Mathematical modeling of Saccharomyces cerevisiae inactivation under high-pressure carbon dioxide

High-pressure carbon dioxide inactivation curves of Saccharomyces cerevisiae at different temperatures were analysed using the modified Gompertz model. Comparable lambda and mu values were obtained under pressure treatment as function of temperature. The phase of disappearance (lambda) and the inactivation rate (mu) of S. cerevisiae were inversely related. Higher mu values were obtained at 50 degrees C than at 40, 30 and 20 degrees C under 10.0 MPa CO2 pressure. Increased pressure and temperature had significant effects on the survival of S. cerevisiae. Arrhenius, linear and square-root models were used to analyse the temperature dependence of the inactivation rate constant. For the Arrhenius model the activation energy (E(mu)) was 56.49 kJ/mol at 10.0 MPa, and 55.70, 53.83 and 52.20 kJ/mol at 7.5, 5.0, and 2.5 MPa, respectively. Results of this study enable the prediction of yeast inactivation exposed to different CO2 pressures and temperatures.     

The activity of plasma membrane H(+)-ATPase is strongly stimulated during Saccharomyces cerevisiae adaptation to growth under high copper stress, accompanying intracellular acidification

For the adaptation of cells of Saccharomyces cerevisiae, a period of latency is necessary before exponential growth is resumed in a medium supplemented with a highly inhibitory concentration of copper. In this work, we have examined some physiological responses occurring during this period of adaptation. The results revealed that plasma membrane H(+)-ATPase (PM-ATPase) activity is strongly stimulated (up to 24-fold) during copper-induced latency in growth medium with glucose, reaching maximal levels when the cells were about to start inhibited exponential growth. This in vivo activation of the ATPase activity by copper was accompanied by the stimulation of the H(+)-pumping activity of the enzyme in vivo and was essentially due to the increase of the apparent V(max) for MgATP. Although the exact molecular basis of the reported plasma membrane ATPase activation was not clarified, no increase in the mRNA levels from the encoding genes PMA1 and PMA2 was apparently detected during copper-induced latency. The physiological response reported here may allow the cells to cope with copper-induced lipid peroxidation and consequent decrease in plasma membrane lipid ordering and increase in the non-specific permeability to protons. The consequences of these copper deleterious effects were revealed by the decrease of the intracellular pH (pH(i)) of the yeast population, from approximately pH(i) 6 to pH(i) 5, during copper-induced latency in growth medium at pH 4.3. The time-dependent patterns of plasma membrane ATPase activation and of the decrease of pH(i) during the period of adaptation to growth with copper correlate, suggesting that the regulation of this membrane enzyme activity may be triggered by intracellular acidification. Consistent with this idea, when exponential growth under copper stress was resumed and the pH(i) of the yeast population recovered up to physiological values, plasma membrane ATPase activity simultaneously decreased from the highly stimulated level attained during the adaptation period of latency.     

高压二氧化碳处理对双孢蘑菇贮藏品质的影响

为了探讨高压二氧化碳处理对双孢蘑菇贮藏品质的影响,分别采用不同高压二氧化碳对双孢蘑菇进行瞬时处理,然后于4℃条件下贮藏8d,测定失重率、硬度、颜色、总酚和抗坏血酸含量以及PPO、PAL和POD活性的变化。结果表明:0.3 MPa高压二氧化碳瞬时处理对双孢蘑菇贮藏保鲜效果最佳,该处理能较好保持双孢蘑菇的硬度、降低其失重率和抑制其褐变,同时还能促进双孢蘑菇多酚和抗坏血酸的积累、降低PPO和POD的活性以及提高PAL的活性。因此,0.3 MPa二氧化碳处理可以较好保持双孢蘑菇的贮藏品质。     

Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains

Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance.     

Application of high power ultrasound in the supercritical carbon dioxide inactivation of Saccharomyces cerevisiae

The objective of the study was to analyze the influence of high power ultrasound (HPU) on the supercritical carbon dioxide (SC-CO₂) inactivation kinetics of Saccharomyces cerevisiae and to determine the effect of the temperature (31–41 °C), pressure (100–350 bar) and composition of the medium (YPD Broth, apple and orange juice) on the process of inactivation. Using a batch-mode SC-CO₂ at 350 bar and 36 °C, a reduction of 6.7 log-cycles was obtained after 140 min of treatment. However, when HPU (40 W ± 5 W and 30 kHz) was applied during the SC-CO₂ treatments, a reduction of 7 log-cycles was achieved after 2 min of treatment for all pressures and temperatures applied. The effect of increasing pressure (from 100 to 350 bar, 36 °C) or temperature (from 31 to 41 °C, 225 bar) did not significantly influence this inactivation level. The application of ultrasound leads to a vigorous agitation and cavitation which could accelerate the SC-CO₂ dissolving in the medium. This accelerates the penetration of CO₂ into cells and its inactivation mechanisms. In batch operations the application of HPU increases the speed of reaching saturation solubility of CO₂ in many liquid media and significantly reduces microbial inactivation times.     

