Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores.

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Inactivation of glucose 6-phosphate dehydrogenase during germination and outgrowth of Bacillus cereus T endospores.

The specific activity and total activity of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) under conditions of complete cell breakage fall 10-20-fold during a 3h period of spore germination and outgrowth. The spores must germinate (lose refractility), but do not have to undergo outgrowth, for the loss of activity to occur. Glucose 6-phosphate dehydrogenase activity from cells as any stage of development is completely stable in extracts at 4 degrees C or 30 degrees C. All of the enzyme activity is found in a soluble (50000g supernatant) fraction and remains completely soluble throughout development. Soluble protein and total cellular protein remain constant for about 2h. Proteinases could not be detected or protein turnover demonstrated during the morphogenetic process. Phenylmethanesuophony fluoride and o-phenanthroline, inhibitors of proteolytic enzymes, do not prevent glucose 6-phosphate dehydrogenase inactivation when added to whole cells. Mixing experiments show no inhibitor of glucose 6-phosphate dehydrogenase to be present in late-stage cells. The enzyme is not excreted into the culture medium. Chloramphenicol and rifampicine immediately stop protein synthesis and development but not the inactivation of glucose 6-phosphate dehydrogenase. NaN3, 2,4-dinitrophenol or anaerobiosis immediately stop development and prevent the loss of enzyme activity. A requirement for metabolic energy is therefore probable. Extracts of spores pre-labelled with L[14C]leucine were made at various stages of morphogenesis and subjected to polyacrylamide-gel electrophoresis. Glucose 6-phosphate dehydrogenase, which was identified by a specific stain, did not lose 14C label, and therefore may not be degraded during the inactivation process.     

Heat killing of bacterial spores analyzed by differential scanning calorimetry.

Thermograms of the exosporium-lacking dormant spores of Bacillus megaterium ATCC 33729, obtained by differential scanning calorimetry, showed three major irreversible endothermic transitions with peaks at 56, 100, and 114 degrees C and a major irreversible exothermic transition with a peak at 119 degrees C. The 114 degrees C transition was identified with coat proteins, and the 56 degrees C transition was identified with heat inactivation. Thermograms of the germinated spores and vegetative cells were much alike, including an endothermic transition attributable to DNA. The ascending part of the main endothermic 100 degrees C transition in the dormant-spore thermograms corresponded to a first-order reaction and was correlated with spore death; i.e., greater than 99.9% of the spores were killed when the transition peak was reached. The maximum death rate of the dormant spores during calorimetry, calculated from separately measured D and z values, occurred at temperatures above the 73 degrees C onset of thermal denaturation and was equivalent to the maximum inactivation rate calculated for the critical target. Most of the spore killing occurred before the release of most of the dipicolinic acid and other intraprotoplast materials. The exothermic 119 degrees C transition was a consequence of the endothermic 100 degrees C transition and probably represented the aggregation of intraprotoplast spore components. Taken together with prior evidence, the results suggest that a crucial protein is the rate-limiting primary target in the heat killing of dormant bacterial spores.     

Alteration by heat activation of enzymes localized in spore coats of Bacillus cereus

Heat activation (70 degrees C for 20 min) resulted in alteration in structural proteins and enzymes found in Bacillus cereus spore coats. The three notable changes were increased glycosylation of coat proteins, alteration in polypeptide pattern on sodium dodecyl sulfate - polyacrylamide gels, and an increase in free SH groups of proteins. About three polypeptides leaked out in small quantities from the spore coats during heat activation. The extraction of five spore coat associated enzyme activities was followed during the coat stripping procedures, which left the cortex and core intact. Two of these activities, L-alanine dehydrogenase and purine nucleoside hydrolase, were solubilized when the undercoat was extracted by 1,4-dithioerythritol (DTE) at pH 9.8. Three other activities, a protease, a corticolytic enzyme, and purine nucleoside phosphorylase, were solubilized by both DTE alone and DTE plus urea at pH 9.8. The DTE plus urea extraction removed the two more insoluble coat layers, the outer cross-patch, and the inner pitted layers. Mutants deficient in the cross-patch layer contained normal amounts of the protease, corticolytic, and purine nucleoside phosphorylase activities suggesting their association with the pitted layer. In intact spores all five enzymes were found to be stable to the heat activation treatment. However, extracted and partially purified preparations of protease, purine nucleoside phosphorylase, and L-alanine dehydrogenase were heat sensitive. Similar preparations of corticolytic enzyme and purine nucleoside hydrolase were stable to the heat activation conditions.     

