Relations between elevated temperatures and fermentation behaviour of Kloeckera apiculata and Saccharomyces cerevisiae associated with winemaking in mixed cultures

One of the important factors affecting wine fermentation is temperature. The influence of elevated temperatures from 10 to 25 °C at 5 °C intervals on yeast growth and fermentation products were studied in mixed cultures of Kloeckera apiculata and Saccharomyces cerevisiae in grape juice. In the experiments carried out at 10 and 15 °C, K. apiculata grew and survived longer compared to trials conducted above 20 °C. In most cases, higher temperatures stimulated the production of higher alcohols but lowered the formation of esters and acetaldehyde. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterization of a <Emphasis Type="Italic">Mastigocladus</Emphasis> species capable of growth,N<Subscript>2</Subscript>-fixation and N-assimilation at elevated temperature

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2–3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2–3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO₃ ⁻ and NH₄ ⁺, respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO₃ ⁻-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH₄ ⁺)-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.     

Heat shock on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> inoculum increases glycerol production in wine fermentation

A heat shock was applied to Saccharomyces cerevisiae: a change from 18°C to 45°C over 5 min and then maintenance at later temperature for 20 min followed by cooling to 18°C. Such a treated inoculum, when used in an alcoholic fermentation of Welsch Riesling grape must at 18°C, gave up to 12 g glycerol l⁻¹ This is a new and easy method for high glycerol production in large scale wine production.     

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.     

Sequential Use of Nitrogen Compounds by Saccharomyces cerevisiae during Wine Fermentation: a Model Based on Kinetic and Regulation Characteristics of Nitrogen Permeases

The efficiency of nitrogen use is a key determinant of the completion of alcoholic fermentation. We analyzed the kinetics of consumption of 18 nitrogen compounds by 14 Saccharomyces cerevisiae strains of various origins in a synthetic medium that mimicked a grape must. The kinetic profiles of total nitrogen consumption were diverse, but the order of nitrogen source consumption was similar for all strains. The nitrogen compounds could be classified into three groups, according to their order of use: prematurely consumed (Lys), early consumed (Asp, Thr, Glu, Leu, His, Met, Ile, Ser, Gln, and Phe), and late consumed (ammonium, Val, Arg, Ala, Trp, and Tyr). The initial concentrations of these compounds did not alter the order in which they were consumed, except for arginine and ammonium. Early consumed amino acids are transported by specific permeases under Ssy1p-Ptr3p-Ssy5 (SPS)-mediated control that are expressed at the beginning of consumption. Most nitrogen compounds consumed late are transported by permeases under nitrogen catabolite repression (NCR), and others (Val, Trp, and Tyr) are transported by SPS-regulated low-affinity permeases. Therefore, the kinetic characteristics of transporters, as well as SPS and NCR, are likely key factors controlling the temporal sequence of consumption of nitrogen compounds and constitute a system highly conserved in S. cerevisiae species. This work sheds new light on the mechanistic basis of the sequential use of different nitrogen compounds in complex environments.     

Analysis of temperature‐mediated changes in the wine yeast Saccharomyces bayanus var uvarum. An oenological study of how the protein content influences wine quality

Saccharomyces bayanus var. uvarum plays an important role in the fermentation of red wine from the D.O. Ribera del Duero. This is due to the special organoleptic taste that this yeast gives the wines and their ability to ferment at low temperature. To determine the molecular factors involved in the fermentation process at low temperature, a differential proteomic approach was performed by using 2D‐DIGE, comparing, qualitatively and quantitatively, the profiles obtained at 13 and 25°C. A total of 152 protein spots were identified. We detected proteins upregulated at 13°C that were shown to be related to temperature stress, the production of aromatic compounds involved in the metabolism of amino acids, and the production of fusel alcohols and their derivatives, each of which is directly related to the quality of the wines. To check the temperature effects, an aromatic analysis by GC–MS was performed. The proteomic and “aromatomic” results are discussed in relation to the oenological properties of S. bayanus var. uvarum.     

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast

Low‐temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low‐temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:776–783, 2014     

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.     

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.     

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.     

Implication of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in the production of pectinolytic enzymes during cocoa fermentation

The role of bacilli in cocoa fermentation is not well known. Their potential of production of pectinolytic enzymes during this process was evaluated. Bacillus growth was monitored and pectinolytic strains were screened for their use of pectin as sole carbon source. Effects of cocoa fermentation parameters susceptible to influence on enzyme production were analysed. Among 98 strains isolated, 90 were positive for pectin degradation and 80% of them presented detectable pectinolytic activities in submerged fermentation. Forty-eight strains produced polygalacturonase (PG), 47 yielded pectin lyase (PL) and 23 strains produced both enzymes. Bacilli growth was not significantly affected during fermentation. PL production was favoured by galactose, lactose, glucose as sugars, and arginine, glutamine, cysteine and ammonium sulphate as nitrogen compounds. Pectin at low concentration (0.05%) and iron stimulated PL production. It was strongly repressed by galacturonic acid (1%), and negatively affected by nitrogen starvation, zinc and temperatures above 45°C. PL yield was very weak below pH 4.0 and in anaerobic conditions. PG production was weakened by sucrose and cation depletion. It was increased slightly by cysteine, ammonium nitrate and nitrogen starvation and significantly above 40°C. PG synthesis was not affected by acidic pH (3.0–6.0) or oxygen availability. As fermentation products, lactate and acetate lowered the production of both enzymes while ethanol had no effect. The high proportion of pectinolytic producers among the strains studied and analysis of factors influencing pectinolytic enzymes production, suggest that Bacillus sp. is liable to produce at least one enzyme during cocoa fermentation.     

