Response of wine yeast (Saccharomyces cerevisiae) aldehyde dehydrogenases to acetaldehyde stress during Icewine fermentation

AIMS: We previously reported that the aldehyde dehydrogenase encoded by ALD3 but not ALD6 was responsible, in part, for the increased acetic acid found in Icewines based on the expression profile of these genes during fermentation. We have now completed the expression profile of the remaining yeast aldehyde dehydrogenase genes ALD2, ALD4 and ALD5 during these fermentations to determine their contribution to acetic acid production. The contribution of acetaldehyde stress as a signal to stimulate ALD expression during these fermentations was investigated for all ALD genes. The expression of glycerol-3-phosphate encoded by GPD2 was also followed during these fermentations to determine its role in addition to the role we already identified for GPD1 in the elevated glycerol produced during Icewine fermentation. METHODS AND RESULTS: Icewine juice (38.5 degrees Brix, 398 +/- 5 g l(-1) sugar), diluted Icewine juice (20.8 degrees Brix, 196 +/- 4 g l(-1) sugar) and the diluted juice with sugar levels equal to the original Icewine juice (36.6 degrees Brix, 395 +/- 6 g l(-1) sugar) were fermented in duplicate using the commercial wine yeast K1-V1116. Acetic acid and glycerol production increased 8.4- and 2.7-fold in the Icewine vs the diluted juice fermentation, respectively, accompanied by a fourfold transient increase in acetaldehyde in the Icewine condition during the first week. Both mitochondrial aldehyde dehydrogenases encoded by ALD4 and ALD5 were expressed, with ALD5 expression highest at the start of all fermentations and ALD4 expression increasing during the first week of each condition. ALD2, ALD4, ALD5 and GPD2 showed no differential expression between the three fermentation conditions indicating their lack of involvement in elevating acetic acid and glycerol in Icewine. When yeast fermenting the diluted fermentation was exposed to exogenous acetaldehyde, the transient spike in acetaldehyde increased the expression of ALD3 but this response alone was not sufficient to cause an increase in acetic acid. Expression of the other aldehyde dehydrogenases was unaffected by the acetaldehyde addition. CONCLUSIONS: The aldehyde dehydrogenases encoded by ALD2, ALD4 and ALD5 do not contribute to the elevated acetic acid production during Icewine fermentation. Expression of GPD2 was not upregulated in high sugar fermentations and does not reflect the elevated levels of glycerol found in these wines. Acetaldehyde at a concentration produced during Icewine fermentation stimulates the expression of ALD3, but has no impact on the expression of ALD2, -4, -5 and -6. Upregulation of ALD3 alone in the dilute fermentation is not sufficient to increase acetic acid in wine and requires additional responses found in cells under hyperosmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work confirms that increased acetic acid and glycerol production during Icewine fermentation follows upregulation of ALD3 and GPD1 respectively, but upregulation of ALD3 alone is not sufficient to increase acetic acid production. Additional responses of cells under osmotic stress are required to increase acetic acid in Icewine.     

絮凝基因FLO1及FLO1c高表达提高工业酿酒酵母乙酸耐受性及发酵性能

乙酸是生物质乙醇发酵过程中酵母细胞面临的重要抑制剂之一,对细胞生长及发酵性能有强烈的抑制作用。增强酵母菌对乙酸胁迫的耐受性对提高乙醇产率具有重要意义。用分别带有完整絮凝基因FLO1及其重复序列单元C发生缺失的衍生基因FLO1c的重组表达质粒分别转化非絮凝型工业酿酒酵母CE6,获得絮凝型重组酵母菌株6-AF1和6-AF1c。同时以空载体p YCPGA1转化CE6的菌株CE6-V为对照菌株。与CE6-V相比,絮凝酵母明显提高了对乙酸胁迫的耐受性。在0.6%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.56倍和1.62倍;在1.0%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.21倍和1.78倍。可见絮凝能力改造能明显提高工业酿酒酵母的乙酸胁迫耐受性及发酵性能,而且FLO1内重复序列单元C缺失具有更加明显的效果。     

A comparative study of the wine fermentation performance of Saccharomyces paradoxus under different nitrogen concentrations and glucose/fructose ratios

