Dependence of synergism of the combined effect of ultrasound and hyperthermia upon intensity of ultrasound

The inactivation of wild-type yeast Saccharomyces cerevisiae was studied after simultaneous treatment with ultrasound and hyperthermia. A temperature range was established within which ultrasound and hyperthermia exert a synergistic action. The effect was shown to depend on ultrasound intensity and the temperature at which the treatment takes place. The temperature range enhancing the ultrasound effect shifted forward higher temperature with increasing ultrasound intensity. For every intensity value, an optimal temperature exists at which the synergetic effect is maximum. The biophysical interpretation of the results obtained is based on the assumption that synergism is due to an additional lethal damage, which arises from the interaction of some sub-lesions induced by both agents. These sublesions are considered non-lethal if the agents are applied separately.     

Effects of low electric treatment on yeast microflora

AIMS: To contribute to the understanding of phenomena related to different intensity electric current treatments on the growth and metabolism of selected micro-organisms using laboratory samples of pure and co-cultures (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermondii strain 465). METHODS AND RESULTS: Low electric current (10, 30, 50 and 100 mA) was applied to prepared samples. Parameters, such as polarity, treatment duration (18-48 h) and type of inoculum yeast, were varied one at a time to highlight their cause-effect relationships. The effects on cell activity as well as microflora viability were assessed. Bioindicators capable of describing the phenomena caused by the electric current on the microflora were identified. CONCLUSIONS: Results demonstrated that a low voltage treatment using graphite electrodes had a greater effect on the viable S. cerevisiae strain 404 microflora. There was less bactericidal activity in the S. cerevisiae strain 404 than in the H. guilliermondii strain 465. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may be of significant importance in the development of new technological processes in the fields of agriculture and food, particularly new fermenting process controls.     

Relationship between sublethal injury and inactivation of yeast cells by the combination of sorbic acid and pulsed electric fields

The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log10 5 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.     

Scaling-up in industrial winemaking using low electric current as an alternative to sulfur dioxide addition

AIMS: To better understand the outcome of employing low electric current (LEC) technology as a new preservation and alternative in wine technology, and to contribute to its development. It is used in industrial-scale winemaking with commercial yeast (Saccharomyces cerevisiae) during the grape must fermentation. METHODS AND RESULTS: LEC (200 mA, time 16 days) was applied to fresh grape must as an alternative method to the usual sulfur dioxide addition used in the industrial process; two tanks, each 30,000 l, were employed for parallel fermentations. The results show that LEC decreased the survival time and increased the death rate of apiculate yeasts, whereas it did not affect the growth and survival of S. cerevisiae. A comparison was made of the main chemical and sensory parameters of the wines obtained. CONCLUSIONS: The results have demonstrated that the low-voltage treatment had a positive effect on the grape juice fermentation (yeast microflora) during the early stages of winemaking. SIGINIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing, for 'biological wine', new winemaking technologies for an innovative control process of yeast fermentation.     

Controlling grape must fermentation in early winemaking phases: the role of electrochemical treatment

AIMS: To contribute to an understanding of the phenomena related to the effect of low electric current (LEC) in grape must fermentation during laboratory and pilot plant scale winemaking, with selected co-culture yeasts (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermodii strain 465). METHODS AND RESULTS: LEC (10, 30, 50 and 100 mA) was applied to fresh grape must as an alternative method to the usual addition of SO2. Parameters such as polarity, treatment duration (24-96 h) and type of inoculum yeast were varied one at a time. LEC decreased the survival time and increased the death rate of H. guilliermondii strain 465 in co-cultures, whereas it did not affect the growth and survival of S. cerevisiae strain 40. A final comparison was made of the main physico-chemical parameters on wine obtained after the different tests. CONCLUSIONS: The results have demonstrated that the low voltage treatment using a pair of graphite electrodes had a positive effect on grape juice fermentation (yeast microflora) during the early stages of winemaking, even with the potential of being an alternative method to the usual addition of SO2. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing new winemaking technologies for an innovative yeast fermentation control process for 'biological wine'.     

Role of DNA damage in the inactivation of Saccharomyces cerevisiae yeasts by 313-nm ultraviolet light

The relative contribution of respiration photoinhibition and DNA damage in the lethal effect induced by 313 nm ultraviolet light (UV) has been investigated in some strains of the yeast Saccharomyces cerevisiae. It has been shown that cells inactivation is essentially due to photo-induced damage to DNA. By photoreactivation experiments it has been found that dimers of the pyrimidine bases are the main lethal photoproducts induced in the DNA by 313 nm ultraviolet light.     

