Co-existence of two types of chromosome in the bottom fermenting yeast,Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.     

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     

Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed cultures.     

CONTROLLED PRODUCTION OF CIDER BY INDUCTION OF ALCOHOLIC FERMENTATION AND MALOLACTIC CONVERSION

Characterization experiments involving lactic bacteria allowed a strain of Leuconostoc oenos to be selected in terms of growth capacity at variable pH, temperature, ethanol and sulphite concentrations, malic to lactic conversion yield, acetic acid uptake and dextran production capacity. Cider was produced under controlled conditions where the effect of Kloeckera apiculata yeasts on the development of Saccharomyces cerevisiae and production of major non-volatile compounds influencing end product quality was studied. The apiculate yeast was found to produce large amounts of acetic acid and use the other organic acids; also, it hindered fermentation to a certain extent. A study of the effect of the inoculation time with L oenos as an inducer of malolactic fermentation revealed sequential inoculation of the lactic bacterium once most major sugars in the must had been depleted to be the most favourable. Using yeast cell walls boosted fermentation development, as well as degradation of malic acid and synthesis of succinic and acetic acid.     

XVI. Yeast sequencing reports. A new essential gene GCR4 located in the upstream region of GCR1

A new essential gene of Saccharomyces cerevisiae was found upstream of GCR1. Its cloning and sequencing predict a 280 amino acid protein (32 577 Da). The predicted protein is fairly hydrophobic, and a search of the database did not identify any homologous proteins. A LEU2 disruption at codon 104 was lethal, but disruption at codon 221 showed a temperature-sensitive conditional growth phenotype. Abnormalities were observed in some glycolytic enzyme levels. The sequence has been submitted to GenBank-EMBL-DDBJ under Accession Number D29645.     

Ochratoxin A Removal by Yeasts after Exposure to Simulated Human Gastrointestinal Conditions

Yeasts can remove ochratoxin A (OTA) in foods; however, we do not know if they can experience this ability in the gut. Thus, the aim of this paper was to study OTA binding by 3 wine strains of Saccharomyces cerevisiae (W13, W28, and the commercial isolate BM45) and S. cerevisiae var. boulardii ATCC MYA‐796 (a probiotic yeast) after exposure to conditions simulating the passage through human gastrointestinal tract. Yeasts removed OTA by up to 44%, with a better ability found after gastrointestinal and sequential assays. However, the phenomenon was reversible, because ca. 18% to 28% of toxin was not stably bound. After the evaluation of the net amount of OTA bound by yeasts (that is, after toxin release), S. cerevisiae W13 and S. cerevisiae var. boulardii ATCC MYA‐796 were selected as the most promising strains for further studies (for example, clinical trials) due to their ability to remove 21% of OTA.     

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.     

Effect of Initial PH on Growth Characteristics and Fermentation Properties of Saccharomyces cerevisiae

As the core microorganism of wine making, Saccharomyces cerevisiae encounter low pH stress at the beginning of fermentation. Effect of initial pH (4.50, 3.00, 2.75, 2.50) on growth and fermentation performance of 3 S. cerevisiae strains Freddo, BH8, Nº.7303, different tolerance at low pH, chosen from 12 strains, was studied. The values of yeast growth (OD₆₀₀, colony forming units, cell dry weight), fermentation efficiency (accumulated mass loss, change of total sugar concentration), and fermentation products (ethanol, glycerol, acetic acid, and l ‐succinic acid) at different pH stress were measured. The results showed that the initial pH of must was a vital factor influencing yeast growth and alcoholic fermentation. Among the 3 strains, strain Freddo and BH8 were more tolerant than Nº.7303, so they were affected slighter than the latter. Among the 4 pH values, all the 3 strains showed adaptation even at pH 2.50; pH 2.75 and 2.50 had more vital effect on yeast growth and fermentation products in contrast with pH 4.50 and 3.00. In general, low initial pH showed the properties of prolonging yeast lag phase, affecting accumulated mass loss, changing the consumption rate of total sugar, increasing final content of acetic acid and glycerol, and decreasing final content of ethanol and l ‐ succinic acid, except some special cases. Based on this study, the effect of low pH on wine products would be better understood and the tolerance mechanism of low pH of S. cerevisiae could be better explored in future.     

