Gaseous environments modify physiology in the brewing yeast Saccharomyces cerevisiae during batch alcoholic fermentation

Aims:  To investigate the impact of different gaseous atmospheres on different physiological parameters in the brewing yeast Saccharomyces cerevisiae BRAS291 during batch fermentation.
Methods and Results:  Yeasts were cultivated on a defined medium with a continuous sparging of hydrogen, helium and oxygen or without gas, permitting to obtain three values of external redox. High differences were observed concerning viable cell number, size and metabolites produced during the cultures. The ethanol yields were diminished whereas glycerol, succinate, acetoin, acetate and acetaldehyde yields were enhanced significantly. Moreover, we observed major changes in the intracellular NADH/NAD⁺ and GSH/GSSG ratio.
Conclusions:  The use of gas led to drastic changes in the cell size, primary energy metabolism and internal redox balance and E _h . These changes were different depending on the gas applied throughout the culture.
Significance and Impact of the Study:  For the first time, our study describes the influence of various gases on the physiology of the brewing yeast S. cerevisiae . These influences concern mainly yeast growth, cell structure, carbon and redox metabolisms. This work may have important implications in alcohol-related industries, where different strategies are currently developed to control better the production of metabolites with a particular attention to glycerol and ethanol.     

Development of a kinetic model for the alcoholic fermentation of must

We Propose a kinetic expression which accounts for the temperature dependence of ethanol yield losses in batch alcoholic fermentation. Moreover, the characteristic parameters of the microbial growth equation have been calculated for Saccharomyces cerevisiae under typical wine industry conditions. A substrate consumption equation is established which minimizes possible model deviations in the latter process stages. Experimental data were obtained in the laboratory and the proposed equations were then applied at an industrial level (2.5 x 10(4) L) where they described the data well.     

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.     

Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery

AIMS: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. METHODS AND RESULTS: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-delta PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. CONCLUSION: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.     

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.     

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.     

Loss of Rim15p in shochu yeast alters carbon utilization during barley shochu fermentation

ABSTRACT

Rim15p of the yeast Saccharomyces cerevisiae is a Greatwall-family protein kinase that inhibits alcoholic fermentation during sake brewing. To elucidate the roles of Rim15p in barley shochu fermentation, RIM15 was deleted in shochu yeast. The disruptant did not improve ethanol yield, but altered sugar and glycerol contents in the mash, suggesting that Rim15p has a novel function in carbon utilization.     

Critical role of RPI1 in the stress tolerance of yeast during ethanolic fermentation

Stress tolerance of yeast Saccharomyces cerevisiae during ethanolic fermentation is poorly understood due to the lack of genetic screens and conventional plate assays for studying this phenotype. We screened a genomic expression library of yeast to identify gene(s) that, upon overexpression, would prolong the survival of yeast cells during fermentation, with the view to understand the stress response better and to use the identified gene(s) in strain improvement. The yeast RPI1 (Ras-cAMP pathway inhibitor 1) gene was identified in such a screen performed at 38 °C; introducing an additional copy of RPI1 with its native promoter helped the cells to retain their viability by over 50-fold better than the wild type (WT) parent strain, after 36 h of fermentation at 38 °C. Disruption of RPI1 resulted in a drastic reduction in viability during fermentation, but not during normal growth, further confirming the role of this gene in fermentation stress tolerance. This gene seems to improve viability by fortifying the yeast cell wall, because RPI1 overexpression strain is highly resistant to cell lytic enzyme zymolyase, compared with the WT strain. As the RPI1 overexpression strain substantially retains cell viability at the end of fermentation, the cells can be reused in the subsequent round of fermentation, which is likely to facilitate economical production of ethanol.     

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.     

Influence of Saccharomyces cerevisiae strains on fermentation and flavor compounds of white wines made from cv. Emir grown in Central Anatolia,Turkey

The effect of inoculation with selected Saccharomyces cerevisiae strains was studied on fermentation and flavor compounds of wines made from Vitis vinifera L. cv. Emir grown in Central Anatolia, Turkey. Flavor compounds were analysed and identified by GC-FID and GC-MS, respectively. The total concentrations of flavor compounds did not increase with the addition of indigenous and commercial wine yeasts, but differences were noted in individual volatile compounds. Cluster and factor analyses of flavor compounds also showed that wines produced were different depending on the wine strain used. Wines were completely fermented to less than 1.4 g/l residual sugar. Yeasts other than S. cerevisiae survived longer than previously reported. Inoculation with selected strains increased the ethanol level. Journal of Industrial Microbiology & Biotechnology (2002) 29, 28–33 doi:10.1038/sj.jim.7000258 Received 11 July 2001/ Accepted in revised form 27 March 2002     

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

A CAN1/can1Δ heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.     

Influence of oxygen on the biosynthesis of cellular fatty acids,sterols and phospholipids during alcoholic fermentation by Saccharomyces cerevisiae and Torulaspora delbrueckii

Saccharomyces cerevisiae and Torulaspora delbrueckii were grown under different O₂ availabilities on grape must. Oxygen requirements for the two yeasts were different: under anaerobic conditions, S. cerevisiae produced a higher percentage of unsaturated fatty acids, and had a greater cell yield and fermentation activity than T. delbrueckii. Addition of ergosterol (25mg/l) and oleic acid (31mg/l) caused total recovery of cellular growth and the fermentation activity of S. cerevisiae in anaerobiosis, but not of T. delbrueckii. However a short period of aeration to a 48 h culture in anaerobiosis, led to total recovery of the cellular growth and fermentation activity in both yeasts. Likewise, the effect of a short aeration period on unsaturated fatty acid biosynthesis was similar for both species.