Effect of Several Environmental Conditions on the “Thermal Death Rate” of Endospores of Aerobic, Thermophilic Bacteria

The composition of the recovery medium affected the apparent heat resistance of Bacillus stearothermophilus when the pH of the medium was 7.0 but not when the pH was 6.5. The rate of thermal death at 110 C was exponential. Deviations from exponential rates of thermal death during the initial phases of heating at 96 C were observed with endospores of B. coagulans under different conditions of sporulation. Additionally, the apparent heat resistance was influenced by the composition of the media used for sporulation and recovery and by the composition of the suspending menstruum. The presence of 0.001 m sorbic acid in the suspending menstruum at pH 7.0 and the temperature of incubation of the cultures after heating did not affect the apparent heat resistance of B. coagulans. Several explanations are discussed for the observed deviations from exponential thermal death rates and the effect of the environment on the apparent heat resistance of B. coagulans.     

Kinetics of Thermal Death of Bacteria

Experimental observations on thermal injury and death of bacteria in the stationary phase can be explained by assuming that death results from inactivation of (X(L)) of N critical sites. It is assumed: (i) that inactivation of individual sites occurs at random and follows first-order kinetics, (ii) that the critical sites are identical and of equal heat resistance, and (iii) the bacterial population is homogeneous in heat resistance. A method is described for calculating k (the rate constant for inactivation of individual sites), N (sites per cell), and X(L) (the number which must be inactivated to cause death under the experimental conditions used) from experimental data. Theoretical curves calculated by using this model are identical with experimental curves, providing support for the assumptions used. Calculated values of N and X(L) were 130 and 21.7 for Pseudomonas viscosa and 175 and 2.7 for Salmonella anatum. There is considerable uncertainty in the absolute values of N, but they are probably > 100. It is predicted that X(L) will vary depending on the recovery medium used after heating. This theory is consistent with all experimental observations on thermal injury and death of bacteria.     

Influence of pH and sodium chloride on kinetics of thermal inactivation of the bacteriocin-like substance P34

Aims: To investigate the kinetics of thermal inactivation of the bacteriocin‐like substance P34 at different pH and sodium chloride concentration. Methods and Results: Samples of bacteriocin were treated at different time–temperature combinations in the range of 0–300 min and 90–120°C and the kinetic parameters for bacteriocin inactivation were calculated. For all treatments, the thermal inactivation reaction fitted adequately to first‐order model. D‐ and k‐values were smaller and higher, respectively, for pH 4·5 than for 6·0 or 7·0, indicating that bacteriocin P34 was less thermostable at lower pH. At 120, 115 and 100°C, the addition of sodium chloride decreased thermal stability. For other temperatures, addition of NaCl increased stability of the peptide. The presence of greater amount of the salt (50 g l^?1) resulted in a higher thermal stability of bacteriocin P34, suggesting that the reduction in water activity of the solution interfered on the stability of the peptide. Conclusions: Based on an isothermal experiment in the temperature range of 90–120°C, and by thermal death time models, bacteriocin P34 is less heat stable at low pH and has increased thermal stability in the presence of NaCl. Addition of NaCl improved the stability of the peptide P34 at high temperatures. Significance and Impact of the Study: Studies on kinetics of thermal inactivation of bacteriocins are essential to allow their proper utilization in the food industry.     

Heat resistance of Byssochlamys ascospores.

Ascospores from 25 strains of Byssochlamys were studied for their ability to resist heat treatment in a standard defined medium. Seven of these were able to survive heating at 90 degrees C for 25 min or longer, when initial numbers were frequently near 10(6)/ml. Ascospores from five resistant strains suspended in the medium at pH 5.0 were usually more resistant than those at pH 3.6. Rapid heat inactivation occurred for one strain at pH 6.6. Nonlogarithmic heat death rate was observed in all strains tested.     

Heat resistance of Byssochlamys ascospores.

Ascospores from 25 strains of Byssochlamys were studied for their ability to resist heat treatment in a standard defined medium. Seven of these were able to survive heating at 90 degrees C for 25 min or longer, when initial numbers were frequently near 10(6)/ml. Ascospores from five resistant strains suspended in the medium at pH 5.0 were usually more resistant than those at pH 3.6. Rapid heat inactivation occurred for one strain at pH 6.6. Nonlogarithmic heat death rate was observed in all strains tested.     

Effects of pulsed electric fields on inactivation kinetics of Listeria innocua

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40 degrees C, a 3. 0-V/microm PEF treatment at an inlet temperature of 40 degrees C resulted in > or = 6.3 log inactivation of strain NCTC 11289 at 49.5 degrees C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.     

Stress-adaptive responses by heat under the microscope of predictive microbiology

AIMS: In previous studies the microbial kinetics of Escherichia coli K12 have been evaluated under static and dynamic conditions (Valdramidis et al. 2005, 2006). An acquired microbial thermotolerance following heating rates lower than 0.82 degrees C min(-1) for the studied micro-organism was observed. Quantification of this induced physiological phenomenon and incorporation, as a model building block, in a general microbial inactivation model is the main outcome of this work. METHODS AND RESULTS: The microbial inactivation rate observed (k(obs)) under time-varying temperature conditions is studied and expressed as a function of the heating rate (dT/ dt). Hereto, a model building block related to the microbial physiology (k(phys)) under stress conditions is developed. Evaluation of the performance of the developed mathematical approach depicts that physiological adaptation is an essential issue to be considered when modelling microbial inactivation. CONCLUSIONS: Consideration, at a mathematical level, of microbial responses resulting in physiological adaptations contribute to the reliable quantification of the safety risks during food processing. SIGNIFICANCE AND IMPACT OF THE STUDY: By taking into account the physiological adaptation, the microbiological evolution during heat processing can be accurately assessed, and overly conservative or fail dangerous food processing designs can be avoided.     

