Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

The timing of diammonium phosphate supplementation of wine must affects subsequent H2S release during fermentation

Aims:  The aim of this study was to evaluate the impact of supplementation by diammonium phosphate (DAP) on hydrogen sulfide (H₂S) production, when DAP given either prior to fermentation or during the early stationary growth phase of yeast. Methods and Results:  Three contrasting Saccharomyces cerevisiae wine strains were used to ferment synthetic grape juice (GJ) containing 67 mg l^?1 of initial yeast assimilable nitrogen (YAN), supplied either as DAP or as mixture of amino acids. Sufficient DAP was added either prior to or 72 h after the initiation of fermentation to achieve a final YAN concentration of 267 mg l^?1. Supplementation prior to fermentation stimulated H₂S production. The results obtained in model solutions were validated using natural GJ. Conclusion:  The timing of DAP supplementation is critical for ensuring that fermentation proceeds without excessive release of H₂S. Significance and Impact of the Study:  This result has important implications for the wine‐making industry, because it highlights the value of determining the initial nitrogen level of a GJ. It raises awareness of the dependence of wine quality on the correct timing of DAP supplementation.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Occurrence of hydrogen sulfide in wine and in fermentation: influence of yeast strain and supplementation of yeast available nitrogen

Hydrogen sulfide (H₂S) is a powerful aroma compound largely produced by yeast during fermentation. Its occurrence in wines and other fermented beverages has been associated with off-odors described as rotten egg and/or sewage. While the formation of hydrogen sulfide (H₂S) during fermentation has been extensively studied, it is the final H₂S content of wine that is actually linked to potential off-odors. Nevertheless, factors determining final H₂S content of wine have received little attention, and it is commonly assumed that high H₂S-forming fermentations will result in high final concentrations of H₂S. However, a clear relationship has never been established. In this report, we investigated the contribution of yeast strain and nitrogen addition to H₂S formation during fermentation and its consequent occurrence the resulting wines. Five commercial Saccharomyces cerevisiae wine yeast strains were used to ferment a Chardonnay juice containing 110 mg/l of YAN (yeast assimilable nitrogen), supplemented with di-ammonium phosphate (DAP) to increase YAN concentration to moderate (260 mg/l) and high (410 mg/l) levels. In contrast to the widely reported decrease in H₂S production in response to DAP addition, a non-linear relationship was found such that moderate DAP supplementation resulted in a remarkable increase in H₂S formation by each of the five wine yeasts. H₂S content of the finished wine was affected by yeast strain, YAN, and fermentation vigor. However, we did not observe a correlation between concentration of H₂S in the finished wines and H₂S produced during fermentation, with low-forming fermentations often having relatively high final H₂S and vice versa. Management of H₂S in wine through nitrogen supplementation requires knowledge of initial YAN and yeast H₂S characteristics.     

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.     

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.     

Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.     

Crabtree effect in aerobic fermentations using grape juice for the production of alcohol reduced wine

Summary Aerobic fermentations of grape juice to alcohol reduced wine were carried out by technical strains of wine yeast (S. cerevisiae var. ellipsoideus) at a temperature of 25 °C and an aeration rate of 1 vvm using a two-stage batch and fed-batch process. In the fed-batch phase of each fermentation Crabtree Effect [CE] limits between 0.2 and 0.5 g glucose/L have been detected.     

Biotechnological impact of stress response on wine yeast

Wine yeast deals with many stress conditions during its biotechnological use. Biomass production and its dehydration produce major oxidative stress, while hyperosmotic shock, ethanol toxicity and starvation are relevant during grape juice fermentation. Most stress response mechanisms described in laboratory strains of Saccharomyces cerevisiae are useful for understanding the molecular machinery devoted to deal with harsh conditions during industrial wine yeast uses. However, the particularities of these strains themselves, and the media and conditions employed, need to be specifically looked at when studying protection mechanisms.     

The effect of Debina grapevine indigenous yeast strains of <Emphasis Type="Italic">Metschnikowia</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> on wine flavour

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

添加芦荟汁对红葡萄酒发酵的抑菌效果

二氧化硫（SO₂）危害人体健康。为了寻找用于葡萄酒酿造的SO₂替代品,在赤霞珠葡萄酒自然发酵和接种发酵中比较了添加芦荟汁（80 g/L）与SO₂（60 mg/L）的抑菌和发酵效果。结果表明,添加芦荟汁促进接种酵母和内源酵母生长,SO₂抑制内源酵母生长,接种酵母、添加芦荟汁和添加SO₂显著抑制细菌和霉菌生长,SO₂比芦荟汁对细菌和霉菌生长的抑制作用大。添加芦荟汁和添加SO₂同样提高乙醇产量和减小酸度下降,降低葡萄酒的残糖量和褐变度。添加芦荟汁提高了挥发性酸度和高级醇产量,降低了接种发酵的葡萄酒色度和提高乙酸产量,降低了自然发酵的乙醛产量,而SO₂作用相反。添加芦荟汁具有减少SO₂用量的可能性。     

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...     

