CINNAMIC ACID AS THE BASIS OF A MEDIUM FOR THE DETECTION OF WILD YEASTS

Cinnamic acid (100 μg ml^?1) incorporated in a solid medium was found to inhibit the growth of brewing strains (Pof^?) of yeast while permitting the growth of Pof+ wild yeast contaminants. Typically, colonies of Saccharomyces cerevisiae var. diastaticus (Pof+) mixed with brewing yeast (S. cerevisiae NCYC 240) were visible after 5d incubation at 25°C. The incubation time required to detect a selection of brewery wild yeast isolates was found to vary from 3–12 d.     

DISTRIBUTION OF ESTERS PRODUCED DURING SUGAR FERMENTATION BETWEEN THE YEAST CELL AND THE MEDIUM

The distribution of the esters formed during sugar fermentations between the yeast cells and the medium was investigated in fermentations by 5 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum (carlsbergensis). The esters studied included the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₂ fatty acids. All of both acetates appeared in the medium. The proportion of the fatty acid ethyl esters transferred to the medium decreased with increasing chain length: all in the medium for ethyl caproate, 54–68% for ethyl caprylate, 8–17% for ethyl caprate, and all remaining in the yeast cell for ethyl laurate. A higher proportion of the esters formed appeared to remain in the cells of the S. uvarum strains than in cells of S. cerevisiae.     

Isolation of glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae and their use in baker's yeast production

 Glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae 0511 were obtained by mutation under specific conditions. A total of 3388 strains were isolated and among them were found 46 mutants sensitive to methylglyoxal. The intracellular glutathione concentration of mutant strain S. cerevisiae 3033 was 0.0172 g/g dry weight, which was a decrease of >76% compared to that of the parent. The growth of mutant strains S. cerevisiae 3033 and S. cerevisiae 1116 in the medium with glutathione present and absent was compared to that of the parent strain. The sensibility of the baker's yeast strains studied to antifoaming agents, butanol and acetic acid was also investigated. The relationship between glutathione presence in the cell and the sensitivity of strain S. cerevisiae 3033 to antifoaming agents and butanol was ascertained, while such a connection with the presence of acetic acid in the molasses medium used for baker's yeast cultivation was not observed. The higher sensitivity of strain S. cerevisiae 3033 to some chemical compounds in the molasses nutrition medium was shown. Received: 2 November 1999 / Revised version: 15 February 2000     

The ecology and evolution of non‐domesticated Saccharomyces species

Yeast researchers need model systems for ecology and evolution, but the model yeast Saccharomyces cerevisiae is not ideal because its evolution has been affected by domestication. Instead, ecologists and evolutionary biologists are focusing on close relatives of S. cerevisiae, the seven species in the genus Saccharomyces. The best‐studied Saccharomyces yeast, after S. cerevisiae, is S. paradoxus, an oak tree resident throughout the northern hemisphere. In addition, several more members of the genus Saccharomyces have recently been discovered. Some Saccharomyces species are only found in nature, while others include both wild and domesticated strains. Comparisons between domesticated and wild yeasts have pinpointed hybridization, introgression and high phenotypic diversity as signatures of domestication. But studies of wild Saccharomyces natural history, biogeography and ecology are only beginning. Much remains to be understood about wild yeasts' ecological interactions and life cycles in nature. We encourage researchers to continue to investigate Saccharomyces yeasts in nature, both to place S. cerevisiae biology into its ecological context and to develop the genus Saccharomyces as a model clade for ecology and evolution. © 2014 The Authors. Yeast published by John Wiley & Sons Ltd.     

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

FORMATION OF HIGH CONCENTRATIONS OF ALCOHOL BY VARIOUS YEASTS*

The presence of Aspergillus oryzae-proteolipid (PL) at the early stage off fermentation was essential to obtain yeast cells capable of producing high concentrations of alcohol, when the fermentation tests were carried out by stepwise addition of sucrose to the synthetic media. High concentrations of alcohol were produced by strains of Saccharomyces cerevisiae Hansen and Saccharomyces uvarum (carlsbergensis) as well as Saccharomyces sake which produced more than 20 percent alcohol in the PL-supplemented medium at 20°C.     