Effects of electroconductive heat treatment and electrical pretreatment on thermal death kinetics of selected microorganisms

Suspensions of yeast cell (zygo Saccharomyces bailii) in a phosphate buffer solution were subjected to conventional (hot water) and ohmic (electric current) heating under identical temperature histories. Experiments were also conducted with cells of Escherichia coli to compare the lethal effect of combination of sublethal electrical preteatment and conventional heating with conventional heating. The kinetic parameters (D,Z,K and E(a)) were determined for both organisms during different treatments. There was no significant difference in the death rate of yeast cells during conventional and ohmic heating at the voltage range used in this study. Results of electrical pretreatment and conventional heating on E. coli indicated differences under certain conditions when compared with pure conventional heating. Thus it is concluded that microbial death during ohmic heating was due primarily to thermal effects with no significant effect of electric current per se. Sublethal electrical pretreatment appears to offer potential for increased bacterial inactivation in certain cases.     

Dependence of the degree of synergism in the simultaneous action of UV light and hyperthermia on yeast cells on the intensity of the UV light

In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae it was shown that the definite temperature interval revealed the synergistic effect under simultaneous action of UV radiation and hyperthermia. The correlation between the degree of synergistic interaction and UV light intensity and irradiation temperature was estimated: the temperature interval synergistically enhancing the UV light effect was shifted towards higher temperatures as the UV light intensity was increasing, the optimal temperature to achieve the most synergistic effect existing for every intensity examined.     

Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo

A DNA fragment from yeast (Saccharomyces cerevisiae) was identified by its homology to a chicken β-tubulin cDNA and cloned. The fragment was shown to be unique in the yeast genome and to contain the gene for yeast β-tubulin, since it can complement a benomyl-resistant conditional-lethal mutation. A smaller subfragment, when used to direct integration of a plasmid to the benomyl resistance locus in a diploid cell, disrupted one of the β-tubulin genes and concomitantly created a recessive lethal mutation, indicating that the single β-tubulin gene of yeast has an essential function. Determination of the nucleotide sequence reveals extensive amino acid sequence homology (more than 70%) between yeast and chicken brain β-tubulins.     

Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF). METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli. This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water. However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators. PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods. Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.     

The role of the repair of double-stranded DNA breaks in the radioresistance of yeast cells

The role of DNA double-strand break (DSB) repair in radioresistance of Saccharomyces cerevisiae G1 cells is discussed. The contribution of rapid and slow DNA DSB repair to radioresistance of diploid yeast has been estimated. The contribution of the DNA DSB repair involving no homologous chromosome interaction is shown to be insignificant in comparison with the recombinational repair. The rapid DNA DSB repair efficiency calculation method based on the proposed yeast radiation inactivation model is given. The calculations are in a satisfactory agreement with the experimental data. Possible mechanisms of radiation induction of lethal sectoring in yeast are discussed. This phenomenon is supposed to be due to the DNA DSB processing during vegetative division of irradiated cells. A general scheme of radiation inactivation of yeast cells is proposed.     

Mechanism of the Synergistic Inactivation of Escherichia coli by UV-C Light at Mild Temperatures

UV light only penetrates liquid food surfaces to a very short depth, thereby limiting its industrial application in food pasteurization. One promising alternative is the combination of UV light with mild heat (UV-H), which has been demonstrated to produce a synergistic bactericidal effect. The aim of this article is to elucidate the mechanism of synergistic cellular inactivation resulting from the simultaneous application of UV light and heat. The lethality of UV-H treatments remained constant below ∼45°C, while lethality increased exponentially as the temperature increased. The percentage of synergism reached a maximum (40.3%) at 55°C. Neither the flow regimen nor changes in the dose delivered by UV lamps contributed to the observed synergism. UV-H inactivation curves of the parental Escherichia coli strain obtained in a caffeic acid selective recovery medium followed a similar profile to those obtained with uvrA mutant cells in a nonselective medium. Thermal fluidification of membranes and synergistic lethal effects started around 40 to 45°C. Chemical membrane fluidification with benzyl alcohol decreased the UV resistance of the parental strain but not that of the uvrA mutant. These results suggest that the synergistic lethal effect of UV-H treatments is due to the inhibition of DNA excision repair resulting from the membrane fluidification caused by simultaneous heating.     

Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.     

Modification of Combined Effect of Radiation and Sodium Chloride on Microorganisms by Chemical Agents during Irradiation

Effects of various chemical agents on the synergistic action of NaCl to the radiation inactivation of bacteria and yeast were studied. The remarkable modification of the radiation lethal effect by some reagents is considered to be a strong evidence for an indirect nature of NaCl synergistic action during irradiation. Most of these modification effects were restricted to the actions during irradiation, supporting the free radical hypothesis in which the short-life active species formed by radiation were considered to attack bacterial cells. Furthermore, pre-irradiation effects under various conditions suggest that the enhancement of radiation lethal effect by NaCl may involve the intracellular events.     

