Induction of flocculation of brewer's yeast strains of Saccharomyces cerevisiae by changing the calcium concentration and pH of culture medium

Strains of Saccharomyces cerevisiae (NCYC 1190, 1214 and 1364) behaved as nonflocculent in defined culture medium, as well as the strain NCYC 1214 in rich medium. Flocculation was induced by Ca²⁺ addition and/or by correcting the pH to a suitable value. Since the free Ca²⁺ concentration is pH-dependent, these two factors (total Ca²⁺ concentration and pH) cannot be dissociated and are the critical parameters governing flocculation, in culture medium, of the brewing strains studied.     

Sequence and genetic organization of the 19.3-kb erythromycin- and dalfopristin-resistance plasmid pLME300 from Lactobacillus fermentum ROT1

Lactobacillus fermentum ROT1 was isolated from a raw milk dairy product. It is resistant to novobiocin, tetracycline, erythromycin and dalfopristin. A chromosomal tetracycline-resistance determinant was identified as tetM. A 19,398-bp plasmid (pLME300), present in several erythromycin-resistant strains of Lb. fermentum, was isolated from strain ROT1 and completely sequenced. Based on putative open reading frames, pLME300 contains at least four different functional regions. In region I, ORF1 shows high homologies to replication proteins of different theta-replicating plasmids. In addition, a tandem repeat of a 22-bp sequence appears 4.5 times. In region II, ORF3 may code for a methylase, and ORF4 has homologies to Mrr restriction system proteins of Deinococcus radiodurans and Escherichia coli suggesting a restriction-modification system. Region III harbours antibiotic-resistance genes, coding for a macrolide-lincosamide-streptogramin B (MLS) methylase Erm(LF) and the streptogramin A acetyltransferase Vat(E), which is identical to Vat(E) from Enterococcus faecium. Furthermore, region III shows a 91% nucleotide sequence identity to an erm-vat linkage of E. faecium. Region IV carries ORFs that appear to be involved in plasmid mobilization as characterized by a putative origin of transfer and a mobilization protein. pLME300 is the largest completely sequenced multi-resistance plasmid isolated from any Lactobacillus strain so far.     

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.     

Improvement on laboratory fermentation equipment to study Saccharomyces cerevisiae metabolism

Beside being an ordinary fermenter, the present equipment was conceived to sample the medium, to store the samples and to record photographs of the yeasts. Ten sensors were used to measure gas exchanges. During the growth of ScM1 (a Saccharomyces cerevisiae strain) on glucose, we could observe two different linear decreases of CO₂ production rates (18.17±0.12 mmol CO₂ h^–2 (g biomass)^–1 and 8.67±0.12 mmol CO₂ h^–2 (g biomass)^–1), together with a sudden variation of slope during the respiro-fermentative phase. Nomenclature F_in InletairFlowl h ^–1 F_out OutletgasFlowl h ^–1 _in Inletairtemperature°C_out Outletgastemperature°CP _atm AtmosphericPressuremmHgP _in InletairOverPressuremmHgP _out OutletgasOverPressuremmHgDODissolvedO ₂ mg l^–1 pO₂ PartialPressureO ₂ in Outlet gas % (v/v) pCO₂ PartialPressureCO ₂ in Outlet gas % (v/v) Int(t) Whole number of hours     

Effect of different starvation conditions on the flocculation of Saccharomyces cerevisiae

AIMS: To study the effect of different starvation conditions on the flocculation of an ale brewing yeast of Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was assessed by a micro-flocculation technique (Soares and Mota 1997). Carbon-starved cells of a NewFlo phenotype strain did not lose flocculation during a 48 h period. Cells incubated only in the presence of fermentable carbon sources (glucose, galactose and maltose at 2%, w/v), showed a progressive flocculation loss. The incubation of cells in 4% (v/v) ethanol did not induce a flocculation loss. The simultaneous incubation of cells in the presence of 2% (w/v) glucose and 15 microg ml(-1) cycloheximide hindered flocculation loss. The presence of 0.1 mmol l(-1) PMSF or 10 mmol l-1 EDTA prevented partially or completely, respectively, the loss of flocculation in the presence of glucose. CONCLUSIONS: Fermentable sugars induced a flocculation loss, which seems to require de novo protein synthesis and the involvement of different proteases. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the role of nutrients on the physiological control of yeast flocculation.     

Addition of nutrients induce a fast loss of flocculation in starved cells of Saccharomyces cerevisiae

Incubation of flocculent cells of a brewing strain of Saccharomyces cerevisiae of the NewFlo phenotype for 8–12 h, in the absence of either carbon or nitrogen source, did not induce a loss of flocculation, although an increase (about two times) in the number of cells occurred in nitrogen starved cells. The addition of glucose or ammonium sulphate to carbon or nitrogen starved cells, respectively, triggered a rapid loss of flocculation.     