Aims:  The main goal of the present study is to determine the effects of different nitrogen concentrations and glucose/fructose ratios on the fermentation performance of Saccharomyces paradoxus , a nonconventional species used for winemaking.
Methods and Results:  Ethanol yield, residual sugar concentration, as well as glycerol and acetic acid production were determined for diverse wine fermentations conducted by S. paradoxus . Experiments were also carried out with a commercial Saccharomyces cerevisiae wine strain used as control. The values obtained were compared to test significant differences by means of a factorial anova and the Scheffé test. Our results show that S. paradoxus strain was able to complete the fermentation even in the nonoptimal conditions of low nitrogen content and high fructose concentration. In addition, the S. paradoxus strain showed significant higher glycerol synthesis and lower acetic acid production than S. cerevisiae in media enriched with nitrogen, as well as a lower, but not significant, ethanol yield.
Conclusions:  The response of S. paradoxus was different with respect to the commercial S. cerevisiae strain, especially to glycerol and acetic acid synthesis.
Significance and Impact of the Study:  The present study has an important implication for the implementation of S. paradoxus strains as new wine yeast starters exhibiting interesting enological properties.     

Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana

AIM: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. METHODS AND RESUlts: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3.5-4.0%, 0.1-0.3% and 0.2-0.4% respectively, whilst the alcohol contents of samples collected within the day were between 1.4% and 2.82%; palm wine which had accumulated over night, 3.24% to 4.75%; and palm wine held for 24 h, over 7.0%. CONCLUSION: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and influence the composition of the product.     

Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains

While unfermented grape must contains approximately equal amounts of the two hexoses glucose and fructose, wine producers worldwide often have to contend with high residual fructose levels (>2 gl(-1)) that may account for undesirable sweetness in finished dry wine. Here, we investigate the fermentation kinetics of glucose and fructose and the influence of certain environmental parameters on hexose utilisation by wine yeast. Seventeen Saccharomyces cerevisiae strains, including commercial wine yeast strains, were evaluated in laboratory-scale wine fermentations using natural Colombard grape must that contained similar amounts of glucose and fructose (approximately 110 gl(-1) each). All strains showed preference for glucose, but to varying degrees. The discrepancy between glucose and fructose utilisation increased during the course of fermentation in a strain-dependent manner. We ranked the S. cerevisiae strains according to their rate of increase in GF discrepancy and we showed that this rate of increase is not correlated with the fermentation capacity of the strains. We also investigated the effect of ethanol and nitrogen addition on hexose utilisation during wine fermentation in both natural and synthetic grape must. Addition of ethanol had a stronger inhibitory effect on fructose than on glucose utilisation. Supplementation of must with assimilable nitrogen stimulated fructose utilisation more than glucose utilisation. These results show that the discrepancy between glucose and fructose utilisation during fermentation is not a fixed parameter but is dependent on the inherent properties of the yeast strain and on the external conditions.     

过表达tRNA基因tL(CAA)K提高酿酒酵母乙酸耐受性

乙酸是木质纤维素类生物质水解液中的常见毒性抑制物,选育乙酸耐受性好的酿酒酵母菌株,有利于高效利用木质纤维素类生物质,发酵生产生物燃料和生物基化学品.目前对酿酒酵母抗逆性的研究多集中在转录水平,但对转运RNA (Transfer RNA,tRNA)在耐受性中的作用研究较少.在对酿酒酵母抗逆性研究过程中发现,一些转运RNA...     

Biochemical study of idebenone effect on mitochondrial metabolism of yeast

The aim of this work was to study the effect of the drug idebenone on the growth of a strain of Saccharomyces cerevisiae yeast and its respiratory-deficient mutant (rho(0)). We took this yeast as a model system of the interaction of the drug with mammalian cells. The effect of idebenone was evaluated in rich and minimal media. In the S288c strain, idebenone exerted a growth inhibitory effect in concentrations higher than 50 microM in media containing a carbon source consumed at mitochondrial level. In conditions of low oxygen supply, idebenone allows yeast to keep a cellular yielding comparable with conditions of normal oxygen supply. Also, the presence of idebenone in the growth media increased by 50% the fluorescence signal of rhodamine 123, indicating a higher mitochondrial membrane potential. The results could explain the effect of idebenone in the treatment of diseases in which oxygen deficiency alters the energetic metabolism of the cell.     

