Thermal Inactivation Characteristics of Bacillus subtilis Spores at Ultrahigh Temperatures

The thermal inactivation characteristics of Bacillus subtilis A spores suspended in skim milk with the use of large-scale ultrahigh temperature (UHT) processing equipment were investigated in terms of survival as measured with two plating media. Data on survival immediately after UHT treatments were recorded in temperature-survivor curves, time-survivor curves, and decimal reduction time (DRT) curves. The temperature-survivor curves emphasized that inactivation is accelerated more by increases in the treatment temperature than by increases in the exposure time. Time-survivor curves and DRT curves were not linear. Generally, exceedingly concave time-survivor curves were observed with the standard plating medium; however, only slightly concave curves were observed when CaCl(2) and sodium dipicolinate were added to the medium. For a given UHT sample, larger D values were obtained by use of the medium with the added CaCl(2) and sodium dipicolinate. The DRT curves of all data were concave and appeared to have two discrete slopes (z(D) values). The z(D) values observed in the upper UHT range (above 260 F; 127 C) were twice those observed at lower test temperatures.     

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.     

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.     

Effect of Various Gas Atmospheres on Destruction of Microorganisms in Dry Heat

The heat resistance of dry bacterial spores was tested in various gases at temperatures ranging from 121.1 to 160 C (250 to 320 F). Spores of Clostridium sporogenes (PA 3679) were heated in air, carbon dioxide, and helium; spores of Bacillus subtilis 5230 were heated in these gases and also in oxygen and in nitrogen. The surrounding gas influenced the heat resistance, but the differences among gases were small. D values were about 7 min at 148.9 C (300 F); z values were about 18.3 C (33 F) for B. subtilis, and about 21.7 C (39 F) for C. sporogenes. The resistance of B. subtilis in carbon dioxide was about the same as in air, but lower than in all other gases; resistance in helium and nitrogen was about the same, and was higher than in all other gases. C. sporogenes had the least resistance in air; the resistance was about the same in carbon dioxide and helium. For B. subtilis, the gases in order of increasing heat resistance were carbon dioxide, air, oxygen, helium, and nitrogen, and for C. sporogenes, air, carbon dioxide, and helium. Neither oxygen content nor molecular weight of the gas appeared to have a marked influence on dry-heat resistance of the spores, whereas the more inert gases seemed to yield larger D values.     

Inactivation of bacillus spores in dry systems at low and high temperatures.

A plot of the thermal resistance of Bacillus subtilis var. niger spores (log D value) against temperature was linear between 37 and 190 degrees C (z = 23 degrees C), provided that the relative humidity of the spore environment was kept below a certain critical level. The corresponding plot for Bacillus stearothermophilus spores was linear in the range 150 to 180 degrees C (z = 29 degrees C) but departed from linearity at lower temperatures (decreasing z value). However, the z value of 29 degrees C was decreased to 23 degrees C if spores were dried before heat treatment. The straight line corresponding to this new z value was consistent with the inactivation rate at a lower temperature (60 degrees C). The data indicate that bacterial spores which are treated in dry heat at an environmental relative humidity near zero are inactivated mainly by a drying process. By extrapolation of the thermal resistance plot obtained under these conditions for B. subtilis var. niger spores, the D value at 0 degrees C would be about 4 years.     

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.     

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.     

Sterilization of soybean cotyledons by boiling in lactic acid solution

F. RUÍZ-TERÁN AND J.D. OWENS. 1996. The effect of pH on the heat resistance of Bacillus stearothermophilus spores at 100°C in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 sodium phosphate buffer was examined. At pH values of 7.0 and 6.0 spores survived 60 min exposure unharmed but at pH 4.3 and 3.0 they died with decimal reduction times (DRTs) of 27 min and 2.8 min, respectively. Death rates were similar in the presence or absence of hydrated soybean cotyledons. In the presence of phosphate buffer and cotyledons at mean pH 3.6 the DRT was 118 min but in the presence, in addition, of lactic acid it was 11 min. It is suggested that the enhanced death rate was due to toxic effects of undissociated lactic acid. Rhizopus oligosporus NRRL 2710 grew well on cotyledons, having pH values from 7.0 to 3.7, prepared by boiling for 60 min in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 phosphate buffer.     

