The heat resistance of ascospores of four Saccharomyces spp. isolated from spoiled heat-processed soft drinks and fruit products

The heat resistance ( D and z values) of four Saccharomyces spp., viz. S. bailii, S. cerevisiae, S. chevalieri, S. uvarum , was investigated by a previously described method (Put et al. 1977). The highest heat resistance was observed in ascospores of S. cerevisiae 175 showing a decimal reduction time or death rate constant at 60°C of 22.5 minutes. The average D 60°C values of the remaining species were: S. bailii 10 min; S. chevalieri 13 min; and S. uvarum 1.5 min; while z values varied between 4.0 and 6.5°C. It can be postulated that there may exist some comparison between the mechanism of yeast ascospore and bacterial endospore heat resistance. However, (i) washed Saccharomyces ascospores cannot be stored at 5°C for a longer period than 2–3 weeks without loss of heat resistance; and (ii) ascospore heat survival curves are essentially not linear over the whole range, which may be due to some heterogeneity of the ascospore population tested. Besides, it was observed that the heat resistance ( D 60°C values) of the Saccharomyces ascospores proved to be 50–150-fold higher than the D _60°C values of the corresponding vegetative cells.     

An electron microscopical study of the development of peroxisomes during formation and germination of ascospores in the methylotrophic yeast Hansenula polymorpha

Ascospore formation was studied in liquid cultures of the yeast Hansenula polymorpha, previously grown under conditions in which the synthesis of alcohol oxidase was repressed (glucose as growth substrate) or derepressed (methanol, glycerol and dihydroxyacetone as growth substrates and after growth on malt agar plates). In ascospores obtained from repressed cells, generally one small peroxisome was present. The organelle probably originated from the small peroxisome, originally present in the vegetative cells. They had no crystalline inclusions and cytochemical experiments indicated the presence of catalase, urate oxidase and amino acid oxidase activities in these organelles. In ascospores obtained from derepressed cells, generally 1–3 crystalline peroxisomes were observed. These organelles also originated from the peroxisomes originally present in the vegetative cells by means of fragmentation or division. They contained, in addition to the enzymes characteristic for peroxisomes in spores from repressed cells, also alcohol oxidase. The latter enzyme is probably responsible for the crystalline substructure of these peroxisomes.Peroxisomes had no apparent physiological function in the process of ascosporogenesis. A glyoxysomal function of the organelles during germination of the ascospores was also not observed. Germination of mature ascospores in media containing different sources of carbon and nitrogen showed that the function of the peroxisomes present in ascospores of Hansenula polymorpha is probably identical to that in vegetative haploid cells. They are involved in the oxidative metabolism of different carbon and nitrogen sources. Their enzyme profile is a reflection of that of peroxisomes of vegetative cells and their presence may enable the formation of cells which are optimally adapted to environmental conditions extant during spore germination.     

The basis of differential staining of ascospores and vegetative cells of yeasts

Summary An investigation of the staining reactions of ascospores from 10 species of yeasts with 19 different dyes, showed that differentiation of spores from vegetative cells by staining depends on their relative permeability properties. Three possible staining mechanisms are discussed and examined for consistency with the data. It was also discovered that ascospores of Hansenula saturnus, which are not stained by the malachite green method, could be differentiated very satisfactorily from the vegetative cells by staining with hot rose bengal, followed by counter-staining with cold victoria blue B.     

Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis.

The influence of different sporulation temperatures (30, 37, 44 and 52 degrees C) upon heat resistance of Bacillus subtilis was investigated. Heat resistance was greater after higher sporulation temperatures. Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested. Heat resistance increased about tenfold in the range of 30-44 degrees C. Sporulation at 52 degrees C did not show any further increase in heat resistance. This effect was constant over all the range of heating temperatures tested (100-120 degrees C). z value remained constant (z = 9 degrees C). Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature. Spores obtained from cells incubated at 32 or 52 degrees C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis

The influence of different sporulation temperatures (30, 37, 44 and 52°C) upon heat resistance of Bacillus subtilis was investigated.
Heat resistance was greater after higher sporulation temperatures. Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested. Heat resistance increased about tenfold in the range of 30–44°C. Sporulation at 52°C did not show any further increase in heat resistance.
This effect was constant over all the range of heating temperatures tested (100–120°C). z value remained constant ( z = 9°C).
Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature. Spores obtained from cells incubated at 32 or 52°C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.     

Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates carrying plasmid genes versus chromosomal enterotoxin genes

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringens isolates associated with food poisoning carry a chromosomal cpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (D values) at 55 degrees C than vegetative cells of plasmid cpe isolates exhibited. Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100 degrees C than the spores of plasmid cpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomal cpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.     