The release of dipicolinic acid during heating and its relation to the heat destruction of Bacillus stearothermophilus spores

The rates of dipicolinic acid (Dpa) release and the rates of death were studied for spores of five strains of Bacillus stearothermophilus. It was observed that a highly significant relationship exists between the rate of Dpa release and rate of spore death for the four out of five strains tested and for all test temperatures. At 115 degrees C the rate of Dpa release was found to be faster than the rate of death, equal at 120 degrees C and slower at 125 degrees C. The role of Dpa in heat resistance was considered and a theory is proposed to explain the mechanism by which the heat resistance of bacterial spores is overcome.     

Involvement of the spore coat in germination of Bacillus cereus T spores

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

Involvement of the spore coat in germination of Bacillus cereus T spores.

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105 degrees C, 120 degrees C, and 131 degrees C, respectively. The estimated Z values were 6.3 degrees C, 6.1 degrees C, and 9.7 degrees C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108 degrees C, 121 degrees C, and 131 degrees C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.     

Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus

The influence of sporulation temperature (20, 30 and 37 °C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 °C were the most heat-resistant. However, spores sporulated at 20 °C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 °C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 °C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 °C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 °C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 °C, respectively.     

High-pressure-mediated survival of Clostridium botulinum and Bacillus amyloliquefaciens endospores at high temperature

Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120 degrees C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110 degrees C) accelerated the inactivation of C. botulinum spores. However, incubation at 100 degrees C, 110 degrees C, or 120 degrees C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120 degrees C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120 degrees C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.     

Pressure inactivation of Bacillus endospores

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80 degrees C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70 degrees C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60 degrees C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.     

Properties and developmental roles of the lysyl- and tryptophanyl-transfer ribonucleic acid synthetases of Bacillus subtilis: common genetic origin of the corresponding spore and vegetative enzymes

The lysyl-transfer ribonucleic acid synthetase (LRS) and tryptophanyl-transfer ribonucleic acid synthetases (TRS) (l-lysine:tRNA ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) have been purified 60- and 100-fold, respectively, from vegetative cells and spores of Bacillus subtilis. There are no significant differences between the corresponding spore and vegetative enzymes with respect to their elution characteristics from columns of phosphocellulose or hydroxylapatite, their molecular weight (~130,000 for LRS and ~87,000 for TRS as determined by gel filtration), their kinetic constants for substrates (in the amino acid-dependent adenosine triphosphate-pyrophosphate exchange reaction), and the kinetics of inactivation by heat and by antibody. The Mg(2+) requirement for optimal enzyme activity of the corresponding spore and vegetative enzyme differ slightly. Mutants having defective (temperature sensitive) vegetative LRS or TRS activities produce spores in which these enzymes are also defective. The mutant spores are more heat sensitive than the parental type, but contain normal levels of dipicolinic acid. They germinate normally at the restrictive temperature (43 C), but are blocked at specific developmental stages in outgrowth. No modification in temperature sensitivity phenotype occurs during outgrowth, nor is there a change in molecular weight of the two enzymes. The implication is that the LRS and TRS activities of the vegetative and spore stages are each coded (at least in part) by the same structural gene. The temperature sensitivity of mutant spores is discussed with respect to those factors which are involved in the formation of the heat-resistant state.     