Methods of assaying Bcl-2 and Bax family proteins in yeast

Bcl-2 family proteins play an evolutionarily conserved role in regulating the life and death of the cell. Certain proapoptotic members of the Bcl-2 family, Bax and Bak, have intrinsic cytotoxic activities in that they not only induce or sensitize mammalian cells to undergo apoptosis but also display a lethal phenotype when ectopically expressed in two yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. Furthermore, the antiapoptotic Bcl-2 and Bcl-XL proteins can protect yeast against Bax-mediated lethality, suggesting that the death-regulatory functions of these Bcl-2 family proteins are well preserved in yeast. These observations provide the opportunity to study the function of Bcl-2 family proteins in genetically tractable yeast and to apply classical yeast genetics and functional cloning approaches to the dissection of programmed cell death pathway regulated by Bcl-2 family proteins. We describe here methods used in our laboratory to express and to study the functions of Bcl-2 family proteins in both the budding yeast S. cerevisiae and the fission yeast S. pombe.     

The influence of dissolved CO(2) concentration on the death kinetics of Saccharomyces cerevisiae

AIMS: The effects of temperature and concentration of dissolved CO(2) on the inactivation of Saccharomyces cerevisiae were investigated using a plug-flow system. METHODS AND RESULTS: Several combinations of pressure (4, 6, 8, 10 mega-Pa (MPa)) and temperature (30, 34, 36, 38 degrees C) were used. The D-values obtained were 0.14 min at 8 MPa and 38 degrees C, and 0.15 min at 10 MPa and 36 degrees C. The log D-values were related linearly to the treatment temperature and to the dissolved CO(2) concentration. The thermal resistance constant (zCO(2)(T)) was 9.5 degrees C in the media, including significant levels of CO(2), and the CO(2) resistance constant was z(temp.)(gamma)=7.2 gamma. CONCLUSION: This work has shown that inactivation followed first-order death kinetics, and the effects of temperature and CO(2) concentration were consistent through the critical temperature and pressure of CO(2). Therefore, it is feasible to estimate D-values at any temperature and any CO(2) concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Non-thermal inactivation of micro-organisms in acidic beverages could be realized by the present technique.     

Kinetics of inactivation of Bacillus subtilis spores by continuous or intermittent ohmic and conventional heating

Bacillus subtilis spores were suspended in 0.1% NaCl solution (ca. 10(7) CFU/mL) and treated by conventional or ohmic heating under identical temperature histories. Temperatures tested were in the range of 88 to 99 degrees C. Survival curves and calculated D values showed significantly higher lethality for spores by ohmic than conventional heating. The z or Ea values corresponding to the two heating methods, however, were not significantly different. Spores of B. subtilis were suspended in nutrient broth and treated with conventional and ohmic heating through a single- or a double-stage treatment. In case of double-stage treatment, heating was interrupted by a 20 min of incubation at 37 degrees C to induce a Tyndallization effect. Spore inactivation during double-stage treatment was greater for ohmic than conventional heating. The enhanced spore inactivation by ohmic, compared with conventional, heating resulted from a greater rate of spore death during the first stage of heating and greater decrease in count of viable spores immediately after the incubation period that intervened the heating process. Thus it is concluded that spore inactivation during ohmic heating was primarily due to the thermal effect but there was an additional killing effect caused by the electric current.     

Studying phospholipid metabolism using yeast systematic and chemical genetics

Most phospholipid metabolic pathways in the budding yeast Saccharomyces cerevisiae are analogous to their mammalian counterparts. The biological tractability of yeast provides for an opportunity to rapidly determine functions of specific lipids or lipid metabolic pathways using both classical and chemical-genetic techniques. The recent generation of the yeast genome deletion collection revealed that approximately 75% of yeast genes are not essential for life. Coupling analysis of the yeast deletion collection with automation using high-throughput robotics enables yeast genetic screens to be more thorough and bypasses the requirement for library screens to identify genes of interest. Two high-throughput yeast genetic methods are described, systematic synthetic lethality and chemical genetics. Systematic synthetic lethality is based on the principle that inactivation of two genes separately has minimal effects on cell growth whereas inactivation of both genes simultaneously results in growth defects due to their shared requirement in a particular cellular process. Chemical genetics is the analysis of bioactive compounds to determine processes that regulate susceptibility to the compound under study, and provides powerful data regarding precise targets and mechanism of action that regulate action of the compound.     

Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains

AIMS: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. METHODS AND RESULTS: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0.2 and 6.7 g l(-1)) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. CONCLUSIONS: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.