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.     

The utility of a fungal bibonuclease for reducing the nucleic acid content of permeabilized yeast cells

The main drawback of yeast biomass as a source of protein for human consumption is its high nucleic acid content. The present study deals with the development of a process for reducing the nucleic acid content of strains of Saccharomyces cerevisiae, Candida utilis, C.tropicalis and C.lipolytica by treating with an RNase of Aspergillus candidus strain Ml6a. The cells were permeabilised either by heat‐treatment at 95°C for 5 min. or by treatment with chloroform for 6h followed by a heat treatment at 65°C for 3 min. The former pretreatment was sufficient for C. utilis and C.tropicalis strains whereas S.cerevisiae required the latter treatment. The optimum conditions for the enzymatic treatment were a pH of 4.5–5.0, temperature of 45–55°C, incubation period of 60–90 min and an enzyme to cell ratio of 1:6,000 (w/v). Crude enzyme preparations showed a better activity than the pure enzyme. Under optimal conditions 80–85% of the total nucleic acid could be removed from yeast cells by the enzymatic treatment without any significant concomitant loss of protein.     

Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pH_i) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (μ_max) of 0.58–1.50 h⁻¹. At pH 2.5, slow growth was observed for the isolates from mawè (μ_max of 0.06–0.80 h⁻¹), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pH_i homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.     

Tolerance to thermal and reductive stress in Saccharomyces cerevisiae is amenable to regulation by phosphorylation-dephosphorylation of ubiquitin conjugating enzyme 1 (Ubc1) S97 and S115

Ubiquitin conjugating enzyme 1 (Ubc1) is a member of the E2 family of enzymes that conjugates ubiquitin to damaged proteins destined for degradation by the ubiquitin proteasomal system. It is necessary for stress tolerance and is essential for cell survival in Saccharomyces cerevisiae. Ubc1 has five serine residues that are potential substrates for phosphorylation by kinases. However, no data are available to indicate that Ubc1 function or stress tolerance in S. cerevisiae is regulated by serine phosphorylation of Ubc1. We demonstrate that Ubc1 is phosphorylated in serine residue(s). Furthermore, expression of Ubc1 mutants that are ‘constitutively phosphorylated’ or ‘dephosphorylated’ in mitogen‐activated protein (MAP) kinase serine residues (S97 and S115) affected tolerance to thermal and reductive stress in S. cerevisiae. Specifically, expression of Ubc1S97A and S115D increased thermo‐tolerance in both BY4741 and TetO₇‐UBC1ura3Δ cells. Serine phosphorylation of Ubc1 was decreased in BY4741 cells following exposure at 40 °C. Tolerance to reductive stress in the same strains correlated with the expression of Ubc1S97A. Ubc1 phosphorylation did not show significant alteration under similar conditions. Both hog1Δ and slt2Δ cells expressing Ubc1S115D and Ubc1S115A were rendered tolerant to thermal and reductive stress respectively. Ubc1 phosphorylation was higher in BY4741 cells compared to hog1Δ cells at 30 °C and was significantly reduced in BY4741 cells upon exposure at 40 °C. Taken together, the cell survival assays and Ubc1 phosphorylation status in strains and under conditions as described above suggest that tolerance to thermal and reductive stress in S. cerevisiae may be regulated by MAP kinase‐mediated phosphorylation of Ubc1S97 and S115. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

An improved protocol for the preparation of yeast cells for transformation by electroporation

Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6–67-fold in wild-type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15–300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures. Both DTT and lithium acetate were necessary for maximal transformation frequencies. Pretreatment with lithium and DTT also resulted in an ∼3·5-fold increase in the electroporation transformation frequency of the pathogenic fungus Candida albicans. © 1998 John Wiley & Sons, Ltd.     

