Effect of Cell Moisture on the Thermal Inactivation Rate of Bacterial Spores

Thermal inactivation rates were determined for two strains of Bacillus subtilis var. niger spores after equilibration to various relative humidity (RH) levels. In these tests, small thin stainless-steel squares were each inoculated with a drop of spore suspension and equilibrated to 11, 33, or 85% RH. Following equilibration, the squares were placed on a hot plate preheated to 108, 125, 136, 164, or 192 C for various exposure times and then assayed for surviving organisms. The results revealed that spores of the A strain of B. subtilis were least resistant if preequilibrated to 11% RH and most resistant if preequilibrated to 85% RH. The same trend was obtained at all temperatures except 192 C, at which, no difference was noted, probably because the rapid kill time approaches the heat-up time of the stainless-steel square. The B strain of B. subtilis spores showed an opposite RH effect; that is, the cells preequilibrated to 11% RH were the most resistant. Because the two strains of spores were grown on different media, further studies were conducted at 136 C after subculturing the cells on different media. When the B strain was subcultured on the A strain medium, the pattern was reversed; the cells preequilibrated to low RH were then least resistant. Although it was not possible to reverse these cells to the original pattern by subculturing on the original B strain medium again, the pattern was altered to the point that there was no significant difference in heat resistance of these cells regardless of the preequilibration RH. The same result was obtained when the A strain was grown on the B strain medium; that is, the thermal resistance could not be reversed, but it was altered from the point where the low RH equilibrated cells were least resistant initially to the point where there was no significant difference in any of the cells regardless of what RH was used for preequilibration. The thermal resistance of spores seemed to be dependent on (i) the medium on which the spores are grown, (ii) the RH on which they are exposed before heating, and (iii) some genetic characteristic of the cell.     

Thermal inactivation and injury of Bacillus stearothermophilus spores.

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Thermal inactivation and injury of Bacillus stearothermophilus spores

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Heat stability of Bacillus cereus enzymes within spores and in extracts.

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.     

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.     

A Combined Model for Growth and Subsequent Thermal Inactivation of Brochothrix thermosphacta

A mathematical technique for integrating growth and thermal inactivation models of microorganisms into a smooth combined model that can be applied to circumstances under which the temperature gradually rises from growth to inactivation regions is described. For the death part of the model, a correction term is introduced to allow for additional resistance of the cells gained during slow heating. The model was validated with Brochothrix thermosphacta heated in broth at rising temperatures.     

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.     

Heat resistance of Salmonella senftenberg 775W at various sucrose concentrations in distilled water

Summary The heat resistance of Salmonella senftenberg 775 W, NCTC 9959, has been determined in distilled water pH 6.5 at sucrose concentrations up to 2.20 mol l^–1 at temperatures between 63 and 70°C. Surviving cells were counted on minimal and enriched agar media to investigate the influence of the various nutrients on the recovery of heat injured cells. At various sucrose concentrations and temperatures multiphasic exponential parts of inactivation curves were found. Systematic differences between the recovery media depended on sucrose concentration, temperature and phase of exponential inactivation. At 60°C and sucrose concentrations between 0.52 and 1.82 mol l^–1 the relationship between inactivation rate and sucrose concentration could be described by the equation ln k₅=ln k₀-_T [sucrose]. The activation energy of thermal inactivation reactions, substantially decreased when sucrose (1.82 mol l^–1) was added to the heating menstruum. The activation energies in different recovery agars were of the same order, which suggests that the critical sites in heat inactivation are not key enzymes of the synthetic pathways of amino-acids and nucleotides. The differences between activation energies, calculated for cells of the various exponential phases of inactivation in both non-sucrose and 1.82 mol sucrose per 1 heating media, were also small, further suggesting that these critical sites are the same in cells from the various phases. Compared to published data on the heat resistance of S. senftenberg 775 W, we found a decreased resistance in a non-sucrose medium but an equal or increased resistance, depending on the phase of exponential inactivation, at a sucrose concentration of 1.82 mol l^–1.     

