Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae

Summary This paper describes the characteristics of the structural and functional organization of cellular membranes rehydrated after dehydration of the yeast Saccharomyces cerevisiae. It was noted that dehydration and subsequent rehydration of yeast cells causes a considerable increase of cytoplasmic membrane permeability. Addition of CaCl₂, glucose and polyethyleneglycol to the rehydration medium caused a decrease in cell permeability, assessed as the losses of potassium ions, nucleotides, as well as the total losses of intracellular compounds. KCl had a positive effect only at concentrations above 10%. Yeast cells, dried to residual moisture lower than 20%, showed a decrease in membrane permeability as temperatures of the rehydration medium increased up to 38°–43°C. Upon reactivation of viable dehydrated cells in a nutrient medium, a reparation of the structural damages of various intracellular membranes takes place. It was established that at cell dehydration to residual moistures of 8%–12% all the free and a part of bound water is evaporated from cells.     

Survival of Aerobic and Anaerobic Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage

Total and anaerobic counts were ascertained on boneless, cooked, cubed, frozen chicken meat. We determined survival of aerobes and anaerobes in the natural flora after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and at 50, 85, and 100 C for 10 min. Total and anaerobic counts of bacteria in the rehydrated meat were established during storage of samples at 4, 22, and 37 C-until a spoilage odor was detected. Samples were also inoculated with Clostridium sporogenes and were dried and rehydrated at 100 C and stored at 37 C. Approximately 21% of the aerobes and 37% of the anaerobes survived drying and rehydration at room temperature. Many genera of aerobes, anaerobes, and facultative anaerobes survived drying and rehydration at 50 C; only sporeformers survived rehydration at 85 or 100 C. Low-temperature (4 C) storage of rehydrated meat produced ample shelf life (over 20 days), whereas storage at the higher temperature resulted in a shelf life of less than 30 hr. Approximately 81% of the C. sporogenes cells survived rehydration at 100 C and grew to over 10(7) cells within 40 hr. Our study presents additional data for adequate microbiological control in processing of freeze-dehydrated meat. Also, it points out the natural selection for sporeformers at high temperature of rehydration, stressing the need for consumer education in product handling for safety purposes.     

Pyruvate metabolism of the hyperthermophilic archaebacterium Pyrococcus furiosus

The hyperthermophilic anaerobe Pyrococcus furiosus was found to grow on pyruvate as energy and carbon source. Growth was dependent on yeast extract (0.1%). The organism grew with doublings times of about 1 h up to cell densities of 1–2×10⁸ cells/ml. During growth 0.6–0.8 mol acetate and 1.2–1.5 mol CO₂ and 0.8 mol H₂ were formed per mol of pyruvate consumed. The molar growth yield was 10–11 g cells(dry weight)/mol pyruvate. Cell suspensions catalyzed the conversion of 1 mol of pyruvate to 0.6–0.8 mol acetate, 1.2–1.5 mol CO₂, 1.2 mol H₂ and 0.03 mol acetoin. After fermentation of [3-¹⁴C]pyruvate the specific radioactivities of pyruvate, CO₂ and acetate were equal to 1:0.01:1. Cellfree extracts contained the following enzymatic activities: pyruvate: ferredoxin (methyl viologen) oxidoreductase (0.2 U mg^-1, T=60°C, with Clostridium pasteurianum ferredoxin as electron acceptor; 1.4 U mg^-1 at 90°C, with methyl viologen as electron acceptor); acetyl-CoA synthetase (ADP forming) [acetyl-CoA+ADP+Piacetate+ATP+CoA] (0.34 U mg^-1, T=90°C), and hydrogen: methyl viologen oxidoreductase (1.75 U mg^-1). Phosphate acetyl-transferase activity, acetate kinase activity, and carbon monoxide:methyl viologen oxidoreductase activity could not be detected. These findings indicate that the archaebacterium P. furiosus ferments pyruvate to acetate, CO₂ and H₂ involving only three enzymes, a pyruvate:ferredoxin oxidoreductase, a hydrogenase and an acetyl-CoA synthetase (ADP forming).Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - MOPS morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine Part of the work was performed at the Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karlvon-Frisch-Strasse, W-3550 Marburg/Lahn, Federal Republic of Germany     

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 °C, it was carried out for 2 to 3 h at -80 °C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 °C. The applicability of the method was demonstrated using several soybean tissues.     

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.     