Preparation of Free Heat-Resistant Ascospores from Byssochlamys Asci

When Byssochlamys is grown for production of ascospores, some of the asci break up into their constituent ascospores, whereas others do not. For heat resistance studies, it is desirable to prepare a uniform suspension of free ascospores. This was accomplished with the aid of a pressure cell, from which a suspension of asci under high pressure was released to atmospheric pressure through a small orifice. Spores so treated had about the same heat resistance as untreated spores.     

Cyclic-AMP content and trehalase activation in vegetative cells and ascospores of yeast

Addition of glucose to yeast ascospores, glucose-grown vegetative cells from the stationary growth-phase or acetate-grown vegetative cells from the logarithmic growth-phase induces a rapid tenfold increase in the activity of trehalase. Trehalase activation is followed by a period of slow inactivation. It was possible to reverse the inactivation in the presence of glucose in all cell types immediately and completely by subsequent addition of a nitrogen source. This reactivation by nitrogen sources is in disagreement with proteolytic breakdown being responsible for trehalase inactivation in the presence of glucose. The addition of glucose induced in all cell types a rapid transient increase of the cellular cyclic-AMP content. In ascospores the increase of the cyclic-AMP level was about twofold, in glucose-grown stationary-phase vegetative cells four- to fivefold and in acetate-grown vegetative cells about sevenfold. Subsequent addition in the presence of glucose of a nitrogen source caused a new twofold increase of the cyclic-AMP level in ascospores. In the other two cell types however addition of a nitrogen source after the initial transient increase of the cyclic-AMP level did not produce a significant new increase. Although the data obtained for ascospores at first seemed to confirm the crucial role of the increase in the cyclic-AMP level for the activation of trehalase, the data obtained afterwards for vegetative cells indicated that it is possible to activate trehalase in yeast without a concomitant increase of the total cellular cyclic-AMP content.Abbreviations Mes 4-Morpholineethanesulfonic acid - Tris tris(hydroxymethyl)-aminomethane     

Importance of ascospore ornamentation in the taxonomy of Daldinia

Representative specimens of fifteen Daldinia spp. were studied for ultrastructural characteristics of their ascospores by scanning electron microscopy (SEM). The ornamentation of their outermost spore layers was found to be species-consistent, confirming the results of concurrent studies on the morphology of their teleomorphs and anamorphs, secondary metabolite profiles and PCR-based genetic fingerprints. Daldinia spp. may either show smooth or transversally striated ascospores. The spores of the species within the latter group are always ellipsoid-equilateral to ellipsoid-inequilateral with narrowly rounded ends. Smooth, broadly ellipsoid to cylindrical ascospores were observed in all species (D. caldariorum, D. fissa and D. loculata) that are known to produce their stromata on substrates damaged by fire. The ascospores of D. concentrica differed from those of D. childiae (i.e., the cosmopolitan taxon previously regarded as D. concentrica ss. auct.) and other Daldinia spp. in showing a very faint ornamentation, which only became visible at 10000× magnification by SEM. A specimen collected on the isle of Jersey (Channel Islands, UK) showed morphological similarities to the pantropical D. eschscholzii, but its ascospores appeared smooth by SEM, and it may therefore represent a previously undescribed species. Dedicated to Professor Yoshinori Asakawa, Tokushima, Japan, on the occasion of his 60^th birthday PH-R Life Science Center Natural Products     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

Synthesis of a spore-specific surface antigen during sporulation of Saccharomyces cerevisiae

Antisera raised against purified yeast ascospores caused agglutination of both ascospores and vegetative cells. A spore-specific activity was obtained by absorbing out anti-vegetative activity with vegetative cells. The anti-vegetative cell activity was directed against mannan, and was probably due to exposure of some spore coat mannan at the spore surface since concanavalin A and lentil lectin also caused agglutination of ascospores. The spore-specific activity was probably determined by a protein or proteins, since extraction of spores with a mixture of sodium dodecyl sulphate and dithiothreitol markedly affected their agglutination by the spore-specific serum. The spore-specific antigen was synthesized in a soluble form during sporulation several hours before the appearance of the spore surface and the pool of soluble antigen declined as the spore was assembled. Synthesis of the soluble antigen was inhibited by adding cycloheximide at all times up to its first appearance in the sporulating cell.     

Heat and UV light resistance of vegetative cells and spores of Bacillus subtilis Rec-mutants.