The Resistances of Spores of the Genus Bacillus to Phenol, Heat and Radiation

S ummary . The resistance of the spores of 6 species of Bacillus to 5% (w/v) of phenol at 37°, heat and gamma radiation has been determined. Two, and with heat treatment three, different shapes of log survivor time curves were observed with each lethal agent. In relation to the conditions employed in accepted sterilization procedures, all of the strains were highly resistant to phenol. Bacillus stearothermophilus, B. licheniformis and B. subtilis were resistant to gamma radiation, all three having a D value of 0.22 Mrad. Only B. stearothermophilus was heat resistant having a D value of 22.6 min at 115°. When the relative order of resistance to each agent was considered, with the exception of B. stearothermophilus , the spores showed little evidence of any relationship between their resistances to phenol, heat and gamma radiation.     

Heat Resistance of Spores of Marine and Terrestrial Strains of Clostridium botulinum Type C

Resistance to heat of spores of marine and terrestrial strains of Clostridium botulinum type C in 0.067 m phosphate buffer (pH 7.0) was determined. The marine strains were 6812, 6813, 6814, and 6816; the terrestrial strains were 468 and 571. The inoculum level equaled 10(6) spores/tube with 10 replicate tubes for each time-temperature variable. Heating times were run at three or more temperatures to permit survival of some fraction of the inoculum. Survivors were recovered at 85 F (30 C) in beef infusion broth containing 1% glucose, 0.10% l-cysteine hydrochloride, and 0.14% sodium bicarbonate. D values were calculated for each fractional survivor end point after 6 months of incubation. Thermal resistance curves were constructed from the D value data. D(220) (104 C) values for spores of 468 and 571 equaled 0.90 and 0.40 min, respectively. The corresponding values for spores of 6812, 6813, 6814, and 6816 were 0.12, 0.04, 0.02, and 0.08 min. The z values for the thermal resistance curves ranged from 9.0 to 11.5 F (5.0 to 6.2 C).     

Strong and consistently synergistic inactivation of spores of spoilage-associated Bacillus and Geobacillus spp. by high pressure and heat compared with inactivation by heat alone

The inactivation of spores of four low-acid food spoilage organisms by high pressure thermal (HPT) and thermal-only processing was compared on the basis of equivalent thermal lethality calculated at a reference temperature of 121.1°C (F(z)(121.1)(°)(C, 0.1 MPa or 600 MPa)) and characterized as synergistic, not different or protective. In addition, the relative resistances of spores of the different spoilage microorganisms to HPT processing were compared. Processing was performed and inactivation was compared in both laboratory and pilot scale systems and in model (diluted) and actual food products. Where statistical comparisons could be made, at least 4 times and up to around 190 times more inactivation (log(10) reduction/minute at F(T)(z)(121.1)(°)(C)) of spores of Bacillus amyloliquefaciens, Bacillus sporothermodurans, and Geobacillus stearothermophilus was achieved using HPT, indicating a strong synergistic effect of high pressure and heat. Bacillus coagulans spores were also synergistically inactivated in diluted and undiluted Bolognese sauce but were protected by pressure against thermal inactivation in undiluted cream sauce. Irrespective of the response characterization, B. coagulans and B. sporothermodurans were identified as the most HPT-resistant isolates in the pilot scale and laboratory scale studies, respectively, and G. stearothermophilus as the least in both studies and all products. This is the first study to comprehensively quantitatively characterize the responses of a range of spores of spoilage microorganisms as synergistic (or otherwise) using an integrated thermal-lethality approach (F(T)(z)). The use of the F(T)(z) approach is ultimately important for the translation of commercial minimum microbiologically safe and stable thermal processes to HPT processes.     

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.     

Some Observations on Bacterial Thermal Death Time Curves

Thermal death rate data were obtained for spores of Clostridium sporogenes P.A. 3679 and Bacillus subtilis var. niger, and for cells of Salmonella senftenberg 775W. The survival curves for P.A. 3679 were approximately linear, but for B. subtilis var. niger or S. senftenberg 775W they were sigmoidal. Decimal reduction times were derived from the regression slopes of the apparent linear portion of the survival curves, and from these a phantom thermal death time (TDT) curve was constructed. In general, the phantom TDT curves were linear for B. subtilis var. niger and S. senftenberg 775W and nonlinear for P.A. 3679.     

