Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28

We characterized a high malic acid production mechanism in sake yeast strain No. 28. No considerable differences in the activity of the enzymes that were involved in malic acid synthesis were observed between strain No. 28 and its parent strain, K1001. However, compared with strain K1001, which actively took up rhodamine 123 during staining, the cells of strain No. 28 were only lightly stained, even when cultured in high glucose concentrations. In addition, malic acid production by the respiratory-deficient strain of K1001 was 2.5-fold higher than that of the wild-type K1001 and wild-type No. 28. The findings of this study demonstrated that the high malic acid production by strain No. 28 is attributed to the suppression of mitochondrial activity.     

Thermal and surface behavior of yeast protein fractions from Saccharomyces cerevisiae

An easy and inexpensive method of fractionation of a yeast homogenate was proposed and it is based on differential centrifugation steps of insoluble components and subsequent precipitations of soluble fractions. In this fractionation, the effect of addition of protease inhibitor was studied. The procedure, which was performed in mild conditions in order to minimize protein denaturation, produced four fractions that proceed from distinct parts of the yeast cell and with a different chemical composition: Fr I, Fr II, Fr III and Fr IV. Thermal and surface behavior of these samples was also analyzed. Fr I and Fr II, mainly composed by cell wall debris and membrane cell components, respectively, exhibited an adsorption rate (Δγ/Δt^1/2) ten-fold higher than Fr III and Fr IV, composed by nucleoproteins and cytoplasmic proteins. All fractions exhibited a unique DSC endotherm with different peak temperature (T_p) and enthalpy values (ΔH). Fr IV exhibited the highest T_p value (74 °C) and less affected by inhibitor absence. Fr I and Fr II showed the highest ΔH values (27-47 J/g protein) but they were markedly affected reducing their enthalpy values and increasing their surface properties in absence of protease inhibitor.     

Toxicogenomics using yeast DNA microarrays

Development of genomics and bioinformatics enable us to analyze the global gene expression profiles of cells by DNA microarray. Changes in gene expression patterns indicate changes in its physiological conditions. Following the exposure of an organism or cell to toxic chemicals or other environmental stresses, the global genetic responses can be expeditiously and easily analyzed. Baker's yeast, Saccharomyces cerevisiae, is one of the most studied and useful model eukaryotes. The biggest advantage of yeast genomics is the available functional information for each gene and a considerable number of data are accumulating in the field of toxicity assessment using yeast DNA microarray. In this review, we discuss the toxicogenomics of metal ions, alcohols and aldehydes, and other chemicals.     

Sugar induces death of the bottom fermenting yeast Saccharomyces pastorianus

We confirmed that sugar-induced cell death (SICD) occurs in the bottom fermenting yeast Saccharomyces pastorianus under anaerobic conditions and that mitochondrial DNA is only partly required for SICD. Fermentation tests using different ratios of glucose and non-glucose nutrients demonstrated that SICD is influenced by the balance between these nutrients.     

Multifactorial analysis of acetaldehyde kinetics during alcoholic fermentation by Saccharomyces cerevisiae

Acetaldehyde is the terminal electron acceptor in the alcoholic fermentation by Saccharomyces cerevisiae. Quantitatively the most important carbonyl by-product, it has relevance for ethanol production yields as well as product stabilization and toxicology. The aim of this study was to investigate the effect of various enological parameters on acetaldehyde kinetics during alcoholic fermentations. Two commercial yeast strains were tested in two grape musts and the pH, temperature, SO₂ and nutrient addition were varied. All incubations had uniform kinetics where acetaldehyde reached an initial peak value followed by partial reutilization. Peak acetaldehyde concentrations and residual concentrations after 15 days of fermentations ranged from 62 to 119 mg l^− 1 and 22 to 49 mg l^− 1, respectively. A positive linear relationship was found between peak and final acetaldehyde levels in Gewürztraminer, but not Sauvignon Blanc fermentations, where sluggish fermentations were observed. Several factors had a significant effect on peak and/or final acetaldehyde levels. SO₂ addition, grape cultivar and fermentation nutrition were important regulators of peak acetaldehyde production, while final acetaldehyde concentrations were correlated with SO₂ addition, grape cultivar and temperature. The results allowed to estimate the acetaldehyde increase caused by SO₂ addition to 366 ??g of acetaldehyde per mg of SO₂ added to the must. The course of the final fermentation phase was shown to determine acetaldehyde residues. Comparison of acetaldehyde and hexose kinetics revealed a possible relationship between the time of occurrence of peak acetaldehyde concentrations and the divergence of glucose and fructose degradation rates.     

