 17 stored honey samples. The species were identified through 26S rRNA sequencing. Their stress tolerance was determined via the Durham fermentation method and ethanol production ability was determined via flask fermentation. The results demonstrated that all the six strains were Zygosaccharomyces mellis. Their sugar, ethanol, and acid tolerance ranges were 500–700 g/L, 10–12% (v/v), and pH 2.5–4.5, respectively. The SO₂ tolerance was 250 mg/L. Among the six strains, 6-7431 had the best stress tolerance with sugar tolerance of 700 g/L, ethanol tolerance of 12% (v/v), and acid tolerance of pH 2.5. Furthermore, the strain of 6-7431 had the highest percentage of ethanol production at the same initial sugar content as the other strains. Therefore, the selected six yeast strains would be promising fermentation yeasts for wine-making, ethanol production, or other fermentation purposes.     

耐高糖酵母筛选及其高糖胁迫机制的研究进展

耐高糖酵母菌在浓醪酒精发酵过程中起着重要的作用。目前,国内外对耐高酒精度酵母菌有较为广泛深入的研究,而对耐高糖酵母菌的研究还尚有不足。该文介绍了耐高糖酵母的筛选及其高糖胁迫应急机制的研究进展,并对耐高糖酵母的发展趋势进行展望,指出酒精发酵所需酵母菌应生理耐受性较好,能耐高糖度、高酒度、高渗压等,能够抵抗极端不良环境,在工业生产中能够有效提高乙醇的产量。通过筛选耐高糖酵母菌,旨在为酵母菌高糖胁迫机理的研究提供参考。