Production of ethanol at high temperatures in the fermentation of Jerusalem artichoke juice and a simple medium byKluyveromyces marxianus

Summary Temperatures as high as 36°C and 40°C did not negatively affect the ethanol productivity of Jerusalem artichoke (J.a.) juice batch fermentation and the final concentrations of ethanol were close to those produced at lower temperatures. At higher process temperatures (36–40°C), ethanol toxicity inKluyveromyces marxianus was less important during the fermentation of J.a. juice as compared with a simple medium. In simple medium, the heat-sticking of fermentation was observed and the percentage of unfermented sugars steeply increased from 28°C up to 40°C.     

Enhanced production of bioethanol from waste of beer fermentation broth at high temperature through consecutive batch strategy by simultaneous saccharification and fermentation

Malt hydrolyzing enzymes and yeast glycolytic and fermentation enzymes in the waste from beer fermentation broth (WBFB) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A new ‘one-pot consecutive batch strategy’ was developed for efficient bio-ethanol production by simultaneous saccharification and fermentation (SSF) using WBFB without additional enzymes, microbial cells, or carbohydrates. Bio-ethanol production was conducted in batches using WBFB supernatant in the first phase at 25–67 °C and 50 rpm, followed by the addition of 3% WBFB solid residue to the existing culture broth in the second phase at 67 °C. The ethanol production increased from 50 to 102.5 g/L when bare supernatant was used in the first phase, and then to 219 g ethanol/L in the second phase. The amount of ethanol obtained using this strategy was almost equal to that obtained using the original WBFB containing 25% solid residue at 33 °C, and more than double that obtained when bare supernatant was used. Microscopic and gel electrophoresis studies revealed yeast cell wall degradation and secretion of cellular material into the surrounding medium. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) supported the existence of enzymes in WBFB involved in bioethanol production at elevated temperatures. The results of this study will provide insight for the development of new strategies for biofuel production.     

Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads

Yeast immobilized on alginate beads produced a higher ethanol yield more rapidly than did free yeast cells under the same batch-fermentation conditions. The optimal fermentation conditions were 30 °C, pH 5.0, and 10% initial glucose concentration with 2% sodium alginate beads. The fermentation time using reused alginate beads was 10-14 h, whereas fresh beads took 24 h, and free cells took 36 h. All bead samples resulted in nearly a 100% ethanol yield, whereas the free cells resulted in an 88% yield. Transmission electron microscopy (TEM) showed that the shortened time and higher yield with the reused beads was due to a higher yeast population per bead as well as a higher porosity. The ultrastructure of calcium alginate beads and the alginate matrix structure known as the “egg-box” model were observed using TEM.     

Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus

The production of extracellular inulinase (\-1,2-d-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40° C. At both temperatures, the final biomass concentration exceeded 100 g·l^–1 and more than 2 g enzyme. L^–1 of culture supernatant was produced. The biomass yield on O₂ at 40° C was substantially lower than at 30°C. Nevertheless, at 40° C a growth rate of 0.20 h^–1 could be maintained for a longer period than at 30° C. The unexpected higher O₂-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40°C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter.     

Room and low temperature brewing with yeast immobilized on gluten pellets

A biocatalyst prepared by the immobilization of a cryotolerant strain of Saccharomyces cerevisiae on gluten pellets was used for batch and continuous fermentation at low temperatures. The immobilized yeast showed important operational stability in repeated batch fermentations without a decrease of activity even at 0 and 5°C. Repeated batch fermentations using the biocatalyst resulted in improvement of ethanol productivity in comparison with bottom brewing fermentation and free cells using the same yeast strain. At 0 and 10°C, the fermentation rate was four and seven times higher than that of free cells, respectively. For immobilized yeast, diacetyl and polyphenol contents were lower and the alcohol concentration higher at low temperatures (0–7°C) when compared to free cells. Fine clarity was also observed in the beers. Continuous brewing using gluten-supported biocatalyst had an operational stability of 3 months with relatively high productivity and without contamination. Polyphenol and bitterness contents were lower in the continuous process than those of batch fermentations, but at low temperature (5°C) they were higher. The diacetyl content was higher than in batch fermentations and beers had a fine aroma and taste.     

Influence of red wine fermentation oenological additives on inoculated strain implantation

Pure selected cultures of Saccharomyces cerevisiae starters are regularly used in the wine industry. A survey of S. cerevisiae populations during red wine fermentations was performed in order to evaluate the influence of oenological additives on the implantation of the inoculated strain. Pilot scale fermentations (500 L) were conducted with active dry yeast (ADY) and other commercial oenological additives, namely two commercial fermentation activators and two commercial tannins. Six microsatellite markers were used to type S. cerevisiae strains. The methodology proved to be very discriminating as a great diversity of wild strains (48 genotypes) was detected. Statistical analysis confirmed a high detection of the inoculated commercial strain, and for half the samples an effective implantation of ADY (over 80 %) was achieved. At late fermentation time, ADY strain implantation in fermentations conducted with commercial additives was lower than in the control. These results question the efficacy of ADY addition in the presence of other additives, indicating that further studies are needed to improve knowledge on oenological additives’ use.     