Alginate beads and apple pieces as carriers for Saccharomyces cerevisiae var. boulardii,as representative of yeast functional starter cultures

This study focused on two different carriers for Saccharomyces cerevisiae var. boulardii for its targeted release as a starter or as a probiotic: the microencapsulation into alginate beads and the immobilisation on apple pieces. Beads were studied in relation to encapsulation yield (EY), viability of cells throughout the storage, kinetic of cell release, survival under conditions that mimic the transit into the gut and in vivo application (fermentation of grape juice). Concerning the microencapsulation, EY was very high (ca. 90%), cells survived for a long period (upon to 90 days), and the beads assured cell survival and their release under conditions that mimic the gut; moreover, the capsules could be used up to ten times to start a model fermentation of grape juice. Apple pieces were a good system for the immobilisation of S. boulardii; they could be proposed successfully as a reusable carrier for a starter culture, as they assured the start of the fermentation of grape juice for at least seven times.     

Wine vinegar production using base wines made with different yeast species

The influence of base wines obtained by the fermentation of different yeast species on acetic acid bacteria growth and on the analytical profile of vinegars was investigated. Results show that the substrates for wine vinegar production exerted a strong influence on both acetic acid bacteria growth and analytical profile of vinegars. The base wine obtained from the alcoholic fermentation of Saccharomyces cerevisiae was not always the best substrate. The fermentate made with Candida stellata positively influenced the acetic acid bacteria growth and the quality of vinegar, while the wine obtained from the fermentation of Kloeckera apiculata was a good substrate for acetic acid bacteria growth and acetic acid production and could be used for ‘ordinary’ vinegar production. © 1998 Society of Chemical Industry     

SELECTION AND BIOCHEMICAL CHARACTERISATION OF SACCHAROMYCES CEREVISIAE AND KLOECKERA APICULATA STRAINS ISOLATED FROM SPANISH CIDER

The behaviour of different strains of Saccharomyces cerevisiae and Kloeckera apiculata in apple juice fermentation was studied. Ethanol production was higher for Saccharomyces strains while residual sugars and ethyl acetate content was higher for Kloeckera strains. Kl. apiculata fermented products showed the lowest amount of higher alcohols and the lowest content in organic acids with the exception of acetic acid, so this yeast produced an increment in volatile acidity. On the basis of ethyl acetate, hydrogen sulfide, and acetic acid production, fermentative ability, potassium metabisulfite resistance and sporulation percentage, one strain from Sacch. cerevisiae could be employed as starter for making cider.     

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.     

ON THE DIFFERING RATES OF FRUCTOSE AND GLUCOSE UTILISATION IN SACCHAROMYCES CEREVISIAE

Fructose is utilised slower than glucose when the two sugars are fermented separately. This phenomenon occurs in a growth promoting medium as well as in brewers wort when using the brewing yeast Saccharomyces cerevisiae. The use of a fructose adjunct in wort at concentrations of 2% w/v and above, may result in residual concentrations of fructose to remain at the end of fermentation and consequently taint the beer with a sweet off-flavour. Glucose and fructose have no effect on maltose utilisation. Thus they do not exert catabolite repression on the maltose membrane transport system in the particular brewing strain of S. cerevisiae under investigation, when fermented in brewers wort.     

Potential application of Saccharomyces boulardii as a probiotic in goat's yoghurt: survival and organoleptic effects

Potential application of Saccharomyces boulardii in yoghurts could offer an alternative probiotic product to people suffering from antibiotic‐associated diarrhoea whilst on treatment. Three different yoghurts were made using goat's milk. Yoghurt 1 was the control with only starter culture added to the milk. For Yoghurt 2, S. boulardii was added in parallel with the starter culture, and for Yoghurt 3, the yeast was added after the yoghurt was coagulated by the starter. Duplicate counts for lactic acid bacteria (LAB) and S. boulardii alongside organoleptic assessment were carried out every 3 days following manufacture and storage at 6 °C. Our results demonstrate that S. boulardii can survive in high numbers for the period of 4 weeks tested without significant changes in taste. Very important is the fact that the presence of the yeast stimulated the survival of the LAB, which in both yeast yoghurts were significantly higher than in the control.     

