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.     

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.     

Mechanism of the inactivation of bacterial spores by reciprocal pressurization treatment

AIMS: The mechanism of the inactivation of Bacillus subtilis spores by reciprocal pressurization (RP) was unclear. Therefore, the mechanism was investigated. METHODS AND RESULTS: To investigate the effects of RP and continuous pressurization (CP) treatments on the inactivation and injury of B. subtilis spores, spores were treated at 25, 35, 45 and 55 degrees C under 200, 300 and 400 MPa. RP treatment was effective in injuring and inactivating spores. Scanning electron microscopy and transmission electron microscopy observation showed that spores treated by RP treatment were more morphologically and structurally changed than the ones treated by CP treatment. There were significant differences between the release of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by RP and CP treatments. From this result, it was concluded that the core fraction was released into the spore suspension. CONCLUSIONS: The mechanism of RP treatment is believed to work as follows: hydrostatic pressure treatment initiated germination of bacterial spores, and the repeated rapid decompression caused disruption, injury and inactivation of the germinated spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that the physical injury of bacterial spores was effective to inactivate the bacterial spores through the disruption of spores and leakage of their contents.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7.2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of aw during heating. Spore resistance to heat increased in the order: buffer much less than commercial oil less than olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low aw (0.479 and 0.492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values (ca 28 degrees C in oils and 10 degrees-12 degrees C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

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.     

Effects of high hydrostatic pressure on Clostridium sporogenes spores

Spores of Clostridium sporogenes were found to be resistant to ultra high pressure, with treatments of 600 MPa for 30 min at 20 °C causing no significant inactivation. Combination treatments including heat and pressure applied simultaneously (e.g. 400 MPa at 60 °C for 30 min) or sequentially (e.g. 80 °C for 10 min followed by 400 MPa for 30 min) proved more effective at inactivating spores. Pressure cycling (e.g. 60 MPa followed by 400 MPa at 60 °C) also reduced spore numbers. Overall, these pressure treatments resulted in less than a 3 log reduction, and it was concluded that the spores could not be inactivated by pressure alone. This could indicate that for the effective inactivation of bacterial spores, high pressure technology may have to be used in combination with other preservation methods.     

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.     

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°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°C, compared to 160 min at 500 MPa and 20°C and 348 min at atmospheric pressure (0.1 MPa) and 75°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.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7˙2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of a_w during heating. Spore resistance to heat increased in the order: buffer ⋖ commercial oil < olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low a_w (0˙479 and 0˙492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values ( ca 28°C in oils and 10°-12°C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Tailing of survivor curves of clostridial spores heated in edible oils

Tailing of survivor curves was observed for Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores heated whilst suspended in edible oils, but not for the same spores suspended in buffer (pH 7˙2) or mineral oil or for Bacillus cereus F4165/75 spores suspended in buffer or oils. The tailing cannot be ascribed to a genetic or developmental heterogeneity in the resistance of the spore population or to a heterogeneity of the treatment severity during heating. Heat adaptation due to the release of protective factor(s), to the selection for resistant spores or to the diffusion of oil constituents inside the spore protoplast to protect key molecules from heat denaturation was also ruled out. The tailing can be ascribed to spore clumping during the course of heating or to a heterogeneity in heat resistance of germination system(s) within spores, concurrently with the activation of a dormant germination system. It is probably caused by some oleic acid containing triglycerides.     

Tailing of survivor curves of clostridial spores heated in edible oils

Tailing of survivor curves was observed for Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores heated whilst suspended in edible oils, but not for the same spores suspended in buffer (pH 7.2) or mineral oil or for Bacillus cereus F4165/75 spores suspended in buffer or oils. The tailing cannot be ascribed to a genetic or developmental heterogeneity in the resistance of the spore population or to a heterogeneity of the treatment severity during heating. Heat adaptation due to the release of protective factor(s), to the selection for resistant spores or to the diffusion of oil constituents inside the spore protoplast to protect key molecules from heat denaturation was also ruled out. The tailing can be ascribed to spore clumping during the course of heating or to a heterogeneity in heat resistance of germination system(s) within spores, concurrently with the activation of a dormant germination system. It is probably caused by some oleic acid containing triglycerides.     

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

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.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

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.     

Thermal analysis of the spores of Bacillus cereus with special reference to heat activation.

The heat activation of bacterial spores was studied by means of differential thermal analysis in the temperature range 30-110 degrees C using the spores of Bacillus cereus. The thermogram showed three endothermic peaks at 56, 95, and 103 degrees C with one exothermic peak at 105 degrees C during the heating process. The spore coat separated from the native spores also showed a peak at 56 degrees C on its heating thermogram. The peak at 56 degrees C was reversible for both native spores and the spore coat. It was suggested that this peak at 56 degrees C might be related to the heat-activation process that takes place in the spore-coat region. It seems that the peak is due to the denaturation or the structural change of the spore-coat protein that might facilitate either the permeation of germination stimulators or the release of some germination inhibitor into or out of the spores.     

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-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.