A quantitative approach for studying the effect of heat treatment conditions on resistance and recovery of Bacillus cereus spores.

When a bacterial population undergoes an unfavourable transient increase in temperature, a death phase followed by a lag and growth phase are observed for the surviving and cultivable population. The lag phase is of great interest in regard to food safety, but for bacterial spores, very few studies have been carried out on the evolution of lag time versus heat treatment duration. The experiments monitored on spores of two strains of Bacillus cereus showed a biphasic evolution of the lag time for germination of stressed spores with increase in heat treatment duration, at 90 degrees C, 95 degrees C and 100 degrees C and for different recovery conditions in laboratory medium. Initially, the lag time increased with the duration of the thermal stress up to an optimum. Subsequently, the lag time decreased, with increasing stress duration, to a threshold. The evolution of the lag time to germination of surviving spores, under the range of temperature tested, was similar to the evolution of the lag time to growth after mild-temperature treatments observed with vegetative cells of Escherichia coli and Listeria monocytogenes by Breand et al. (1997) [Breand, S., Fardel, G., Flandrois, J.-P., Rosso, L., Tomassone, R., 1997. A model describing the relationship between lag time and mild temperature increase duration. Int. J. Food Microbiol. 38, 157-167]. The mathematical model established from these investigations could also be applied in the case of heat-stressed spores. Moreover, it was observed that there was a significant linear relationship between the D-value and the treatment duration that gave the maximum lag time.     

Modelling the effect of sub(lethal) heat treatment of Bacillus subtilis spores on germination rate and outgrowth to exponentially growing vegetative cells

Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium-tryptone soy broth (TSA)-the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185-209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell.     

Modelling the influence of pH and organic acid types on thermal inactivation of Bacillus cereus spores

A model is proposed to describe the influence pH on the heat resistance of Bacillus cereus spores. In addition to the conventional z value, the effect of pH on the thermal resistance of spores is characterised by a z(pH) value (z(pH) is the distance of pH from a reference pH*, which leads to a 10-fold reduction of D value). The type of organic acid used for acidifying the heating medium, influences the z(pH) value. For nine organic acids, a linear relationship between the calculated z(pH) value and its lower acid pKa is observed. This relationship showed that the acid form (dissociated or undissociated) modifies the thermal spore resistance in addition to the H+ ion. The influence of acetic acid concentration on the D value at pH 7 shows the protective effect of the dissociated acid form on the heat resistance of spores. The acid concentration in the medium modified the heat resistance of spore and the z(pH) value.     

A model describing the relationship between regrowth lag time and mild temperature increase for Listeria monocytogenes

In order to comply with the consumer demand for ready-to-eat and look 'fresh' products, mild heat treatment will be used more and more in the agrofood industry. Nonetheless there is no tool to define the most appropriate mild heat treatment. In order to build this tool, it is necessary to study and describe the response of a bacterial population to a mild increase in temperature, from the dynamic point of view. The response to a mild increase in temperature, defined by stress duration and temperature, consisted in a mortality phase followed by the lag time of the survivors and their exponential growth. The effect of the mild increase in temperature on the mortality phase was described in a previous paper (Bréand et al., Int. J. Food Microbiol., in press). The effect of the stress duration on the lag was presented in a previous paper (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). In particular, the biphasic relationship between the lag and the stress duration was observed and modelled with a four parameter nonlinear model: the primary model (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). The study presented in this paper deals with the effect of the stress temperature on the biphasic relationship between the lag time and the stress duration. The secondary models describing the effect of the stress temperature on this biphasic relationship, were empirically built from our experimental data concerning Listeria monocytogenes. This work pointed out that the higher the stress temperature, the narrower the range of stress duration for which the lag time increased. Since the primary and the secondary models of the lag time were available, the global model describing the effect of the mild increase duration and temperature directly on the lag was fitted. This model allowed an improvement of the parameter estimator precision. The potential contribution in mild heat treatment optimization of this global model and the one built for the mortality phase (Bréand et al., Int. J. Food Microbiol., in press) is discussed.     