葡萄酒酿酒酵母嗜果糖特性的评价方法

[目的]酿酒酵母的嗜果糖性是葡萄酒酵母选育工作的一项重要内容.建立评价菌体发酵果糖能力的方法,是葡萄酒酿酒酵母嗜果糖性研究的基础.[方法]以3株不同果糖发酵能力的酵母菌为研究对象,考察菌体在模拟葡萄汁培养基条件下,发酵情况与单糖利用之间的关系;并通过数学方程拟合单糖动力发酵曲线,得到发酵持续时间、葡萄糖浓度拟为0时的果糖浓度、果糖与葡萄糖曲线面积的差值等参数.[结果]这些参数可以反应出菌体的发酵速率和嗜果糖性.其中后两个参数能显著将3个菌株的嗜果糖特性区分开.[结论]为高果糖利周优良葡萄酒酿酒酵母菌株的筛选和构建,提供了较为全面、客观和有效的评价方法.     

Production of ethanol from guava pulp by yeast strains

Guava pulp used for ethanol production by three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production at the optimum sugar concentration of 10%, at pH 4.1 and 30°C was 1.5%, 3.6% and 3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2, respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were inhibitory for ethanol production by all test cultures. The maximum production of ethanol at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0 by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and 60h fermentation, respectively.     

Influence of yeast quality on performance of gnotobiotically grown Artemia

Using axenically grown Artemia, a model system was developed to evaluate the effect of bacteria on the survival and development of this crustacean. Two strains of baker's yeast (Saccharomyces cerevisiae) were used in all experiments as feed for Artemia: a wild-type strain and its mnn9 mutant, defective in the synthesis of mannoproteins in the outer cell wall. The genetic background, yeast growth phase and growth medium appeared to be important parameters determining the quality of yeast cells as feed for Artemia. A strong positive correlation between Artemia performance and the yeast cell wall chitin and glucan content was obtained, while the mannoprotein content was negatively correlated. Mnn9 yeast cells grown till exponential phase in minimal medium proved to be excellent feed for Artemia, yielding an average 95% survival and 4-mm growth after 6 days at 28 °C, which is comparable to the best results obtained with algal feed. The standard growth test yields highly reproducible results and can become an excellent tool to study the mode of action of bacteria. Furthermore, yeast cell viability and the method used to kill/sterilize the cells are important parameters influencing nauplii performance.     

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206

Saccharomyces cerevisiae T206 K⁺R⁺, a K₂ killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K^-R^- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.     

酵母及与藻类搭配对萼花臂尾轮虫饵料效果的研究

研究了２种酵母单独投喂萼花臂尾轮虫的最适密度、饵料效果及与藻类搭配的投喂效果．结果表明,在５种密度下,面包酵母和啤酒酵母的最适密度分别为１０和５×１０６个·ｍｌ－１．用面包酵母和啤酒酵母培养轮虫所得到的种群密度和瞬时增长率分别是用蛋白核小球藻培养的４０．２％、２６．０％和８５．５％、７６．６％．用面包酵母和蛋白核小球藻适当比例搭配投喂轮虫,其效果接近或超过单一用小球藻在最适密度下的培养效果．     

Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.     

The effect of pyrimethanil on the growth of wine yeasts

Aims:  The toxicity of the fungicide pyrimethanil on the growth of wine yeasts was evaluated using in vivo and in vitro experimentation.
Methods and Results:  The effect of pyrimethanil in the must was studied during the spontaneous wine fermentation of three consecutive vintages and by the cultivation of Hanseniaspora uvarum and Saccharomyces cerevisiae yeasts in a liquid medium. The residues of the fungicide were measured using gas chromatography-mass spectrometry system and the sugar concentration in the must using HPLC-RI. Molecular and standard methods were used for identifying the yeast species. Although the pyrimethanil residues in grapes were below the maximum residue limits, they significantly affected the reduced utilization of sugars in the first days of fermentation. Its residues controlled the growth of H. uvarum during the fermentation and during in vitro cultivation as well.
Conclusions:  The fungicide pyrimethanil had an effect on the course and successful conclusion of spontaneous wine fermentation that was correlated with the initial concentration of yeasts in the must.
Significance and Impact of the Study:  The impact of pyrimethanil on the indigenous mixed yeast flora in fermenting must was investigated for the first time. The results showed that its residues might play an important role in the growth and succession of yeast during spontaneous wine fermentation.