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

The existence of a K⁺/H⁺ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K⁺/H⁺ transport system, which is able to catalyse both K⁺ influx and efflux, is mainly driven by the K⁺ transmembrane gradient and can function even if the plasma membrane H⁺-ATPase is not active. Using the anionic oxonol VI and the cationic DISC₂(5) probes, it was shown that a membrane potential is not created during K⁺ fluxes. Such a dye response argues for the presence of a K⁺/H⁺ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6·8. This reversible transport system presents a high selectivity for K⁺ among other monovalent cations and a higher affinity for the K⁺ influx into the vesicles (exit from cells). The possible role of this K⁺/H⁺ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.     

Relationship between ethanol tolerance, H+ -ATPase activity and the lipid composition of the plasma membrane in different wine yeast strains

Ethanol tolerance, ATPase activity and the lipid composition of the plasma membrane to study potential relationship among them were examined in five different wine yeast strains. Yeast cells were subjected to ethanol stress (4% v/v). Principal component analysis of the results revealed that the wine yeasts studied can be distinguished in terms of ATPase activity and oleic acid (C18:1), and palmitoleic acid (C16:1), in plasma membrane. Multiple regression analysis was used to identify a potential influence of some components of the plasma membrane on ethanol tolerance and ATPase activity. Based on the results, the ergosterol, oleic acid and palmitoleic acid are highly correlated with ATPase activity and ethanol tolerance. Ethanol tolerance and the ATPase activity of the plasma membrane were correlated at the 96.64% level with the oleic acid and ergosterol in plasma membrane. The Saccharomyces cerevisiae var. capensis flor yeast strain, which exhibited the highest ergosterol concentration in plasma membrane when grown in the presence of 4% v/v ethanol, was found to be the most ethanol-tolerant.     

The in vivo activation of Saccharomyces cerevisiae plasma membrane H+-ATPase by ethanol depends on the expression of the PMA1 gene,but not of the PMA2 gene

The expression of the PMA1 and PMA2 genes during Saccharomyces cerevisiae growth in medium with glucose plus increasing concentrations of ethanol was monitored by using PMA1-lacZ and PMA2-lacZ fusions and Northern blot hybridizations of total RNA probed with PMA1 gene. The presence of sub-lethal concentrations of ethanol enhanced the expression of PMA2 whereas it reduced the expression of PMA1. The inhibition of PMA1 expression by ethanol corresponded to a decrease in the content of plasma membrane ATPase as quantified by immunoassays. Although an apparent correspondence could exist between the increase of plasma membrane ATPase activity and the level of PMA2 expression, the maximal level of PMA2 expression remained about 200 times lower than PMA1. On the other hand, ethanol activated the plasma membrane H⁺-ATPase activity from a strain expressing only the PMA1 ATPase but did not activate that from a strain expressing only the PMA2 ATPase. These results provide evidence that in the presence of ethanol it is the PMA1 ATPase which is activated, probably by a post-translational mechanism and that the PMA2 ATPase is not involved.     

海藻糖与酿酒酵母乙醇耐受性相关性的研究进展

为了得到具有高富锌能力的酵母菌,该研究以面包来源的酵母菌DLY28为出发菌株,重复驯化后筛选获得一株优良耐锌酵母,并通过单因素试验及响应面试验对其富锌培养基进行优化。结果表明,通过驯化筛选得到一株优良耐锌酵母S7,其富锌的最优培养基组成为蔗糖含量82 g/L、胰蛋白胨含量26 g/L、锌含量404 mg/L。在此最优条件下,富锌酵母S7的锌吸附量为18.79 mg/g,生物量（OD600 nm值）为1.49。该研究为富锌酵母的生产以及食品有机锌的开发应用提供理论依据。     

ARL1 and membrane traffic in Saccharomyces cerevisiae

To examine the functions of the Arf-like protein, Arl1p, in Saccharomyces cerevisiae, a null allele, arl1delta::HIS3, was constructed in two strains. In one background only, loss of ARL1 resulted in temperature-sensitive (ts) growth (suppressed on high-osmolarity media). Allelic variation at the SSD1 locus accounted for differences between strains. Strains lacking ARL1 exhibited several defects in membrane traffic. First, arl1delta strains secreted less protein as measured by TCA-precipitable radioactivity found in the media of [(35)S]-labelled cells. A portion of newly synthesized carboxypeptidase Y (CPY) was secreted rather than correctly targeted to the vacuole. Uptake of the fluid-phase marker, lucifer yellow, was reduced. All these phenotypes were exacerbated in an ssd1 background. The ts phenotype of the arl1deltassd1 strain was suppressed by YPT1, the yeast Rab1a homologue, suggesting that ARL1 and YPT1 have partially overlapping functions. These findings demonstrate that ARL1 encodes a regulator of membrane traffic.     

