Effect of relative humidity of hot air on the heat resistance of Bacillus cereus spores

The heat resistance to hot air of spores of Bacillus cereus (ATCC 14579) attached to carriers of stainless steel or silicone rubber was investigated in a range from 1% to 100% relative humidity (RH). Apart from an initial stage, linear survivor curves were obtained for all relative humidities. Neither the attachment itself nor the material of the carrier had an influence on the resistance. A distinct maximum of heat resistance was found at 40% RH. At 122°C the rate constants at 40% RH were five orders of magnitude smaller than at 100% RH and two orders of magnitude smaller than at 1% RH. At relative humidities of more than 40% the rate constants were strongly temperature dependent, whereas at lower relative humidities they were less temperature dependent. No significant influence of the relative humidity on the Arrhenius activation energy was found within each humidity range. The mean values were 295 kJ mol^-1 for relative humidities of 60% to 100% RH and 165 kJ mol^-1 for 1% to 20% RH. The occurrence of a maximum is ascribed to the existence of two inactivation mechanisms, the first is retarded and the second is accelerated by a reduction of relative humidity. It is assumed that the first mechanism is a protein denaturation. The second mechanism may be an oxidative process.     

Effect of heat and ultrasonic waves on the survival of two strains of Bacillus subtilis

The combined effect of ultrasonic (20 KHz, 150 W) and heat treatment on the survival of two strains of Bacillus subtilis in three suspending media (distilled water, glycerol and milk) has been studied. When spores suspended in water or milk were subjected to ultrasonic waves before heat treatments a little or no decrease of the heat resistance was observed. However, both sporicidal agents applied simultaneously (thermo-ultrasonication) decreased by 63% (B. subtilis, var. niger-40) and 74% (B. subtilis ATCC 6051) the decimal reduction times for the heat treatment when the spores were suspended in glycerol and by 79% and 40%, respectively when suspended in milk. The thermo-ultrasonication of spores in water markedly reduced the heat resistance of them (between 99.9% and 70%) in the range 70-95 degrees C but the effect of the thermo-ultrasonication significantly diminished as the temperature of the treatment was approached to the boiling point of the water.     

Effect of intracellular carbohydrates on heat resistance of Dictyostelium discoideum spores.

The effect of intracellular trehalose and glycogen on the survival of spores of Dictyostelium discoideum ATCC 25697 after exposure to supraoptimal temperatures was examined. Cells metabolically perturbed by incubation in glucose and inorganic phosphate have intracellular trehalose and glycogen concentrations fivefold and twofold higher, respectively, than those of the controls. These cells were more resistant to the lethal effects of wet heat (45 degrees to 55 degrees C) than were control cells. The presence of 40 mM trehalose in the buffer during heat stress increased the survival of nonperturbed cells to approximately the level of the perturbed cells. No protection was observed when cells were heated in the presence of exogenous glycogen. Glucose or disaccharides other than trehalose when present during heat stress, had no effect on heat resistance. Nonperturbed cells preincubated in 40 mM trehalose and washed before heat stress were more resistant to killing than were controls. Cells perturbed with inorganic phosphate, which has been shown to increase trehalose concentrations but decrease glycogen concentrations, were also more resistant to the lethal effects of wet heat than were controls. The data suggest that trehalose has an effect on the wet-heat resistance of D. discoideum. Some possible mechanisms are suggested.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Effect of water activities of heating and recovery media on apparent heat resistance of Bacillus cereus spores

Spores of Bacillus cereus were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores. The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors. Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity. As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose. In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values. This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose. When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the efficiency of depressors was also less pronounced. It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.     

Effect of lipid materials on heat resistance of bacterial spores

The apparent heat resistance of spores of Bacillus megaterium, B. subtilis, B. cereus, B. stearothermophilus, and Clostridium botulinum type E in lipids was investigated and compared with the resistance of the spores in phosphate buffer solution. The most pronounced increase in heat resistance was noted for B. subtilis and C. botulinum type E, the increase varying with the type of lipid used. A high water content of the lipids used as heating menstruum lowered the heat resistance of the spores. Possible explanations for the high heat resistance of spores in lipids are discussed.     

Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores.

Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.     