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.     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Genetic Basis of Variations in Nitrogen Source Utilization in Four Wine Commercial Yeast Strains

The capacity of wine yeast to utilize the nitrogen available in grape must directly correlates with the fermentation and growth rates of all wine yeast fermentation stages and is, thus, of critical importance for wine production. Here we precisely quantified the ability of low complexity nitrogen compounds to support fast, efficient and rapidly initiated growth of four commercially important wine strains. Nitrogen substrate abundance in grape must failed to correlate with the rate or the efficiency of nitrogen source utilization, but well predicted lag phase length. Thus, human domestication of yeast for grape must growth has had, at the most, a marginal impact on wine yeast growth rates and efficiencies, but may have left a surprising imprint on the time required to adjust metabolism from non growth to growth. Wine yeast nitrogen source utilization deviated from that of the lab strain experimentation, but also varied between wine strains. Each wine yeast lineage harbored nitrogen source utilization defects that were private to that strain. By a massive hemizygote analysis, we traced the genetic basis of the most glaring of these defects, near inability of the PDM wine strain to utilize methionine, as consequence of mutations in its ARO8, ADE5,7 and VBA3 alleles. We also identified candidate causative mutations in these genes. The methionine defect of PDM is potentially very interesting as the strain can, in some circumstances, overproduce foul tasting H₂S, a trait which likely stems from insufficient methionine catabolization. The poor adaptation of wine yeast to the grape must nitrogen environment, and the presence of defects in each lineage, open up wine strain optimization through biotechnological endeavors.     

Effect of ammonium salt on enzymes of ammonium assimilation in maize seedlings

Glutamate dehydrogenase (GDH E.C. 1.4.1.2.4), glutamine synthetase (GS E.C. 6.3.1.2) and glutamate synthase (glutamine oxoglutarate amino transferase, GOGAT E.C. 2.6.1.53) activities, protein and organic nitrogen contents and growth of roots and shoots of maize seedlings raised in dark at 25±2°C in half strength Hoagland’s solution containing different ammonium salts as source of nitrogen, were determined to assess the contribution of alternate pathways in ammonium assimilation. Ammonium nitrate or in some cases ammonium chloride appeared to be the best source for both root and shoot growth and for increase in protein, total nitrogen and the enzymes of ammonium assimilation. In roots, NH₄-nitrogen appeared to be assimilated by both GDH as well as GS-GOGAT pathways specially in the dark grown seedlings, while in shoots it was primarily by GS-GOGAT pathway.     

Selection and fermentation properties of cryophilic wine yeasts

Two cryophilic strains, YM-84 and YM-126, were selected by a double-layer agar fermenting technique from 100 strains of the wine yeast, Saccharomyces cerevisiae. The viability (specific growth rate) and fermentability of the two selected strains at low temperatures (7 and 13°C) were superior to those of wine yeast strains W3 and OC-2, indicating the usefulness of the two strains as cryophilic wine yeasts. Experiments using the two selected strains at intermediate temperatures (22 and 30°C) showed that their fermentation ceased prematurely and their ethanol yields were reduced.     

Genetic and physiological alterations occurring in a yeast population continuously propagated at increasing temperatures with cell recycling

This work investigated the effects of increasing temperature from 30°C to 47°C on the physiological and genetic characteristics of Saccharomyces cerevisiae strain 63M after continuous fermentation with cell recycling in a system of five reactors in series. Steady state was attained at 30°C, and then the temperature of the system was raised so it ranged from 35°C in the last reactor to 43°C in the first reactor or feeding reactor with a 2°C difference between reactors. After 15 days at steady state, the temperature was raised from 37°C to 45°C for 25 days at steady state, then from 39°C to 47°C for 20 days at steady state. Starter strain 63M was a hybrid strain constructed to have a MAT a/α, LYS/lys, URA/ura genotype. This hybrid yeast showed vigorous growth on plates at 40°C, weak growth at 41°C, positive assimilation of melibiose, positive fermentation of galactose, raffinose and sucrose. Of 156 isolates obtained from this system at the end of the fermentation process, only 17.3% showed the same characteristics as starter strain 63M. Alterations in mating type reaction and in utilization of raffinose, melibiose, and sucrose were identified. Only 1.9% of the isolates lost the ability to grow at 40°C. Isolates showing requirements for lysine and uracil were also obtained. In addition, cell survival was observed at 39–47°C, but no isolates showing growth above 41°C were obtained.     

A comparison of the nitrogen metabolic networks of Kluyveromyces marxianus and Saccharomyces cerevisiae

In grape must, nitrogen is available as a complex mixture of various compounds (ammonium and amino acids). Wine yeasts assimilate these multiple sources in order to suitably fulfil their anabolic requirements during alcoholic fermentation. Nevertheless, the order of uptake and the intracellular fate of these sources are likely to differ between strains and species. Using a two-pronged strategy of isotopic filiation and RNA sequencing, the metabolic network of nitrogen utilization and its regulation in Kluyveromyces marxianus were described, in comparison with those of Saccharomyces cerevisiae. The data highlighted differences in the assimilation of ammonium and arginine between the two species. The data also revealed that the metabolic fate of certain nitrogen sources differed, thereby resulting in the production of various amounts of key wine aroma compounds. These observations were corroborated by the gene expression analysis.