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T_m), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T_m from 8.4 ± 1.7 °C (in undried control samples) to 16.5 ± 1.3 °C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T_m, which began approximately 1 day after initial rehydration and plateaued at 12.5 ± 1.8 °C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T_m and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Kinetic behavior of Candida rugosa in the batch fermentation of sugar beet stillage; Temperature dependence of growth and flocculation characteristics

Summary The aerobic fermentation of sugar beet stillages to yeast biomass was carried out byC.rugosa at 20, 30, and 40°C. At 40°C,C.rugosa grew faster with corresponding COD-reduction, and showed better flocculation characteristics.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

Effect of temperatures during pure culture fermentation of Kinema

Kinema was prepared by fermenting whole cooked soybeans with pure culture of Bacillus subtilis KK2:B10 (MTCC 2756) strain at 35°C, 40°C and 45°C for 24h. Temperature, mesophilic plate counts, relative viscosity, water-soluble nitrogen, formal nitrogen contents and reducing sugars of fermenting soybeans were investigated during fermentation. At higher temperatures the growth rate of B. subtilis KK2:B10 was faster. A remarkable increase in the relative viscosity of kinema was observed at 40°C during fermentation. Water-soluble nitrogen and formol nitrogen to total nitrogen contents increased throughout the 24h of fermentation. Reducing sugars increased during the log phase and then decreased sharply. Kinema matured below 10°C for 1 day after the desired fermentation showed a significant increase in relative viscosity. The quality of kinema was maintained with pure culture fermentation by B. subtilis KK2:B10 at 40°C for 20h and matured at 5°C for 1 day.     

Fungal Invertase as an Aid for Fermentation of Cane Molasses into Ethanol

Comparative studies of the fermentation of cane molasses into ethanol by Saccharomyces cerevisiae in the presence or absence of fungal invertase were performed. When cane molasses was fermented by the yeast at 30°C and pH 5.0, the presence of the enzyme had no effect on ethanol production. At pH 3.5, ethanol production was increased by the addition of invertase. At 40°C, the addition of invertase increased ethanol production by 5.5% at pH 5.0 and by 20.9% at pH 3.5.     

Fate of Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage

Total plate counts were determined on boneless cooked, cubed chicken meat obtained from a commercial processor. Survival of the natural flora was determined after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and 50, 85, and 100 C for 10 min. Total counts of bacteria in the rehydrated samples were determined during storage of the meat at 4, 22, and 37 C until spoilage odor was detectable. Meat samples were inoculated with Staphylococcus aureus, then dried, rehydrated, and stored at the same temperatures. Numbers of surviving organisms in the inoculated samples were determined with use of both selective and nonselective media. Representative genera surviving the various rehydration treatments were determined. Approximately 32% of the bacteria in the meat survived during dehydration and rehydration at room temperature. Many numbers and types of vegetative bacteria also survived rehydration at 50 C. When meat was rehydrated at 85 or 100 C, the initial count was less than one per gram. The only organisms isolated from samples rehydrated at 85 or 100 C were of the genus Bacillus. S. aureus in inoculated samples survived dehydration and rehydration at 60 C. Storage of all rehydrated samples at 4 C gave a good shelf life (18 or more days). The study indicates that freeze-dehydrated meat should be produced with adequate microbiological control and that such meat should be rehydrated in very hot water.     

Moisture distribution and texture of spaghetti rehydrated under different conditions

Dried spaghetti was rehydrated to its optimal cooking state, known as al dente, at 60, 80, and 100 °C, in distilled water or 0.1, 1.0, and 2.0 mol/L sodium chloride solutions. Then, the moisture distributions and stress–strain curves were examined to identify the major factors governing the texture of rehydrated spaghetti. The difference in moisture content between the inner and peripheral regions of rehydrated spaghetti and its breaking stress were greater at higher rehydration temperatures; however, rehydration temperature did not affect breaking strain. The sodium chloride concentration of the immersion solution did not affect moisture distribution or breaking stress, while breaking strain was decreased by rehydration at higher sodium chloride concentrations. The results obtained in this study suggest that moisture distribution within spaghetti and its material properties govern its breaking stress and strain, respectively.