Identification and characterization of yeasts in brem, a traditional Balinese rice wine

Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer (ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae(35 strains), Candida glabrata(six strains), Pichia anomala(three strains) and Issatchenkia orientalis(seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Production of higher alcohols,ethyl acetate,acetaldehyde and other compounds by 14Saccharomyces cerevisiae wine strains isolated from the same region (Salnés,N.W. Spain)

Fourteen strains of the yeastSaccharomyces cerevisiae were isolated from three wineries in the Salnés wine region (N.W. Spain) at the three different periods of the natural fermentation. Each wild yeast was screened for production of acetaldehyde, ethyl acetate, isobutanol,n-propanol, amylic alcohol and other important enological compounds during laboratory scale fermentations of grape juice. After 25 days at 20°C, the analytical results evidenced variations in the production of acetaldehyde (from 13.1 to 24.3 mg/l), isobutanol (from 27.7 to 51.1 mg/l), amyl alcohols (from 111 to 183 mg/l) and ethyl acetate (from 19.3 to 43.7 mg/l). Although isolated from the same wine region, differences in the wine composition were observed depending on the particular yeast strain used for the vinification experiments.     

A novel methodology independent of fermentation rate for assessment of the fructophilic character of wine yeast strains

The yeast Saccharomyces cerevisiae has a fundamental role in fermenting grape juice to wine. During alcoholic fermentation its catabolic activity converts sugars (which in grape juice are a near equal ratio of glucose and fructose) and other grape compounds into ethanol, carbon dioxide and sensorily important metabolites. However, S. cerevisiae typically utilises glucose and fructose with different efficiency: glucose is preferred and is consumed at a higher rate than fructose. This results in an increasing difference between the concentrations of glucose and fructose during fermentation. In this study 20 commercially available strains were investigated to determine their relative abilities to utilise glucose and fructose. Parameters measured included fermentation duration and the kinetics of utilisation of fructose when supplied as sole carbon source or in an equimolar mix with glucose. The data were then analysed using mathematical calculations in an effort to identify fermentation attributes which were indicative of overall fructose utilisation and fermentation performance. Fermentation durations ranged from 74.6 to over 150 h, with clear differences in the degree to which glucose utilisation was preferential. Given this variability we sought to gain a more holistic indication of strain performance that was independent of fermentation rate and therefore utilized the area under the curve (AUC) of fermentation of individual or combined sugars. In this way it was possible to rank the 20 strains for their ability to consume fructose relative to glucose. Moreover, it was shown that fermentations performed in media containing fructose as sole carbon source did not predict the fructophilicity of strains in wine-like conditions (equimolar mixture of glucose and fructose). This work provides important information for programs which seek to generate strains that are faster or more reliable fermenters.     

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.     

Fermentation characteristics of hybrids between the cryophilic wine yeast Saccharomyces bayanus and the mesophilic wine yeast Saccharomyces cerevisiae

The cryophilic wine yeasts Saccharomyces bayanus YM-84 and YM-126 were used for hybridization with the mesophilic wine yeast Saccharomyces cerevisiae OC-2. All six hybrids were stable in tetrad analysis and pulsed field gel electrophoresis, even after twenty subcultures over two years. The fermentabilities of these hybrids at a low temperature of 7°C were superior to the mesophilic wine yeast and the same as the cryophilic wine yeasts. Conversely, their fermentabilities at the intermediate temperatures of 28 and 35°C were similar to the mesophilic wine yeast. For laboratory-scale wine-making using Koshu grape juice at 10°C, the fermentability of these hybrids was superior to the mesophilic wine yeast. They also produced higher amounts of malic acid and flavor compounds such as higher alcohols, β-phenylethyl alcohol, isoamyl acetate and β-phenylethyl acetate, and lower amounts of acetic acid than those of OC-2. These results suggest that the cryophilic wine yeast S. bayanus is useful for improving the low temperature fermentation ability of wine yeast strains.