The aromatic volatile composition of Lonicera edulis wines produced with three different strains of Saccharomyces cerevisiae

Three different strains of Saccharomyces cerevisiae – D₁₅, Dibosh and 71B – were evaluated in the fermentation of Lonicera edulis wines. Volatile aromatic components were analysed by gas chromatography–mass spectrometry coupled with headspace solid‐phase microextraction. In all, 81 volatile compounds were identified in L. edulis wines, including 43, 48 and 38 individually found in wines fermented with D₁₅, Dibosh and 71B. There were 17 common volatile aromatic components found in all the three L. edulis wines. The main volatile compounds in wines fermented with D₁₅ and Dibosh yeasts were 2‐methyl‐1‐butanol (24.8%) and hexane (20.6%). Pentanol was the primary volatile aromatic compound in wines produced with S. cerevisiae 71B, accounting for 40.8% of total volatile aromatic compounds. Combining the sensory analysis, S. cerevisiae D₁₅ was suggested to be the most suitable strain for producing L. edulis wine. © 2018 The Institute of Brewing & Distilling     

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of l-lactate during fermentation on glucose-rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the l-lactate produced originates from endogenous l-malate and 25% from exogenous l-malate. Moreover, although a small amount of exogenous l-malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded l-malate was converted into l-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades l-malate. Total malolactic fermentation was obtained in this strain, with most of the l-malate converted into l-lactate and CO₂. Moreover, l-malate was used preferentially by the malolactic enzyme in this strain also.     

Adaptive response and tolerance to weak acids in Saccharomyces cerevisiae boulardii: a metabolomics approach

Weak acids inhibit the growth of probiotics, such as Saccharomyces boulardii. We explored the tolerance of S. boulardii to different weak acids. S. boulardii had better fermentation ability under lactic acid conditions compared with acetic and butyric acid conditions; however, the budding of S. boulardii was significantly stronger than that of Saccharomyces cerevisiae under acetic acid conditions. Although the surface structure of S. boulardii was destroyed, it produced more daughter cells. S. boulardii metabolites were also significantly different from S. cerevisiae under acidic stress. The growth of S. boulardii under weak acid conditions differed significantly from that of S. cerevisiae. S. boulardii-mediated fingerprints under weak acid conditions were identified as latent biomarkers, related to fructose and mannose metabolism, tricarboxylic acid cycle, and the glycolysis pathway. Identified biomarkers will aid in the genetic engineering of S. boulardii and other Saccharomyces strains for improved acid resistance and biomass yield.     

A Comparison of Pulsed Electric Field Resistance for Three Microorganisms with Different Biological Factors in Grape Juice via Numerical Simulation

Pulsed electric field (PEF) is a promising nonthermal food preservation technology that is based on the use of electric field to eradicate spoilage and pathogenic microorganisms in food products. The effect of various biological factors on the transmembrane potential of different microorganisms (Staphyloccocus aureus, Escherichia coli DH5α, and Saccharomyces cerevisiae) was investigated by means of both numerical simulation and experimental method. The PEF resistance of different microorganisms in grape juice was compared by applying field strength of 12–24 kV/cm, treatment time of 30–180 μs, and an initial temperature of 30?ºC. The results showed that S. cerevisiae exhibited the least resistance to PEF treatment, E. coli DH5α the second, and S. aureus the third. The simulation results indicated that larger cells like S. cerevisiae presented the higher values of transmembrane potential and induced field strength around the cells compared to E. coli DH5α and S. aureus, which led to a less resistance to PEF treatment. The effect of cell orientation on the induced transmembrane potential was very slight (1.67 % for E. coli DH5α and 3.43 % for S. cerevisiae). The thicker cell membrane caused concentrated electric field in the cell membrane, which enhanced the sensitivity of microorganism to PEF treatment. However, both transmembrane potential and electric field strength decreased with the thickness of cell wall increasing. According to both experimental and simulation results, it was evident that there was significant difference in the inactivation rate between different microorganisms, which could be largely attributed to the biological factors of different microorganisms.     