d-Xylose consumption by nonrecombinant Saccharomyces cerevisiae: A review

Xylose is the second most abundant sugar in nature. Its efficient fermentation has been considered as a critical factor for a feasible conversion of renewable biomass resources into biofuels and other chemicals. The yeast Saccharomyces cerevisiae is of exceptional industrial importance due to its excellent capability to ferment sugars. However, although S. cerevisiae is able to ferment xylulose, it is considered unable to metabolize xylose, and thus, a lot of research has been directed to engineer this yeast with heterologous genes to allow xylose consumption and fermentation. The analysis of the natural genetic diversity of this yeast has also revealed some nonrecombinant S. cerevisiae strains that consume or even grow (modestly) on xylose. The genome of this yeast has all the genes required for xylose transport and metabolism through the xylose reductase, xylitol dehydrogenase, and xylulokinase pathway, but there seems to be problems in their kinetic properties and/or required expression. Self-cloning industrial S. cerevisiae strains overexpressing some of the endogenous genes have shown interesting results, and new strategies and approaches designed to improve these S. cerevisiae strains for ethanol production from xylose will also be presented in this review.     

LONG-CHAIN FATTY ACID COMPOSITION AS A CRITERION FOR YEAST DISTINCTION IN THE BREWING INDUSTRY

The diversity of yeasts isolated from brewing plants and its role on beer quality makes yeast distinction a major concern in industrial microbiological control. Several approaches have been tried to develop rapid and simple methods to perform such tasks. Among these, stands the utilization of long-chain fatty acid composition of total yeast biomass. In this paper results are reported showing the potential of this technique to characterize yeast flora isolated from industrial plants. Fatty acid profiles of brewing species are clearly differentiated from those of non-Saccharomyces strains using statistical data treatment by principal component analysis (PCA). Distinction between brewing and wild strains of Saccharomyces spp. was not apparent. In comparison, fatty acid profiling showed higher discriminating ability than growth on lysine medium for non-Saccharomyces strains. For distinction of S. cerevisiae var. diastaticus from other Saccharomyces strains, growth on starch medium showed to be necessary.     

AN UPDATE OF ZINC ION AS AN EFFECTOR OF FLOCCULATION IN BREWER'S YEAST STRAINS

Zn⁺⁺ ion was observed to be a strong effector of the flocculation—deflocculation process in an in vitro system for strains of Saccharomyces cerevisiae at ion concentration ranges that are normal in conventional substrates, such as wort. All strains of Saccharomyces uvarum (carlsbergensis) examined in this study did not exhibit any flocculation response to Zn⁺⁺ ion. This test could be employed to distinguish between ale and lager flocculating yeast strains.     

A study on Saccharomyces cerevisiae and Candida utilis cell wall capacity for binding magnesium

The capacity for binding magnesium by bakery's yeast strain Saccharomyces cerevisiae No. 102 (Pure Culture Collection, Faculty Food Technology, Warsaw) and fodder yeast strain Candida utilis (ATCC 9950) was investigated in media supplemented with that element. The capacities of C. utilis (ATCC 9950) and S. cerevisiae (No. 102) biomass for binding magnesium were not statistically different in the first 24 h. In the next 24 h of cultivation the cells of C. utilis (ATCC 9950) were still able to bind magnesium ions, whereas those of S. cerevisiae (No. 102) released a part of previously bound magnesium to the medium. The major part of magnesium bound by the cells of C. utilis (ATCC 9950) was accumulated in cytosole. It was opposite to the cells of bakery yeast S. cerevisiae (No. 102) that accumulated magnesium mainly in the cell wall. The cells of C. utilis (ATCC 9950) yeast were smaller and their cell walls were thinner as compared to those of S. cerevisiae (No. 102) yeast. The thickness of the external mannoprotein layers was similar in both strains analyzed.     

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.     

THE SPORULATION AND MATING OF BREWING YEASTS

A study has been made of the sporulating behaviour of twenty selected brewing strains of yeast, and the mating activity of the products of sporulation. ‘Lager’ yeasts (strains of Saccharomyces carlsbergensis) in general sporulated to a lesser degree and more slowly than ‘ale’ yeasts (strains of Saccharomyces cerevisiae) and produced 1-or 2- spored asci compared with 2-or 3- spored asci for the latter yeasts. Most of the parent strains of S. cerevisiae were shown to be heterozygous for mating type, and they were all probably either triploid or aneuploid. Two of the strains of S. carlsbergensis were apparently homozygous for mating type and also triploid or aneuploid. The compatibility system favours outbreeding of yeasts, ‘ale’ yeasts being more compatible with ‘lager’ yeasts than with other ‘ale’ yeasts.     