Physiological responses of Bacillus amyloliquefaciens spores to high pressure

Pressure inactivation behavior of Bacillus amyloliquefaciens spores was investigated in deionized water. The spores of B. amyloliquefaciens were subjected to 105 degrees C and 700 MPa. The magnitude of the decrease in viability after pressure treatment was similar to that after pressure treatment followed by heat shock. The increase of dipicolinic acid (DPA) release was correlated with the spore inactivation, and the hydrophobicity did not significantly change during the pressure-assisted thermal processing (PATP). Lag phase duration increased with increasing pressure process time. The mechanisms of spore germination and inactivation during the PATP were related to a complex physiological process.     

Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures

AIMS: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition. METHODS AND RESULTS: Bacillus subtilis spores prepared at temperatures from 22 to 48 degrees C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures. As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures. Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox. However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures. In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased. However, this latter change was not due to a reduction in cotA gene expression at higher temperatures. CONCLUSIONS: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The precise conditions for the formation of B. subtilis spores have a large effect on many spore properties.     

Hydroperoxide inactivation of enzymes within spores of Bacillus megaterium ATCC19213

Abstract Hydroperoxide inactivation of the protoplast enzymes enolase, aldolase and glucose-6-phosphate dehydrogenase in intact spores of Bacillus megaterium ATCC19213 was assessed by first treating the cells with lethal levels of H₂O₂, then germinating them in the presence of chloramphenicol prior to permeabilization and enzyme assays. Glucose-6-phosphate dehydrogenase proved to be more sensitive to H₂O₂than enolase or aldolase, in agreement with findings for isolated enzymes. Average D values (time for 90% inactivation) for spores treated with 0.50% H₂O₂ were 173 min for enolase, 67 min for aldolase and 32 min for glucose-6-phosphate dehydrogenase, compared with a D value of 34 min for spore killing. H₂O₂ killing of spores was found to be conditional in that recoveries of survivors were greater on complex medium than on minimal medium. Overall, it appeared that oxidative inactivation of enzymes may be important for hydroperoxide killing of spores.     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .     

Combined effects of high hydrostatic pressure and temperature for inactivation of Bacillus anthracis spores

Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants. To determine inactivation kinetics of spores by high pressure, B. anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75 degrees C. The combination of heat and pressure resulted in complete destruction of B. anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75 degrees C, compared to 160 min at 500 MPa and 20 degrees C and 348 min at atmospheric pressure (0.1 MPa) and 75 degrees C. The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.     

Permeabilization and inhibition of the germination of spores of Aspergillus niger for gluconic acid production from glucose

In this study, the role of citral to permeabilize the spores of Aspergillus niger and replace sodium azide in the bioconversion medium was studied. Further, characterization of glucose oxidase of spores was carried out by exposing both permeabilized and unpermeabilized spores to different pressures (1, 2, 2.7 kb) and temperatures (60, 70, 80, 90 degrees C). Unpermeabilized spores after exposure to high temperatures were permeabilized by freezing before using as catalyst in the bioconversion reaction. Results showed that citral permeabilized the spores and could inhibit spore germination in the bioconversion medium. Rate of reaction was significantly increased from 1.5 to 4.35 g/Lh which was higher than the commercial glucose oxidase 2g/Lh). Glucose oxidase activity of A. niger was resistant to pressure. However, pressure treatment could not permeabilize them. Behaviour of fresh and permeabilized spores to temperature varied significantly. Glucose oxidase activity of fresh spores exposed to high temperature was unaffected at 70 degrees C till 15 min and 84% of relative activity was retained even after 1h at 70 degrees C while permeabilized spore got inactivated at 70 degrees C for 15 min, which followed the same pattern as commercial glucose oxidase. Cellular membrane integrity was lost due to permeabilization by freezing which resulted in heat-inactivation of glucose oxidase when spores were permeabilized before heat treatment. Thus, glucose oxidase of spore remains heat stable when unpermeabilized and active while permeabilized and its reaction rate is higher than the commercial glucose oxidase.     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.