THE ROLE OF DIMETHYL SULPHOXIDE REDUCTASE IN THE FORMATION OF DIMETHYL SULPHIDE DURING FERMENTATIONS

The ability to produce dimethyl sulphide (DMS) during fermentation of wort is apparently a general characteristic of Saccheromyces cerevisiae and Saccharomyces uvarum. Washed suspensions of these yeasts reduce dimethyl sulphoxide (DMSO) but there is no correlation between DMS formation in fermentation and DMSO reduction in whole cells. Although different strains vary widely in their ability to reduce DMSO they all contain similar levels of NADPH—dependent DMSO reductase. This enzyme is a multi-protein system which closely resembles methionine sulphoxide reductase. Spoilage bacteria including Enterobacter cloacae can reduce DMSO more efficiently than can yeast probably because a different enzyme system is involved.     

Solid-state fermentation by S. cerevisiae with high resistance to ferulic acid improves the physicochemical properties of wheat bran and quality of bran-rich Chinese steamed bread

Wheat bran has numerous health benefits, but its poor processing and sensory properties limit its application in the staple food industry. Fermentation by S. cerevisiae changes the performance of wheat bran. However, high levels of ferulic acid (FA) inhibit S. cerevisiae. The effects of solid-state fermentation of S. cerevisiae with high resistance to FA on the physicochemical properties of wheat bran and the quality of bran-rich Chinese steamed bread (CSB) were investigated. The results showed that the growth of S. cerevisiae was inhibited by FA in a dose-dependent manner. Short-term adaptation strategies efficiently improved the tolerance of S. cerevisiae to FA stress. Compared with the parental strain (PS), fermentation of the short-term adapted strains (adapted strains) significantly increased the FA, total phenol, and soluble dietary fiber content in wheat bran. Wheat bran fermented by the adapted strains had a higher antioxidant capacity than wheat bran fermented by PS. In addition, compared with the PS, the wheat bran fermented by the adapted strains can decrease the hardness, improve the specific volume, and the quality of CSB. Thus, solid-state fermentation of the adapted strain is a potentially effective method to improve the nutritional and physicochemical properties of wheat bran as a cereal food ingredient.     

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.     

Scp160p,a new yeast protein associated with the nuclear membrane and the endoplasmic reticulum,is necessary for maintenance of exact ploidy

We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER–nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER–nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.     

Quantitative analysis of yeast gene function using competition experiments in continuous culture

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan^™ analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10–90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype. © 1998 John Wiley & Sons, Ltd.     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Use of PCR‐DHPLC (Polymerase Chain Reaction—Denaturing High Performance Liquid Chromatography) for the Rapid Differentiation of Industrial Saccharomyces pastorianus and Saccharomyces cerevisiae Strains

Strain specific detection and control of Saccharomyces pastorianus and Saccharomyces cerevisiae starter cultures is of great importance for the fermentation industry. The preconditions of strain specific fermentation characteristics can be ensured by periodic analysis and confirmation of the strain identity. With regard to industrial S. pastorianus and S. cerevisiae strains and a focus on brewing strains, the differentiation methods most available are time‐consuming and not very discriminative. In this work PCR‐DHPLC analysis was investigated as a novel approach for the differentiation of industrially used S. pastorianus and S. cerevisiae strains. The PCR‐DHPLC‐system was specific for S. cerevisiae strains and S. pastorianus hybrid strains that contain IGS2 rDNA, which originates from the S. cerevisiae ancestor. For the DNA of 177 strains of 41 non‐target species, which are typical for beverage and fermentation surroundings, the absence of PCR‐amplificates could be confirmed by DHPLC analysis. It was shown that single strains of S. cerevisiae and S. pastorianus could be differentiated. A strain specific differentiation within the group of top‐fermenting Saccharomyces cerevisiae strains could also be performed. For the group of bottom fermenting S. pastorianus brewing strains, strain‐to‐strain specific differences in the DHPLC chromatograms could be observed which can be used to differentiate and to compare two single strains with each other, although the comparison of chromatograms of an unknown S. pastorianus strain with a set of known S. pastorianus chromatograms could only reveal tendencies towards grouping into types. The differential DHPLC chromatogram characteristics (fluorescence intensities, number of peaks/side‐peaks/peak‐shoulders) within S. pastorianus are present, but not as distinctive as for S. cerevisiae. Additionally PCR‐DHPLC has advantages compared to other differentiation methods, such as species specificity, speed (2.5 h for one sample) and precision with the described limits.     

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