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.     

Calorimetric determination of inactivation parameters of micro-organisms

AIMS: This study aimed to apply differential scanning calorimetry (DSC) to evaluate the thermal inactivation kinetics of bacteria. METHODS AND RESULTS: The apparent enthalpy (DeltaH) of Escherichia coli cells was evaluated by a temperature scan in a DSC after thermal pretreatment in the calorimeter to various temperatures between 56 and 80 degrees C. Conventional semilogarithmic survival curve analysis was combined with a linearly increasing temperature protocol. Calorimetrically determined D and z values were compared to those obtained from plate count data collected under isothermal conditions to validate the new approach. CONCLUSIONS: The calculated D values using both apparent enthalpy and viability data for cells heat treated in the DSC were similar to the D values obtained from isothermal treatment. Temperatures for 1 through 10-log microbial population reductions, calculated from plate count and enthalpy data, were in agreement within 0.5-2.4 degrees C at a 4 degrees C min-1 heating rate. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel calorimetric method provides an approach to obtain accurate and reproducible kinetic parameters for inactivation. The calorimetric method here described is time efficient and is conducted under conditions similar to food processing conditions.     

Effects of Pulsed Electric Fields on Inactivation Kinetics of Listeria innocua

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40°C, a 3.0-V/μm PEF treatment at an inlet temperature of 40°C resulted in ≥6.3 log inactivation of strain NCTC 11289 at 49.5°C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.     

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.     

Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating.     

Effect of pre- and post-heat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes

The effect of incubation temperature, before and after a heat shock, on thermotolerance of Listeria monocytogenes at 58°C was investigated. Exposing cells grown at 10°C and 30°C to a heat shock resulted in similar rises in thermotolerance while the increase was significantly higher when cells were grown at 4°C prior to the heat shock. Cells held at 4°C and 10°C after heat shock maintained heat shock-induced thermotolerance for longer than cells held at 30°C. The growth temperature prior to inactivation had negligible effect on the persistence of heat shock-induced thermotolerance. Concurrent with measurements of thermotolerance were measurements of the levels of heat shock-induced proteins. Major proteins showing increased synthesis upon the heat shock had approximate molecular weights of 84, 74, 63, 25 and 19 kDa. There was little correlation between the loss of thermotolerance after the heat shock and the levels of these proteins. Thermotolerance of heat shocked and non-heat shocked cells was described by traditional log-linear kinetics and a model describing a sigmoidal death curve (logistic model). Employing log-linear kinetics resulted in a poor fit to a major part of the data whereas a good fit was achieved by the use of a logistic model.     

Biphasic Thermal Inactivation Kinetics in Salmonella enteritidis PT4

The thermal inactivation kinetics of Salmonella enteritidis PT4 between 49 and 60°C were investigated. Using procedures designed to eliminate methodological artifacts, we found that the death kinetics deviated from the accepted model of first-order inactivation. When we used high-density stationary-phase populations and sensitive enumeration, the survivor curves at 60°C were reproducibly biphasic. The decimal reduction time at 60°C (D_60°C) of the tail subpopulation was more than four times that of the majority population. This difference decreased with decreasing temperature; i.e., the survivor curves became more linear, but the proportion of tail cells remained a constant proportion of the initial population, about 1 in 10⁴ to 10⁵. Z plots (log D versus temperature) for the two populations showed that the D values coincided at 51°C, indicating that the survivor curves should be linear at this temperature, and this was confirmed experimentally. Investigations into the nature of the tails ruled out genotypic differences between the populations and protection due to leakage from early heat casualties. Heating of cells at 59°C in the presence of 5 or 100 μg of chloramphenicol per ml resulted in reductions in the levels of tailing. These reductions were greatest at the higher chloramphenicol concentration. Our results indicate that de novo protein synthesis of heat shock proteins is responsible for the observed tailing. Chemostat-cultured cells heated at 60°C also produced biphasic survivor curves in all but one instance. Cells with higher growth rates were more heat sensitive, but tailing was comparable with batch cultures. Starved cells (no dilution input) displayed linear inactivation kinetics, suggesting that during starvation a rapid heat shock response cannot be initiated.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-dependent conformational transformation in mung bean glyceraldehyde-3-phosphate dehydrogenase

In thermal inactivation at pH 7.3 and below, the tetrameric apo-glyceraldehyde-3-phosphate dehydrogenase of mung beans lost half of its activity more rapidly than the rest, suggesting a pairwise arrangement of subunits (or a C2 symmetry). At pH 8.6, the activity was lost in a single exponential decay, characteristic of functional identity of sites as in a tetrahedral arrangement of subunits (or a D2-type symmetry). At intermittent pH values, the kinetics of thermal inactivation were consistent with the presence of a mixture of C2- and D2-symmetry conformations. In "sudden pH change" experiments, the observed thermal inactivation kinetics were characteristic of the final pH at which the enzyme was heated. Thus, the interconversion of the two conformations is facile and very fast. There is no gross change in molecular weight of the enzyme between pH 7.3 and 8.6.