The effect of age on the thermal resistance of arthroconidia from Chrysosporium inops

J. L. KINDERLERER. 1996. Food-borne members of the genus Chrysosporium have been isolated relatively infrequently. The heat resistance of arthroconidia of the xerophilic fungus, Chrysosporium inops Carmichael, was determined in 0.1% peptone at 66C. The survival curve was sigmoid in shape. The initial lag period was due to the chains of arthroconidia. Thermal inactivation occurred when one viable conidium was left per chain. The presence of chains of arthroconidia was confirmed with the cryo scanning electron microscope. The decimal reduction times were obtained from the regression line of the linear death phase for the heat-sensitive spores. The decimal reduction time (D₆₆) increased with increasing spore age. It was 1.67 min for 3-week-old spores, 1.95 min for 4-week-old spores and 5.49 min for 6-week-old spores. The older spores could recover from thermal death if they were given sufficient time. There was a significant increase in D₆₆ value for 6-week-old spores from 3.97 min to 5.49 when the counts were obtained after 14 d incubation (compared to counts after incubation for 10 d). This effect was not seen for the 3- and 4-week-old spores. There was a small population of heat-resistant spores. The initial population of arthroconidia was greater than log 7 cfu ml^-1. After heating for 1 h at 66C approximately log 2.2 cfu ml^-1 survived. These survivors represented approximately 0.001% of the original population.     

Effect of Microwaves on Escherichia coli and Bacillus subtilis

Suspensions of Escherichia coli and Bacillus subtilis spores were exposed to conventional thermal and microwave energy at 2,450 MHz. The degrees of inactivation by the different energy sources were compared quantitatively. During the transient heating period by microwave energy, approximately a 6 log cycle reduction in viability was encountered for E. coli. This reduction was nearly identical to what is expected for the same time-temperature exposure to conventional heating. Heating of B. subtilis spores by conventional and microwave energy was also carried out at 100 C, in ice and for transient heating. The degree of inactivation by microwave energy was again identical to that by conventional heating. In conclusion, inactivation of E. coli and B. subtilis by exposure to microwaves is solely due to the thermal energy, and there is no per se effect of microwaves.     

Effects of inoculum level and pressure pulse on the inactivation of Clostridium sporogenes spores by pressure-assisted thermal processing

The effects of initial concentration and pulsed pressurization on the inactivation of Clostridium sporogenes spores suspended in deionized water were determined during thermal processing (TP; 105 degrees C, 0.1 MPa) and pressure-assisted thermal processing (PATP; 105 degrees C and 700 MPa) treatments for 40 min and 5 min holding times, respectively. Different inoculum levels (10(4), 10(6), and 10(8) CFU/ml) of C. sporogenes spores suspended in deionized water were treated at 105 degrees C under 700 MPa with single, double, and triple pulses. Thermally treated samples served as control. No statistical significances (p > 0.05) were observed among all different inoculum levels during the thermal treatment, whereas the inactivation rates (k1 and k2) were decreased with increasing the initial concentrations of C. sporogenes spores during the PATP treatments. Double- and triple-pulsed pressurization reduced more effectively the number of C. sporogenes spores than single-pulse pressurization. The study shows that the spore clumps formed during the PATP may lead to an increase in pressure-thermal resistance, and multiple-pulsed pressurization can be more effective in inactivating bacterial spores. The results provide an interesting insight on the spore inactivation mechanisms with regard to inoculum level and pulsed pressurization.     

A vitalistic model to describe the thermal inactivation ofListeria monocytogenes

Summary Thermal inactivation of microorganisms has traditionally been described as log-linear in nature, that is the reduction in log numbers of survivors decreases in a linear manner with time. This is despite a plethora of data that shows consistent deviations from such kinetics for a wide range of organisms and conditions and that cannot be accounted for by experimental artifacts. Existing thermal death models fail to take such deviations into account and also fail to quantify the effects of heating menstruum on heat sensitivity. The thermal inactivation ofListeria monocytogenes ATCC 19115 has been investigated using a factorially-designed experiment comparing 45 conditions of salt concentration, pH value and temperature. Heating was carried out using a Submerged Coil heating apparatus that minimized experimental artifacts. Low pH values increased, whilst high salt concentrations decreased heat sensitivity. Results showed a significant and consistent deviation from log-linear kinetics, particularly at low temperatures. A number of distributions were tested for suitability to describe the variability of heat sensitivity within the population of heated cells (vitalistic approach). The use of the logistic function and log dose (log time) allowed the development of an accurate unifying predictive model across the whole range of heating conditions. It is proposed that this approach should be tested as a generalized modeling technique for death kinetics of vegetative bacteria.     

