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.     

The role of GAP1 gene in the nitrogen metabolism of Saccharomyces cerevisiae during wine fermentation

Aim:  The aim of this study was to analyse the relevance of the general amino acid permease gene ( GAP1 ) of the wine yeast Saccharomyces cerevisiae on nitrogen metabolism and fermentation performance.
Methods and Results:  We constructed a gap1 mutant in a wine strain. We compared fermentation rate, biomass production and nitrogen consumption between the gap1 mutant and its parental strain during fermentations with different nitrogen concentrations. The fermentation capacity of the gap1 mutant strain was impaired in the nitrogen-limited and -excessive conditions. The nitrogen consumption rate between the wild strain and the mutant was different for some amino acids, especially those affected by nitrogen catabolite repression (NCR). The deletion of GAP1 gene also modified the gene expression of other permeases.
Conclusions:  The Gap1 permease seems to be important during wine fermentations with low and high nitrogen content, not only because of its amino acid transporter role but also because of its function as an amino acid sensor.
Significance and Impact of the Study:  A possible biotechnological advantage of a gap1 mutant is its scarce consumption of arginine, whose metabolism has been related to the production of the carcinogenic ethyl carbamate.     

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.     

Saccharomyces cerevisiae biodiversity in spontaneous commercial fermentations of grape musts with 'adequate' and 'inadequate' assimilable-nitrogen content

AIM: To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content. METHODS AND RESULTS: Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity. CONCLUSIONS: The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.     

The vinification of partially dried grapes: a comparative fermentation study of Saccharomyces cerevisiae strains under high sugar stress

AIMS: The study of the fermentation performance of Saccharomyces cerevisiae strains under high sugar stress during the vinification of partially dried grapes. METHODS AND RESULTS: Microvinification of partially dried grape must with sugar concentration of 35 degrees Brix was performed using four commercial strains to carry out alcoholic fermentation. A traditional red vinification without nutrients addition was applied. Yeasts displayed different efficiency to convert sugar in ethanol and varied in glycerol yield. Sugar consumption and ethanol level were attested at 80-87% and 143.5-158.0 g l(-1) respectively. High correlation between sugar and assimilable nitrogen consumption rate was observed. Statistical treatment of data by principal component analysis highlighted the different behaviours that strains exhibited in regard to the production of higher alcohols and other compounds important to wine quality. CONCLUSIONS: Saccharomyces cerevisiae strains displayed appreciable capability to overcome osmotic stress and to yield ethanol fermenting high sugar concentration grape must in winemaking condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provided insights on the strain contribution to wine quality subordinate to stress condition. This investigation is of applicative interest for winemaking and processing industry that use high sugar concentration musts.     

Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations

In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response.     

Isolation and characterization of the HO gene from the yeast Saccharomyces paradoxus

A DNA fragment homologous to the homothallism (HO) gene of Saccharomyces cerevisiae was isolated from Saccharomyces paradoxus and was found to contain an open reading frame that was 90.9% identical to the coding sequence of the S. cerevisiae HO gene. The putative HO gene was shown to induce diploidization in a heterothallic haploid strain from S. cerevisiae. Phylogenetic analysis revealed that the coding and 5'-upstream regulatory regions from five Saccharomyces sensu stricto HO genes have coevolved, and that S. paradoxus is phylogenetically closer to S. cerevisiae than to S. bayanus. Finally, heterothallic haploid strains were isolated from the original homothallic type strain of S. paradoxus by disrupting the S. paradoxus HO gene with the S. cerevisiae URA3 gene.     

Enhanced Glycerol Content in Wines Made with Immobilized Candida stellata Cells

Screening tests carried out for 10 strains of Candida stellata confirmed high levels of glycerol production, although a low fermentation rate and reduced ethanol content were observed. To overcome the poor competition with Saccharomyces cerevisiae, fermentation tests with immobilized C. stellata cells, alone or in combination with S. cerevisiae, have been carried out. The immobilization of C. stellata cells consistently reduced the fermentation length when compared with that obtained with free cells, immobilized cells exhibiting about a 30-and a 2-fold improvement in fermentation rate compared with rates for C. stellata and S. cerevisiae free cells, respectively. Moreover, immobilized C. stellata cells produced a twofold increase in ethanol content and a strong reduction in acetaldehyde and acetoin production in comparison with levels for free cells. The evaluation of different combinations of C. stellata immobilized cells and S. cerevisiae showed interesting results with regard to analytical profiles for practical application in wine making. In fact, analytical profiles of combinations showed, apart from a high glycerol content, a reduction in the amounts of acetic acid and higher alcohols and a consistent increase in succinic acid content in comparison with values for the S. cerevisiae control strain. Sequential fermentation first with immobilized C. stellata cells and then after 3 days with an added inoculum of S. cerevisiae free cells was the best combination, producing 15.10 g of glycerol per liter, i.e., 136% more than the S. cerevisiae control strain produced. Fermentation with immobilized C. stellata cells could be an interesting process by which to enhance glycerol content in wine.     