衔接重复序列对酵母菌絮凝蛋白功能的调控作用

【目的】了解絮凝基因FLO1中重复DNA序列B和D对絮凝蛋白Flo1p功能的影响,为构建遗传稳定的最小絮凝功能基因奠定理论基础。【方法】通过PCR和融合PCR方法分别克隆到完整的絮凝基因FLO1、重复DNA序列B和D分别缺失的衍生基因FLO1b和FLO1d,分析这些基因在非絮凝酵母中表达对细胞絮凝特性的影响。【结果】与完整絮凝基因相比,重复DNA序列B和D分别缺失后对酵母细胞絮凝强度没有明显影响,但不同基因在酵母菌中表达产生的絮凝特性受环境因素,如甘露糖浓度和pH等的影响有明显差异。FLO1中重复DNA序列B和D缺失后,细胞絮凝特性受甘露糖抑制的敏感性降低;同时对环境pH的改变具有更广泛的适应性。【结论】重复DNA序列B和D对絮凝蛋白Flo1p结构和功能具有调控作用,二者缺失后,特别是D缺失后会使絮凝蛋白在极端酸碱环境下更稳定。     

Controlled formation of volatile components in cider making using a combination of Saccharomyces cerevisiae and Hanseniaspora valbyensis yeast species

The effect of pure and mixed fermentation by Saccharomyces cerevisiae and Hanseniaspora valbyensis on the formation of major volatile components in cider was investigated. When the interaction between yeast strains of S. cerevisiae and H. valbyensis was studied, it was found that the two strains each affected the cell growth of the other upon inoculation of S. cerevisiae during growth of H. valbyensis. The effects of pure and mixed cultures of S. cerevisiae and H. valbyensis on alcohol fermentation and major volatile compound formation in cider were assessed. S. cerevisiae showed a conversion of sugar to alcohol of 11.5%, while H. valbyensis produced alcohol with a conversion not exceeding 6%. Higher concentrations of ethyl acetate and phenethyl acetate were obtained with H. valbyensis, and higher concentrations of isoamyl alcohol and isobutyl were formed by S. cerevisiae. Consequently, a combination of these two yeast species in sequential fermentation was used to increase the concentration of ethyl esters by 7.41–20.96%, and to decrease the alcohol concentration by 25.06–51.38%. Efficient control of the formation of volatile compounds was achieved by adjusting the inoculation time of the two yeasts.     

Metabolic diversity of Saccharomyces cerevisiae strains from spontaneously fermented grape musts

One hundred and fifteen Saccharomyces cerevisiae strains from Aglianico del Vulture, a red wine produced in Southern Italy, were characterized for the production of some secondary compounds involved in the aroma and taste of alcoholic beverages. The strains exhibited a uniform behaviour in the production levels of n-propanol, active amyl alcohol and ethyl acetate, whereas isobutanol, isoamyl alcohol and acetaldehyde were formed with a wide variability. Only five strains produced wines close to the reference Aglianico del Vulture wine for the traits considered. Of these, two strains were selected, underwent to tetrad analysis and the single spore cultures were tested in grape must fermentation. The progeny of one strain showed a significant metabolic variability, confirming the necessity to test starter cultures for the segregation of traits of technological interest. Our findings suggest the selection of specific strains for specific fermentations as a function of the vine variety characteristics in order to take the major advantage from the combination grape must/S. cerevisiae strain.     

Quantification of Saccharomyces cerevisiae viability using BacLight

Yeast viability can be accurately quantified using BacLight, a kit which so far has been used only for bacterial analysis. Upon staining, viable cells can be differentiated from non-viable ones by either confocal laser scanning microscopy (CLSM), epifluorescence microscopy, or flow cytometry. Using Saccharomyces cerevisiae as a model, viabilities quantified by CLSM deviated an average of 1.7% from the actual data, and those determined by flow-cytometry by 1.4%.     

On-line determination of flocculating Saccharomyces cerevisiae concentration and growth rate using a capacitance probe

During continuous alcoholic fermentation, Saccharomyces cerevisiae 38A floc concentration was monitored using an on-line impedance probe. Since the sensor response is linear and does not depend significantly on yeast particle size, an automatic technique of determining the yeast growth rate has been developed and validated against the conventional mass balance method.     

Contribution of winery-resident Saccharomyces cerevisiae strains to spontaneous grape must fermentation

The origin of the Saccharomyces cerevisiae strains that are responsible for spontaneous grape must fermentation was investigated in a long-established industrial winery by means of two different approaches. First, seven selected components of the analytical profiles of the wines produced by 58 strains of S. cerevisiae isolated from different sites and phases of the production cycle of a Grechetto wine were subjected to Principal Components Analysis. Secondly, the same S. cerevisiae isolates underwent PCR fingerprinting by means of delta primers. The results obtained by both methods demonstrate unequivocally that under real vinification conditions, the S. cerevisiae strains colonising the winery surfaces are the ones that carry out the natural must fermentation.     