Method for Collecting Naturally Occurring Airborne Bacterial Spores for Determining Their Thermal Resistance

The ability to determine the thermal resistance of naturally occurring air borne bacterial spores associated with spacecraft and their assembly areas has been hindered by lack of an effective collecting system. Efforts to collect and concentrate spores with air samplers or from air filters have not been successful. A fallout method was developed for this purpose and tested. Sterile Teflon ribbons (7.6 by 183 cm) were exposed in pertinent spacecraft assembly areas and subsequently treated with dry heat. Thermal inactivation experiments were conducted at 125 and 113 C. Heating intervals ranged from 1 to 12 h at 125 C and 6, 12, 18, and 24 h at 113 C. Eight hours was the longest heating time yielding survivors at 125 C, whereas survivors were recovered at all of the heating intervals at 113 C. D_125C values were calculated using the fractional-replicate-unit-negative technique of Pflug and Schmidt (1968) and ranged from 25 to 126 min. This variation indicated that the most probable number of survivors at each heating interval did not fall on a straight line passing through the initial spore population. However, the most-probable-number values taken alone formed a straight line suggesting logarithmic thermal destruction of a subpopulation of spores with a D_125C value of 6.3 h.     

Experimental modelling of thermal inactivation of urease

Thermal inactivation of jack bean urease (EC 3.5.1.5) was investigated in a 0.1 M phosphate buffer with pH 7. An injection flow calorimetry method was adapted for the measurement of the enzyme activity. The inactivation curves were measured in the temperature range of 55 to 87.5 degrees C. The curves exhibited a biphasic pattern in the whole temperature range and they were well fitted with a biexponential model. A simultaneous fit of all inactivation data was based on kinetic models that were derived from different inactivation mechanisms and comprised the material balances of several enzyme forms and the enthalpy balance characterizing the initial heating period of enzyme solution. The multitemperature evaluation revealed that an adequate model had to incorporate at least three reaction steps. It was concluded that the key reaction steps at urease thermal inactivation were the reversible dissociation/denaturation of native form into an inactive denatured form, and irreversible association reactions of both the denatured and native forms.     

C. botulinum inactivation kinetics implemented in a computational model of a high‐pressure sterilization process

High‐pressure, high‐temperature (HPHT) processing is effective for microbial spore inactivation using mild preheating, followed by rapid volumetric compression heating and cooling on pressure release, enabling much shorter processing times than conventional thermal processing for many food products. A computational thermal fluid dynamic (CTFD) model has been developed to model all processing steps, including the vertical pressure vessel, an internal polymeric carrier, and food packages in an axis‐symmetric geometry. Heat transfer and fluid dynamic equations were coupled to four selected kinetic models for the inactivation of C. botulinum; the traditional first‐order kinetic model, the Weibull model, an nth‐order model, and a combined discrete log‐linear nth‐order model. The models were solved to compare the resulting microbial inactivation distributions. The initial temperature of the system was set to 90°C and pressure was selected at 600 MPa, holding for 220 s, with a target temperature of 121°C. A representation of the extent of microbial inactivation throughout all processing steps was obtained for each microbial model. Comparison of the models showed that the conventional thermal processing kinetics (not accounting for pressure) required shorter holding times to achieve a 12D reduction of C. botulinum spores than the other models. The temperature distribution inside the vessel resulted in a more uniform inactivation distribution when using a Weibull or an nth‐order kinetics model than when using log‐linear kinetics. The CTFD platform could illustrate the inactivation extent and uniformity provided by the microbial models. The platform is expected to be useful to evaluate models fitted into new C. botulinum inactivation data at varying conditions of pressure and temperature, as an aid for regulatory filing of the technology as well as in process and equipment design. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effect of microwave radiation on inactivation of Clostridium sporogenes (PA 3679) spores.