Concentration of amino acids in wine after the end of fermentation by Saccharomyces cerevisiae strains

Both quantitative and qualitative differences in the utilization and release of assimilable nitrogen by two wine strains of Saccharomyces cerevisiae (cerevisiae and capensis) under different conditions of oxygen were observed. These differences were influenced by the presence of oxygen at the beginning of the fermentation, and by the strain of S cerevisiae. The release of some amino acids post‐fermentation may be the result of reoxidation of NAD(P)H in order to maintain a normal redox balance. Copyright © 2003 Society of Chemical Industry     

Variations of internal pH in typical Italian sourdough yeasts during co-fermentation with lactobacilli

The effects of organic acids (lactic and acetic) and extracellular pH (pHex) on the intracellular pH (pHi) of Saccharomyces cerevisiae and Candida milleri during co-fermentation with lactobacilli were investigated by using Fluorescence-Ratio-Imaging-Microscopy (FRIM). Yeasts were grown in a system that partially mimics sourdough composition, using individual fermentation and combinations with lactic acid bacteria. Fermentations were carried out at 25 °C for 22 h at an initial pH of 5.3. The two yeast species grew equally well during the co-fermentations with lactobacilli. Our results reveal large differences in pHi values between the two yeast species, primarily in relation with pHex changes, while the concentration of organic acids did not seem to affect the pHi. Moreover, the pHi of S. cerevisiae seemed to be affected by maltose consumption. The pH gradient (difference between internal and external pH) of S. cerevisiae remained rather constant, ranging from 2.0 to 2.5. C. milleri instead exhibited a higher pHi, that remained constant throughout the experiments and was unaffected by pHex and/or sugar consumption. Thus, the pH gradient of C. milleri varied much more than that of S. cerevisiae, ranging from 2.3 to 3.8. Our results suggest that the two yeast species have different pHi regulation mechanisms.     

MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE

Lambic and Gueuze are special Belgian beers obtained by spontaneous fermentation. Micro-organisms involved in this fermentation were counted and differentiated using several selective growth media. Micro-organisms were also isolated from samples of Lambic of different age and originating from different casks and brews and identified by classical tests. The following general pattern of microbial development was observed. After 3 to 7 days the fermentation started with the development of wort Enterobacteriaceae and strains of Kloeckera apiculata. These organisms were overgrown after 3 to 4 weeks by strains of Saccharomyces cerevisiae and S. bayanus. These were responsible for the main fermentation, lasting for 3 to 4 months. This was followed by a strong bacterial activity. This period was dominated by the growth of strains of Pediococcus cerevisiae. These reached their maximal numbers during the summer months and were responsible for a fivefold increase in lactic acid concentration. In some casks they caused ropiness. After the main fermentation period Lambic is very sensitive to spoilage by acetic acid bacteria of the genus Acetomonas. The presence of air may be the determining factor for their development. After 8 months a new increase in yeast cells was noted. These belonged now mainly to the genus Brettano-myces bruxellensis and Br. lambicus. They caused a further slow decrease in residual extract and the appearance of special flavours. Oxidative yeasts of the genera Candida, Cryptococcus, Torulopsis and Pichia were also detected and may be responsible for the formation of a flim on the beer surface after the main fermentation.     

A spheroplast rate assay for determination of cell wall integrity in yeast

The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1–5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5–15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls. © 1998 John Wiley & Sons, Ltd.     

The effect of ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae grown anaerobically in a chemostat

The effects of produced ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. The cells adapted to increased concentrations of produced ethanol by increasing the proportion of ergosterol at the expense of lanosterol, by increasing the proportion of phosphatidylinositol at the expense of phosphatidylcholine, and by increasing the amount of C_18:0 fatty acids in total phospholipids at the expense of C_16:0 fatty acids. The produced ethanol had no effect on the phospholipid content nor on the proportion of unsaturated fatty acids in the phospholipids. The specific growth rate had no effect on the phospholipid content, the sterol composition, the phospholipid composition, the fatty acid composition of total phospholipids, or on the proportion of unsaturated fatty acids in the phospholipids of S. cerevisiae. It was not possible to separate the effects of produced ethanol and growth rate on the ergosterol content of the chemostat-grown S. cerevisiae cells.