Effect of Sodium Nitrite, Sodium Chloride, and Sodium Nitrate on Germination and Outgrowth of Anaerobic Spores

The effects of meat-curing agents on germination and outgrowth of putrefactive anaerobe 3679h (PA 3679h) spores were studied in microcultures. Nitrite concentrations up to 0.06% at pH 6.0 or between 0.8 and 1% at pH 7.0 allowed emergence and elongation of vegetative cells but blocked cell division. The newly emerged cells then lysed. With more than 0.06% nitrite at pH 6.0 or more than 0.8 to 1% at pH 7.0, the spores lost refractility and swelled, but vegetative cells did not emerge. Even as much as 4% nitrite failed to prevent germination (complete loss of refractility) and swelling of the spores. Sodium chloride concentrations above 6% prevented complete germination (i.e., the spores retained a refractile core). In the presence of 3 to 6% sodium chloride, most of the spores germinated and produced vegetative cells, but cell division was often blocked. Sodium nitrate had no apparent effect on germination and outgrowth at concentrations up to 2%.     

Sporicidal Action of Auto-oxidized Ascorbic Acid for Clostridium

Neutralized ascorbic acid (AA), buffered or unbuffered and autoclaved or filter-sterilized, was sporicidal for Clostridium. A 0.2% concentration of AA was generally employed, and spore counts were made in a soft-agar modification of Wynne's medium in Prickett tubes. Spores of Clostridium botulinum 115B were less susceptible than those of C. sporogenes PA 3679, whereas C. bifermentans spores were by far the most sensitive. At 75 C, spores of PA 3679 were killed at a rate of about 9% at 0 min (warm-up) to 99+% at 100 min. The lower the temperature, the longer the time needed for a given lethality. The percentage of killing increased with increasing concentrations of AA, and the rate of killing was lower at a higher concentration of spores. At least two mechanisms were operative: a major mechanism involving a product(s) of AA auto-oxidation, and a minor mechanism involving copper-ascorbate toxicity. AA reduced in natural gas was not sporicidal after 18.5 hr at 25 C, whereas 92% of the spores were killed by oxidized AA. Although H(2)O(2) per se was sporicidal, catalase did not reverse lethality of fresh or oxidized AA. Dehydroascorbate was as sporicidal as any AA preparation. Added copper (0.00001%) increased the rate of lethality of freshly prepared AA from 66 to 83% but was not effective with thoroughly oxidized AA. Ethylenediaminetetraacetic acid, NH(4) (+), and phosphate partially reversed AA toxicity, deionized water had no effect, and complex media, as well as thioglycolate, eliminated AA lethality. Since the percentage of killing was affected by spore concentration, AA did not seem to stimulate "lethal germination."     

Influence of Spore Moisture Content on the Dry-Heat Resistance of Bacillus subtilis var. niger

The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (z(D) = 21 C), but an unusually low z for spores on paper (z(D) = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (z(D) = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (a(w)) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below a(w) 0.2 or above a(w) 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature.     

Effect of Temperature and Gas Velocity on the Dry-Heat Destruction Rate of Bacterial Spores

Spores of Bacillus subtilis were dried in vacuo for use in dry-heat thermal destruction tests. Survivor curve tests were conducted in a specifically designed dry-heat oven. This oven provided accurate temperature control and permitted air or nitrogen to be passed over the spores during the lethal treatment. Experiments were carried out at various flow rates of the two gases (air and nitrogen) and various temperatures, and the data were expressed as survivor curves from which the decimal reduction time (D value) was obtained. Linear regression analysis methods were used to compute the slope of the survivor curves. The results indicated that as the flow rate of gas is increased, the effect of temperature on the destruction rate of the spores is lessened, the z value becoming very large. It is believed that the higher flow rates of dry gas cause greater dehydration of the spores and that spore moisture loss is one of the major factors in determining the dry-heat thermal destruction rate of bacterial spores.     

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.