Three techniques for studying effects of microwave radiation on microorganisms were introduced. Spores of Clostridium sporogenes (PA 3679) were chosen as a test organism because the kinetic parameters for thermal inactivation are well known and because of the importance of the genus Clostridium to the food industry. For the first technique, a specially designed kinetics vessel was used to compare inactivation rates of microwave-heated and conventionally heated spores at steady-state temperatures of 90, 100, and 110 degrees C. Rates were found to be similar at the 95% confidence level. The second and third techniques were designed to study the effect of relatively high power microwave exposure at sublethal temperatures. In the second approach, the suspension was continuously cooled via direct contact with a copper cooling coil in a well-mixed vessel, outside the microwave oven. The suspension was pumped through a Teflon loop in the oven, where it continuously absorbed approximately 400 W of microwave power. Inactivation occurred in both irradiated and unirradiated samples. It was suspected that copper ions entered the suspension from the copper coil and were toxic to the spores. The fact that the results were similar, however, implied the absence of nonthermal microwave effects. In the third approach, the copper coil was replaced with a silicone tubing loop in a microwave transparent vessel. The suspension was continuously irradiated at 150 W of microwave power. No detectable inactivation occurred. Results indicated that the effect of microwave energy on viability of spores was indistinguishable from the effect of conventional heating.     

Comparison of TDT and Arrhenius models for rate constant inactivation predictions of Bacillus stearothermophilus heated in mushroom-alginate substrate

Bacillus stearothermophilus spores were heated in a mixture of mushroom puree with alginate, in the temperature range 110–130°C. Both Arrhenius and the traditional Bigelow models were used to describe the dependence of the constant inactivation rate ( K ) or ( D ) with the temperature. Results showed that both are very good linear regression models, but a discrepancy between 20 and 45% was found in the constant inactivation rate predicted by both models at high temperatures (125–140°C). Despite this discrepancy, the Arrhenius model was a better predictor of the inactivation rate constants at temperatures of 125 and 130°C for B. stearothermophilus spores heated in an alginate-mushroom mixture.     

Spores of microorganisms XVI

The heat inactivation of spores ofBacillus cereus irradiated and non-irradiated with X-rays was investigated with respect to their dipicolinic acid (DPA) content. Spores with a high DPA content (98 μg./mg. dry weight) displayed a biphasic heat inactivation curve. This biphasic character of the curve was preserved for the heat inactivation of previously irradiated spores even if the length of the initial lag of inactivation rate was reduced. Spores with a lower DPA content (24 μg./mg. dry weight) display a single-phase logarithmic order of dying from the beginning of heating. In both types of spores differing in the DPA content a pronounced sensitization of radiation-surviving spores toward subsequent heating was observed.     

Mechanisms of heat inactivation in Salmonella serotype Typhimurium as affected by low water activity at different temperatures

AIMS: To determine the effect of reduced water activity (a(w)) on thermal inactivation of Salmonella serotype Typhimurium at different temperatures and its mechanism. METHODS AND RESULTS: D-value determinations at a range of different temperatures showed that heating at reduced a(w) (0.94, produced by addition of glucose or sodium chloride to nutrient broth) was protective at temperatures above 53-55 degrees C but sensitizing below this temperature. Using selective enumeration media to determine injury, it was shown that at lower heating temperatures cells survived at high a(w) with cytoplasmic injury whereas at low a(w) these cells were killed. At higher temperatures ribosome degradation was a more important cause of death and was inhibited by low a(w) heating media thereby providing greater heat resistance. CONCLUSIONS: The observed change in behaviour reflects the different reactions responsible for thermal death at different temperatures and their different response to reduced a(w). SIGNIFICANCE AND IMPACT OF THE STUDY: This work qualifies the previous assumption that reduced a(w) is protective and suggests that the efficacy of low temperature pasteurization regimes may be increased by reduced a(w).