Evaluation of bacterial contamination in a fed-batch alcoholic fermentation process

Alcoholic fermentation by a commercial baker's yeast in a fed-batch process with cell recycling and high-test molasses as substrate was strongly inhibited by Lactobacillus fermentum CCT 1407 after a few recycles. When total acidity (mainly lactic acid) exceeded 4.8 g/l broth it seriously interfered with yeast bud formation and viability and above 6.0 g/l it decreased alcoholic efficiency.     

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.     

Sex pheromones of the yeast Hansenula wingei and their relationship to sex pheromones in Saccharomyces cerevisiae and Saccharomyces kluyveri

The yeast, Hansenula wingei has two mating types designated 5 and 21. Cells of each mating type were found to produce mating type-specific sex pheromone which induces sexual agglutinability of the opposite mating type. Crude fractions of these pheromones were prepared by using an Amberlite CG 50 (H⁺ type) column. The agglutinability-inducing action of the pheromones required glucose as carbon source, but no external nitrogen source. The action of the pheromones was inhibited by 5 g/ml cycloheximide. The optimum pH for the pheromone action was 4.0. Pheromones of Saccharomyces cerevisiae and Saccharomyces kluyveri induced sexual agglutinability of 5 mating type cells but did not that of 21 mating type cells. a Pheromones of the Saccharomyces yeasts had no effect on both 5 and 21 mating type cells. The sex pheromones of H. wingei had no effect on the sexual agglutinability of inducible a cells of S. cerevisiae. From the experimental results obtained so far, we propose to call 5 and 21 mating types in H. wingei a and mating types, respectively.     

Mutagenic effect of freezing on mitochondrial DNA of Saccharomyces cerevisiae

Although suggested in some studies, the mutagenic effect of freezing has not been proved by induction and isolation of mutants. Using a well-defined genetic model, we supply in this communication evidence for the mutagenic effect of freezing on mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae. The cooling for 2 h at +4 degrees C, followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C resulted in induction of respiratory mutations. The immediate freezing in liquid nitrogen was without mutagenic effect. The study of the stepwise procedure showed that the induction of respiratory mutants takes place during the freezing at -10 and -20 degrees C of cells pre-cooled at +4 degrees C. The genetic crosses of freeze-induced mutants evidenced their mitochondrial rho- origin. The freeze-induced rho- mutants are most likely free of simultaneous nuclear mutations. The extracellular presence of cryoprotectants did not prevent the mutagenic effect of freezing while accumulation of cryoprotectors inside cells completely escaped mtDNA from cryodamage. Although the results obtained favor the notion that the mutagenic effect of freezing on yeast mtDNA is due to formation and growth of intracellular ice crystals, other reasons, such as impairment of mtDNA replication or elevated levels of ROS production are discussed as possible explanations of the mutagenic effect of freezing. It is concluded that: (i) freezing can be used as a method for isolation of mitochondrial mutants in S. cerevisiae and (ii) given the substantial development in cryopreservation of cells and tissues, special precautions should be made to avoid mtDNA damage during the cryopreservation procedures.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Fermentation of brined turnip roots using Lactobacillus plantarum and Leuconostoc mesenteroides starter cultures

The controlled fermentation of turnip slices using Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures led to earlier acid production and earlier and more pronounced inhibition of Enterobacteriaceae than with uninoculated (natural) fermentation. Unlike the natural fermentation, the controlled fermentations did not show a yeast secondary fermentation and also had a better colour. Due to its ability to produce higher amounts of acid, the use of Lact. plantarum is more desirable than of Leuc. mesenteroides.     

Metabolism of prebiotic products containing beta(2-1) fructan mixtures by two Lactobacillus strains

The capacity of two probiotic strains, isolated from human breast milk, to use several beta(2-1) fructan mixtures as carbon and energy source in in vitro cultures has been tested. Results showed that both strains, Lactobacillus gasseri CECT5714 and Lactobacillus fermentum CECT5716, reached higher growth levels on culture media containing fructooligosaccharide mixtures produced by enzymatic synthesis, compared to those obtained by inulin hydrolysis. Furthermore, the shortest beta(2-1) fructan, kestose, was the only prebiotic compound in the mixtures significantly metabolized in all growth media tested. Analysis of short-chain fatty acid production showed no correlation between the fatty acid profile produced and the carbon source used in each experiment. These data could serve to select appropriate beta(2-1) fructans to be used as prebiotics for L. gasseri CECT5714 and L. fermentum CECT5716 and to design suitable symbiotic food products containing the mentioned lactobacilli.