Meta-analysis of the influence of Saccharomyces cerevisiae supplementation on ruminal parameters and milk production of ruminants

The effects of yeast supplementation on intake, production, and rumen fermentation characteristics have been widely studied, but results are inconsistent between different studies. A quantitative meta-analysis was applied to 110 papers, 157 experiments, and 376 treatments dealing with yeast supplementation in ruminants. The objective was first to highlight the major quantitative effects of live yeast supplementation on intake, rumen fermentation, and milk production, and second, to identify major differences in experimental conditions between studies that can affect the response to treatment. Some of these experimental conditions are referred to as interfering factors. Yeast supplementation increased rumen pH (+0.03 on average) and rumen volatile fatty acid concentration (+2.17 mM on average), tended to decrease rumen lactic acid concentration (−0.9 mM on average), and had no influence on acetate-to-propionate ratio. Total-tract organic matter digestibility was also increased by yeast supplementation (+0.8% on average). Yeast supplementation increased dry matter intake (DMI; +0.44 g/kg of body weight; BW), milk yield (+1.2 g/kg of BW), and tended to increase milk fat content (+0.05%), but had no influence on milk protein content. Dose effects of yeast supplementation, expressed as log₁₀ [1+(cfu per 100 kg of BW)], globally confirmed the qualitative effects observed in the first analysis. The positive effect of yeast supplementation on rumen pH increased with the percentage of concentrate in the diet and with the DMI level. It was negatively correlated with the level of dietary neutral detergent fiber (NDF). The positive effect of yeast supplementation on rumen volatile fatty acid concentration increased with DMI and crude protein levels. The positive effect of yeast supplementation on organic matter digestibility increased with the percentage of concentrate and NDF in the diet. The negative effect of yeast supplementation on lactic acid concentration tended to decrease when the DMI level and the percentage of concentrate in the diet increased. The effects of interfering factors were globally similar when either dose effect or qualitative effect of yeast was taken into account. Although rumen fermentation efficiency per se was not measured, these results suggest an improvement in rumen fermentation by yeast supplementation. This effect could, however, be modulated by several different factors such as DMI, percentage of concentrate or NDF in the diet, or species.     

High-cell-density fermentation of Saccharomyces cerevisiae for the optimisation of mead production

Mead is a traditional drink that contains 8%–18% (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Mead fermentation is a time-consuming process and the quality of the final product is highly variable. Therefore, the present investigation had two main objectives: first, to determine the adequate inoculum size of two commercial wine-making strains of Saccharomyces cerevisiae for the optimisation of mead fermentation; and second, to determine if an increase in yeast pitching rates in batch fermentations altered the resulting aroma profiles. Minor differences were detected in the growth kinetics between the two strains at the lowest pitching rate. With increasing pitching rates net growth of the strain ICV D47 progressively decreased, whereas for the QA23 the increasing inoculum size had no influence on its net growth. The time required to reach the same stage of fermentation ranged from 24 to 96 h depending on the inoculum size. The final aroma composition was dependent on the yeast strain and inoculum size. Fourteen of the twenty-seven volatile compounds quantified could contribute to mead aroma and flavour because their concentrations rose above their respective thresholds. The formation of these compounds was particularly pronounced at low pitching rates, except in mead fermented by strain ICV D47, at 10⁶ CFUs/mL. The esters isoamyl acetate, ethyl octanoate and ethyl hexanoate were the major powerful odourants found in the meads. The results obtained in this study demonstrate that yeast strain and inoculum size can favourably impact mead's flavour and aroma profiles.     

Effect of feeding yeast culture on performance, health, and immunocompetence of dairy calves

Objectives were to determine effects of feeding a culture of Saccharomyces cerevisiae on performance, health, and immunocompetence of calves in the first 70 d of age. Holstein calves (n = 512) at 2 ± 1 d of age were randomly assigned to yeast culture (YC, 218 females and 37 males) or control (223 females and 34 males). Yeast culture was fed at 2% of the grain dry matter. All calves received colostrum during the first 24 h, pasteurized milk thereafter until 60 d of age, and grain was fed ad libitum for the first 70 d of age. Calves were housed in individual hutches, and grain intake was measured 5 d/wk. Body weight was measured at 5, 30, and 68 d of age, and attitude and fecal consistency were scored daily. Incidence and duration of health disorders and treatments were recorded. Neutrophil phagocytic and killing activities and antibody response to immunization with ovalbumin were measured. Concentrations of glucose and 3-hydroxybutyrate were measured in plasma. Grain intake did not differ between treatments and averaged 908 g/d throughout the study. Body weight change, concentrations of glucose, and 3-hydroxybutyrate did not differ between YC and control. Minor effects on neutrophil function were observed, and YC tended to increase the number of phagocytized bacteria and killing of phagocytized bacteria but did not influence humoral immune response. Attitude scores were similar between treatments throughout the study. Almost all calves experienced mild diarrhea during the study, but feeding YC improved fecal scores, reduced days with watery feces, incidence of fever and diarrhea, and risk of health disorders. Because of the high incidence of diarrhea, mortality preweaning was also high, but YC improved survival of calves by decreasing mortality rate past 13 d of age. Income at the end of the study was improved by $48/calf with YC. Feeding yeast culture in grain improved health, minimized frequency of health treatments, and reduced risk of morbidity and mortality in dairy calves.     

Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang,China

The evolution of yeast species and Saccharomyces cerevisiae genotypes during spontaneous fermentations of Muscat blanc planted in 1957 in Jingyang region of China was followed in this study. Using a combination of colony morphology on Wallerstein Nutrient (WLN) medium, sequence analysis of the 26S rDNA D1/D2 domain and 5.8S-ITS-RFLP analysis, a total of 686 isolates were identified at the species level. The six species identified were S. cerevisiae, Hanseniaspora uvarum, Hanseniaspora opuntiae, Issatchenkia terricola, Pichia kudriavzevii (Issatchenkia orientalis) and Trichosporon coremiiforme. This is the first report of T. coremiiforme as an inhabitant of grape must. Three new colony morphologies on WLN medium and one new 5.8S-ITS-RFLP profile are described. Species of non-Saccharomyces, predominantly H. opuntiae, were found in early stages of fermentation. Subsequently, S. cerevisiae prevailed followed by large numbers of P. kudriavzevii that dominated at the end of fermentations. Six native genotypes of S. cerevisiae were determined by interdelta sequence analysis. Genotypes III and IV were predominant. As a first step in exploring untapped yeast resources of the region, this study is important for monitoring the yeast ecology in native fermentations and screening indigenous yeasts that will produce wines with regional characteristics.     

Stable disruption of ethanol production by deletion of the genes encoding alcohol dehydrogenase isozymes in Saccharomyces cerevisiae

We analyzed the effects of the deletions of genes encoding alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae. The decrease in ethanol production by ADH1 deletion alone could be partially compensated by the upregulation of other isozyme genes, while the deletion of all known ADH isozyme genes stably disrupted ethanol production.     

Determination of the antigenotoxic potencies of some luteolin derivatives by using a eukaryotic cell system, Saccharomyces cerevisiae

In this study, we aimed to examine the mutagenic and antimutagenic potencies of three luteolin derivatives (luteolin-7-O-glucoside, luteolin-7-O-rutinoside and luteolin-7-O-glucuronide) by using a eukaryotic cell system, Saccharomyces cerevisiae (RS112).     

Effect of oxygen and lipid supplementation on the volatile composition of chemically defined medium and Chardonnay wine fermented with Saccharomyces cerevisiae

Oxygen or lipids are required to complete stressful alcoholic fermentation. Lack of these nutrients can inhibit sugar uptake and growth, which leads to incomplete or ‘stuck’ fermentation. Oxygen or lipids supplementation not only restores yeast fermentative activity and also affects formation of yeast volatile metabolites. To clarify the effect of oxygen and lipid supplementation on the formation of flavour active metabolites during wine fermentation, we evaluated the addition of these two nutrients to chemically defined grape juice and filter clarified Chardonnay must. Lipid addition increased the concentration of esters, higher alcohols and volatile acids, whereas oxygen increased the concentration of higher alcohols and altered the proportion of acetate to ethyl esters and the proportion of branch-chain acids to medium-chain fatty acids. Combined addition of lipids and oxygen showed an additive effect on concentration of higher alcohols whereas oxygen suppressed the enhancing effect of lipids on formation of esters and volatile acids. Our results demonstrate the potential of lipid and oxygen supplementation for the manipulation of wine aroma in white wine fermentation.     

Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain

Succinate dehydrogenase (SDH) of Saccharomyces cerevisiae consists of four subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. We determined the effect of SDH deficiency on the productivity of organic acids in a sake yeast strain Kyokai no. 9. The SDH activity of single disruptants was retained at 30-90% of that of the wild-type strain, but the activity disappeared in double disruptants of the SDH1 and SDH2 or SDH1b (the SDH1 homologue) genes. Two double disruptants showed no growth on a medium containing glycerol as the sole carbon source, while the single disruptants could utilize glycerol. These results indicate that double disruption of the SDH1 and SDH2 or SDH1b genes is required for complete loss of SDH activity and that the SDH1b gene compensates for the function of the SDH1 gene. The sdh1 sdh1b disruptant showed a marked increase in succinate productivity of up to 1.9-fold along with a decrease in malate productivity relative to the wild-type strains under shaking conditions. Under both static and sake brewing conditions, the productivity of these organic acids in the disruptants was virtually unchanged from that in the wild-type strain. Furthermore, SDH activity was undetectable in the wild-type and the disrupted strains under static conditions. These results suggest that SDH activity contributes to succinate production under shaking conditions, but not under static and sake brewing conditions.