The heat and UV light resistance of spores and vegetative cells of Bacillus subtilis BD170 (rec+) were greater than those of B. subtilis BD224 (recE4). Strain BD170 can repair DNA whereas BD224 is repair deficient due to the presence of the recE4 allele. Spores of a GSY Rec+ strain were more heat resistant than spores of GSY Rec- and Uvr- mutants. The overall level of heat and UV light resistance attained by spores may in part be determined by their ability to repair deoxyribonucleic acid after exposure to these two physical mutagens.     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

Oxylipin-coated hat-shaped ascospores of Ascoidea corymbosa

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

Properties of fructose 1,6-diphosphate aldolases from spores and vegetative cells of Bacillus cereus

Fructose 1,6-diphosphate aldolase from cells of Bacillus cereus appears to be typical Class II aldolase as judged by its functional and physical properties. Spore and vegetative cell aldolase had similar enzymatic, immunochemical, and heat resistance properties in the absence of calcium, but they differed in their thermal stabilities in the presence of calcium, their Stokes' radii, their mobility in acrylamide gel electrophoresis, and their molecular weights. The pH optimum for both enzymes was 8.5, and their K(m) with respect to substrate was 2 x 10(-3)m. Highly purified spore and vegetative cell aldolases were both heat labile with half-lives of 4 min at 53 C and pH 6.4. In the presence of 3 x 10(-2)m solution of calcium ions, the stability of the spore protein increased 12-fold but the vegetative form became more heat labile. The enhanced stability of the spore aldolase was not diminished by dialysis or gel filtration but was lost after chromatography on diethylaminoethyl cellulose at pH 7.4. Aldolase from vegetative cells exists in an equilibrium mixture of two molecular weights, 115,000 and 79,000 in the approximate ratio of 1:4, respectively. The molecular weight of spore aldolase is 44,000. Spore aldolase was more mobile during electrophoresis than its vegetative cell counterpart because of its smaller size.     

The radiation resistance of ascospores and sclerotia of Pyronema domesticum

The Food and Drug Administration has become aware of several instances where supposedly sterile medical surgical products made of Chinese cotton have been found to contain viable Pyronema domesticum. The aim of this research was to determine the gamma and electron beam radiation resistance values for the two dormant phases (ascospores and sclerotia) of P. domesticum. The resistance values were obtained by developing a standardized system to cultivate, purify, and harvest biological indicators containing sclerotia or ascospores. Ascospores were more resistant to radiation than sclerotia. The D ₁₀ values for sclerotia were 0.79 and 1.09 kGy for strains 32030 and 14881, respectively. The resistance value for wild type ascospores was 2.83 kGy. The current standard for assuring radiation sterilization of medical devices is ISO 11137. This standard was developed to address the propensity for highly radiation-resistant organisms such as P. domesticum. Prior to the standard, biological indicators such as Bacillus pumilus, having a nominal D ₁₀ value or 1.7 kGy, were used to determine the sterility of many medical devices. Journal of Industrial Microbiology & Biotechnology (2002) 29, 51–54 doi:10.1038/sj.jim.7000267 Received 09 October 2001/ Accepted in revised form 08 April 2002     

Nonisothermal heat resistance determinations with the thermoresistometer Mastia

Aims:  To design and build a thermoresistometer, named Mastia, which could perform isothermal and nonisothermal experiments.
Methods and Results:  In order to evaluate the thermoresistometer, the heat resistance of Escherichia coli vegetative cells and Alicyclobacillus acidoterrestris spores was explored. Isothermal heat resistance of E. coli was characterized by D _60°C = 0·38 min and z =  4·7°C in pH 7 buffer. When the vegetative cells were exposed to nonisothermal conditions, their heat resistance was largely increased at slow heating and fast cooling rates. Isothermal heat resistance of A. acidoterrestris was characterized by D _95°C = 7·4 min and z =  9·5°C in orange juice. Under nonisothermal conditions, inactivation was reasonably well predicted from isothermal data.
Conclusions:  The thermoresistometer Mastia is a very suitable instrument to get heat resistance data of micro-organisms under isothermal and nonisothermal treatments.
Significance and Impact of the Study:  The thermoresistometer Mastia can be a helpful tool for food processors in order to estimate the level of safety of the treatments they apply.     

Thermal destruction of dried vegetative yeast cells and dried bacterial spores in a convective hot air flow: strong influence of initial water activity

Thermal treatment of Bacillus subtilis spores and Saccharomyces cerevisiae cells dried on glass beads was performed at various initial water activities (in the range 0.10-0.90). Experiments were carried out at 150 degrees C, 200 degrees C and 250 degrees C for 5-120 s. Significant destruction of up to 10(7) vegetative cells and up to 10(5) spores g(-1) was achieved, depending upon treatment conditions. This study demonstrated that the initial water activity (a(w)) value of a sample is very important in the destruction or survival of microorganisms treated with hot air stresses. As described previously, the heat resistance of spores and vegetative cells was strongly enhanced by low initial a(w) values until an optimal a(w) value between 0.30 and 0.50, with maximal viability at 0.35 for both S. cerevisiae and B. subtilis. However, our results highlighted for the first time that very low initial a(w) values (close to 0.10) greatly improved the destruction of spores and vegetative cells. Factors and possible mechanisms involved in the death of vegetative cells and spores are discussed.