Yeasts Isolated from the Alcoholic Fermentation of Agave duranguensis During Mezcal Production

Mezcal is a spirit produced in some regions of México. In the state of Durango, mezcal is produced via traditional fermentation of the Agave duranguensis plant. To better understand traditional fermentation processes, it is necessary to know which yeast species are present in fermentations in different producer regions. The aim of this research was to study yeasts involved in traditional mezcal fermentation in Durango, México, and investigate the phylogeny of the native Saccharomyces cerevisiae strains involved in this process. The 5.8S-ITS genomic region was analyzed to identify strains present in the fermentation process samples in this study. To differentiate strains belonging to the genus Saccharomyces, different molecular techniques were used, including analysis of mitochondrial DNA and δ elements and sequencing of the 5.8S-ITS genomic region. Although a high diversity of microorganisms was found at the beginning of fermentation, Saccharomyces cerevisiae was the only yeast present at the end of fermentation in region I, while Torulaspora delbrueckii was found in a higher number than S. cerevisiae at the end of fermentation in the region II. Molecular techniques demonstrated that Saccharomyces cerevisiae isolated in Durango are phylogenetically independent from the strains isolated in other regions of Latin America and Europe.     

Composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay

There is a lack of knowledge about the composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay cultivars. The objectives were to determine the relative abundance of indigenous and commercial S. cerevisiae strains in spontaneous fermentations at three wineries from the two cultivars and to compare the composition of the S. cerevisiae strains between cultivars and wineries. Three fermentation vessels were sampled at three stages of fermentation for each cultivar at each winery. Isolates were identified to the strain level using seven microsatellite loci. Commercial S. cerevisiae strains were isolated at a frequency higher than that of the indigenous strains at each winery for both cultivars. The composition of S. cerevisiae strains was different for each cultivar and at each winery. Our results illustrate the clear influence of inoculated commercial active dry yeast strains on the composition of S. cerevisiae strains in spontaneous fermentations at wineries conducting both inoculated and spontaneous fermentations.     

Yeasts isolated from New Zealand vineyards and wineries

Background and Aims: The yeast flora from a range of New Zealand commercial wineries was surveyed to estimate the incidence of yeast species in grape juice. Methods and Results: Molecular analysis of the internal transcribed spacer region was performed for 1279 yeast colonies isolated from 17 different fresh grape juices sampled in eight New Zealand wineries between 2003 and 2009. The 17 juices contained at least 25 different species of yeast from nine genera. Microsatellite fingerprinting of Saccharomyces cerevisiae showed that some strains were identical to known commercial yeast varieties, but we also found evidence for local populations of S. cerevisiae common to individual wineries or regions. Five genotypes from Central Otago, New Zealand, were very closely related to a single sequenced strain derived from Chile, which in turn is related to European wine isolates. Conclusions: The yeast flora found in New Zealand grape juices is broadly similar to that found in wineries elsewhere around the world. Genotyping of S. cerevisiae suggests recent dispersal of both commercial and non-commercial yeast strains from Europe to New Zealand. Significance of the Study: These data are consistent with two human-mediated modes for the international dispersal of S. cerevisiae: one via the escape of strains traded commercially, and another via long distance dispersal of non-commercial strains.     

DIFFERENTIATION OF BREWERY YEAST STRAINS BY RESTRICTION ENDONUCLEASE ANALYSIS OF THEIR MITOCHONDRIAL DNA

Mitochondrial DNA (mtDNA) was isolated from different strains of brewery yeast and digested with various restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. Of the twenty restriction endonucleases used, only two—Aval and Haelli—produced different restriction fragment patterns when applied to the mtDNA from two strains of Saccharomyces uvarum. The restriction fragment patterns produced by the other eighteen enzymes were identical. Analysis of mtDNA from a strain of Saccharomyces cerevisiae with the same twenty restriction endonucleases revealed several differences with respect to Saccharomyces uvarum. Taken together, these results indicate that restriction endonuclease fragmentation patterns of mtDNA are useful as diagnostic tools for distinguishing strains of ale and larger yeast.     

SYNTHESIS OF HIGHER ALCOHOLS IN THE GENUS ZYMOMONAS

Several strains of bacteria of the genus Zymomonas were examined with respect to their potential for higher alcohol synthesis. All strains studied were able to produce higher alcohols during growth in a simple medium containing glucose and yeast extract. The higher alcohols produced were mainly n-propanol and iso-amyl alcohol. In contrast to Saccharomyces cerevisiae, only trace amounts of higher alcohols were produced from glucose by resting cells. When amino acids or other precursors were added to the fermentation medium, the resting cells formed higher alcohols. The stimulation of n-propanol synthesis by precursors was the most pronounced. The results obtained indicate that, with minor differences, the mechanisms of higher alcohol synthesis are comparable to these used by yeasts.     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.