Effects of minerals on resistance of Bacillus subtilis spores to heat and hydrostatic pressure

Among Bacillus subtilis IFO13722 spores sporulated at 30, 37, and 44 degrees C, those sporulated at 30 degrees C had the highest resistance to treatments with high hydrostatic pressure (100 to 300 MPa, 55 degrees C, 30 min). Pressure resistance increased after demineralization of the spores and decreased after remineralization of the spores with Ca(2+) or Mg(2+), whereas the resistance did not change when spores were remineralized with Mn(2+) or K(+), suggesting that former two divalent ions were involved in the activation of cortex-lytic enzymes during germination.     

Mineralization and heat resistance of bacterial spores.

The heat resistances of the fully demineralized H-form spores of Bacillus megaterium ATCC 19213, B. subtilis var. niger, and B. stearothermophilus ATCC 7953 were compared with those of vegetative cells and native spores to assess the components of resistance due to the mineral-free spore state, presumably mainly from dehydration of the spore core, and to mineralization. Mineralization greatly increased heat resistance at lower killing temperatures but appeared to have much less effect at higher ones.     

Heat shock proteins do not influence wet heat resistance of Bacillus subtilis spores

Spores of Bacillus subtilis are significantly more resistant to wet heat than are their vegetative cell counterparts. Analysis of the effects of mutations in and the expression of fusions of a coding gene for a thermostable β-galactosidase to a number of heat shock genes has shown that heat shock proteins play no significant role in the wet heat resistance of B. subtilis spores.     

A note on the effect of sporulation conditions on the resistance of Bacillus spores to heat and chemicals

Spores of Bacillus subtilis SA22 harvested after 22 d incubation on nutrient agar at 30°C were more resistant to 0–04% peracetic acid at 20°C than spores harvested following 2 d incubation. Similarly, spores of B. subtilis globigii B17, harvested after 7 d incubation on a sporulation agar were up to 10 times less resistant to 0.04% peracetic acid at 20°C than spores harvested after 35 d incubation. An increase in resistance to heating at 100°C and to exposure to 17.7% hydrogen peroxide at 20°C occurred as the age of B. subtilis SA22 spores prior to harvesting increased, whereas differences in resistance were not observed with spores of B. subtilis globigii B17.     

Influence of the incubation temperature after heat treatment upon the estimated heat resistance values of spores of Bacillus subtilis

S. CONDÓN, A. PALOP, J. RASO AND F.J. SALA. 1996. The influence of the incubation temperature on the estimated heat resistance for survivors after heat treatment was investigated. The survival curves and the D _t values of spores of Bacillus subtilis heated at different temperatures in pH 7 buffer, obtained after incubating survivors at different temperatures (30, 37, 44 or 51°C), were compared. The incubation temperature influenced the profile of survival curves. Lower incubation temperatures led to bigger D _t values and longer shoulders. D _t values obtained after incubating at 30°C were higher (x3 approx.) than those obtained by incubating at 51°C. The incubation temperature did not modify z values ( z = 9.1). These results show that shoulders are not only due to the activation of dormant spores but also to heat damage repair mechanisms. From the profile of survival curves at different incubation temperatures it would seem that heat damage is accumulative. Cells can repair the initial heat injury, but the accumulation of injuries would eventually make the damage irreversible.     

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro

AIM: To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. METHODS AND RESULTS: The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. CONCLUSIONS: POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.     

Gamma radiation resistance of Bacillus anthracis spores

The aim of the presented study was determined the effectiveness of action the gamma radiation on water suspension B. anthracis spores. The irradiation was performed using a Cobalt 60 (Co 60) source, by using single and fractionary irradiation doses. In the investigations was used B. anthracis stain "Sterne" 34F2. The obtained results show, that gamma radiation effectively inactivates B. anthracis spores. On the efficiency of sterilization process influence the irradiation's method and the number of spores in 1 ml suspension. In the suspension 1.5 x 10(9) spore in 1 ml, sporicidal doses gamma radiation amount to 25.0 kGy (single dose) or 41.5 kGy (fractionary dose). The volume suspension about definite inoculum of spores, subjected working the gamma rays has not influence on sporicidal effectiveness of radiation sterilization.