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.     

Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics

We report the isolation of multiple strains of Saccharomyces cerevisiae and Saccharomyces paradoxus from a natural woodland site in southeastern Pennsylvania, USA, using enrichment culturing in a medium containing 7.6% (v/v) ethanol. The method was applied to bark and flux material collected from broad-leaved trees (mostly Quercus spp.) and to associated soils. Many candidate wild strains of Saccharomyces were isolated using this method, most of them from soils associated with oaks. Matings to genetically marked tester strains of S. cerevisiae and S. paradoxus identified roughly equal numbers of these two species within this collection. The S. paradoxus isolates showed significant partial reproductive isolation from a conspecific European strain, whereas the S. cerevisiae isolates did not. Variability in both chromosome size and Ty1 element hybridization profiles was observed within both populations at this site. We discuss the relevance of our data to current debates concerning whether S. cerevisiae is a wild species or a domesticated species.     

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations

AIM: To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. METHODS AND RESULTS: Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. CONCLUSIONS: Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.     

Assimilable nitrogen utilisation and production of volatile and non-volatile compounds in chemically defined medium by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeasts

Surveys conducted worldwide have shown that a significant proportion of grape musts are suboptimal for yeast nutrients, especially assimilable nitrogen. Nitrogen deficiencies are linked to slow and stuck fermentations and sulphidic off-flavour formation. Nitrogen supplementation of grape musts has become common practice; however, almost no information is available on the effects of nitrogen supplementation on wine flavour. In this study, the effect of ammonium supplementation of a synthetic medium over a wide range of nitrogen values on the production of volatile and non-volatile compounds by two high-nitrogen-demand wine fermentation strains of Saccharomyces cerevisiae was determined. To facilitate this investigation, a simplified chemically defined medium that resembles the nutrient composition of grape juice was used. Analysis of variance revealed that ammonium supplementation had significant effects on the concentration of residual sugar, L-malic acid, acetic acid and glycerol but not the ethanol concentration. While choice of yeast strain significantly affected half of the aroma compounds measured, nitrogen concentrations affected 23 compounds, including medium-chain alcohols and fatty acids and their esters. Principal component analysis showed that branched-chain fatty acids and their esters were associated with low nitrogen concentrations, whereas medium-chain fatty esters and acetic acid were associated with high nitrogen concentrations.     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

The effect of acetic acid bacteria upon the growth and metabolism of yeasts during the fermentation of grape juice

The influence of species of Acetobacter and Gluconobacter upon growth of the wine yeasts Saccharomyces cerevisiae, Kloeckera apiculata and Candida stellata was examined during mixed culture in grape juice. Acetobacter pasteurianus, A. aceti and Gluconobacter oxydans grew in conjunction with yeasts during juice fermentation. As determined by viable counts, yeast growth was only slightly impaired by the presence of bacteria. However, as judged by the concentrations of glucose, fructose, ethanol, glycerol, acetaldehyde, ethyl acetate, iso -amyl alcohol and organic acids in the fermented juice, acetic acid bacteria significantly influenced the alcoholic fermentation by yeasts.     

Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production.     

Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose–xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L⁻¹ acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L⁻¹) in all cultures. Xylose fermentation in acetic –acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.     

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.     

Determination of Growth and Glycerol Production Kinetics of a Wine Yeast Strain Saccharomyces cerevisiae Kalecik 1 in Different Substrate Media

Summary Glycerol has been known as an important by-product of wine fermentations improving the sensory quality of wine. This study was carried out with an endogenic wine yeast strain Saccharomyces cerevisiae Kalecik 1. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose in a batch system. Depending on the determined values of Monod constants, glucose (K_s = 28.09 g/l) was found as the most suitable substrate for the yeast growth. Initial glucose, fructose and sucrose concentrations necessary for maximum specific yeast growth rate were determined as 175 g, 100 l, and 200 g/l, respectively. The yeast produced glycerol at very high concentrations in fructose medium. Fructose was determined as the most suitable substrate for glycerol production while the strain showed low tendency to use it for growth. S. cerevisiae Kalecik 1 could not produce glycerol below 200 g/l initial sucrose concentration. When natural white grape juice was used as fermentation medium, maximum glycerol concentration and dry weight of the yeast were determined as 9.3 g/l and 11.8 g/l, respectively.     

过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性

木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。