Transport and toxicity of sulphur dioxide in Saccharomyces cerevisiae var ellipsoideus

The kinetics of radioactive SO₂ uptake by S. cerevisiae var. ellipsoideus, the effect of temperature on the uptake, the irreversible thermal inactivation of the uptake and the specificity for the molecular form of SO₂ in the process indicate that mediated transport is operative in this uptake. Both the transport and toxicity of SO₂ in this yeast are strongly pH-dependent and the dependence is based on the determining effect pH exerts on the distribution of the forms which SO₂ assumes in solution.     

酿酒酵母对木质纤维素水解液抑制物耐受性的研究进展

在木质纤维素转化为燃料乙醇的过程中,预处理产生的多种抑制物对酿酒酵母产生毒害作用,抑制细胞内相关代谢关键酶的活性,影响细胞生长和发酵过程,降低发酵过程中糖的转化率。该文介绍了木质纤维素常用的三种预处理方式及在不同预处理过程中抑制物形成的规律和作用机制,对提高酿酒酵母耐受性的方法进行了综述,并展望了未来通过基因工程和代谢工程相互耦合共同提高酿酒酵母性能的研究趋势,为提高酿酒酵母抑制物耐受性和纤维素乙醇发酵效率提供参考和借鉴。     

H+-ATPase activity in Bifidobacterium with special reference to acid tolerance

The acid tolerance of 17 strains of nine species of bifidobacteria was compared using brief exposures to acidic conditions (pH 2-5). In addition, because it has been hypothesized that the acid tolerance of bifidobacteria depends on H+-ATPase activity, the activity of this enzyme in various strains and species was compared. In general, the acid tolerance of bifidobacteria was found to be weak, with the exception of Bifidobacterium lactis and Bifidobacterium animalis. High numbers of all strains of B. lactis and B. animalis survived exposure to pH 3-5 for 3 h. The H+-ATPase activity of the acid-tolerant strains B. lactis LKM512 and JCM 10602T, and B. animalis JCM 1190T, JCM 1253, JCM 7117, and JCM 7124 was higher at pH 4 than at pH 5. In contrast, the H+-ATPase activity of nonacid-tolerant strains was lower at pH 4 than at pH 5.     

发酵工业中酿酒酵母耐性机制的研究进展

由于工业生产条件和成本的限制,发酵过程中酿酒酵母会经受多种不同环境胁迫因子的影响。研究不良环境中酿酒酵母的耐性机制,旨在为提高产品质量、降低生产成本以及相应的基因改良提供理论依据。文中主要综述了酿酒酵母耐受高渗、氧化和乙醇等胁迫因素的机理以及海藻糖对酵母细胞的保护作用。      

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum in orange juice using ultra high-pressure homogenisation

Yeasts and lactic acid bacteria are the usual contaminants in orange juice and responsible for decreasing the shelf life of the product. Ultra high-pressure homogenisation has been shown to be an alternative to the traditional thermal pasteurisation of pumpable foods. The product was pumped through a homogeniser valve at 100 MPa, 150 MPa, 200 MPa, 250 MPa and 300 MPa using two synchronized overlapped intensifiers at a flow rate of approximately 270 mL/min. The inlet temperature was kept at 10 °C, pH at 4.1 and soluble solids at 10.0 °Bx. After processing, the product was immediately cooled to 4 °C and the microbiological count was determined. The study showed that Lactobacillus plantarum and Saccharomyces cerevisiae are sensible to ultra high-pressure homogenisation treatment. The results indicated that pressures higher than 250 MPa were able to completely destroy initial loads of 1.2 × 107 UFC/mL of L. plantarum and 2.9 × 105 UFC/mL of S. cerevisiae in orange juice, making this technology a promising way to nonthermally process orange juices.Industrial relevanceThis paper deals with inactivation of microorganism contaminants of orange juice using dynamic ultra high process technology. It is of industrial interest and relevance to evaluate the use of this non-thermal emerging technology to process fluid foods that may result into better taste, optimum product functionality, safety and quality characteristics.     

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.     

酿酒酵母的乙醇耐受性分子机理与全转录工程

研究酿酒酵母对乙醇耐受性的机理,对于发展乙醇生产有重要意义.酿酒酵母乙醇耐受性涉及到基因组水平上许多基因的复杂的相互作用,已知许多影响细胞膜的完整性和通透性、细胞壁结构、蛋白质构象,以及糖和氨基酸等的吸收等基因都与乙醇耐受性有关,与乙醇诱导相关的基因往往也与其他的环境因素如渗透压、热激、化学毒性、氧化压力等诱导的基因有关或重叠.因此,从基因转录动力学研究酿酒酵母乙醇耐受性并通过全转录工程构建乙醇耐受性工程菌己成为重要的研究热点.该文对近年来酿酒酵母乙醇耐受性分子机理以及全转录工程构建工程菌的研究作一综述,旨在为了解酵母乙醇耐受性机理和培育乙醇耐受性高产酵母菌株提供参考.