Lag time variability in individual spores of Clostridium botulinum

Quantifying lag times from individual spores and the associated variability is an important part of understanding the hazard associated with spore-forming pathogens such as Clostridium botulinum. Knowledge of the underlying distribution would allow greater refinement of risk assessments. To date most studies have either examined lag time indirectly by measuring time to growth or have only examined the first stage of lag, germination. Recent studies have attempted to quantify the variability of spores during the different stages of lag phase and to examine the relationships between these stages. The effect of incubation temperature (22 °C, 15 °C, 10 °C or 8 °C), heat treatment (unheated or 80 °C for 20 s) and sodium chloride concentration in both the sporulation medium (0 or 3% w/v) or growth medium (0 or 2% w/v) on growth from individual spores has been examined. These studies found spores within a single population are very heterogeneous with large variability in all stages of lag. The duration and variability of times for germination, outgrowth and first doubling depended on both the historic treatment of the spores and the prevailing growth conditions, and the stage of lag most affected was treatment dependant.     

Modelling the influence of the sporulation temperature upon the bacterial spore heat resistance, application to heating process calculation

Environmental conditions of sporulation influence bacterial heat resistance. For different Bacillus species a linear Bigelow type relationship between the logarithm of D values determined at constant heating temperature and the temperature of sporulation was observed. The absence of interaction between sporulation and heating temperatures allows the combination of this new relationship with the classical Bigelow model. The parameters zT and zT(spo) of this global model were fitted to different sets of data regarding different Bacillus species: B. cereus, B. subtilis, B. licheniformis, B. coagulans and B. stearothermophilus. The origin of raw products or food process conditions before a heat treatment can lead to warm temperature conditions of sporulation and to a dramatic increase of the heat resistance of the generated spores. In this case, provided that the temperature of sporulation can be assessed, this model can be easily implemented to rectify F values on account of possible increase of thermal resistance of spores and to ensure the sterilisation efficacy.     

Predicting heat inactivation of Listeria monocytogenes under nonisothermal treatments

The aim of this study was to find a model that accurately predicts the heat inactivation of Listeria monocytogenes (ATCC 15313) at constantly rising heating rates (0.5 to 9 degrees C/min) in media of different pH values (4.0 to 7.4). Survival curves of L. monocytogenes obtained under isothermal treatments at any temperature were nearly linear. Estimations of survival curves under nonisothermal treatments obtained from heat resistance parameters of isothermal treatments adequately fit experimental values obtained at pH 4.0. On the contrary, survivors were much higher than estimations at pH 5.5 and 7.4. The slower the heating rate and the longer the treatment time, the greater the differences between the experimental and estimated values. An equation based on the Weibullian-like distribution, log S(t) = (t/delta)p, accurately described survival curves of L. monocytogenes obtained under nonisothermal conditions within the range of heating rates investigated. A nonlinear relationship was observed between the scale parameter (delta) and the heating rate, which allowed the development of an equation capable of predicting the inactivation rate of L. monocytogenes under nonisothermal treatments at pH 5.5 and 7.4. The model predictions were a good fit to the measured data independent of the magnitude of the thermotolerance increase. This work might contribute to the increase in safety of those food products that require long heating lag phases during the pasteurization process.     

COMBINED EFFECT OF HEAT AND ALKALI IN STERILIZING SUGARCANE BAGASSE

ABSTRACT— Bacterial spores contained in sugarcane bagasse were subjected to various combinations of heat exposure and alkali concentration and the rate of destruction determined for each set of conditions. A series of survival, thermal death time and alkaline destruction curves revealed a different mode of death by heat exposure than alkali treatment. Addition of alkali into the heating menstruum caused the death rates of bacterial spores to be much greater than with heat alone at a given temperature. Exposure of the spores to a temperature of 75°C for 130 min was required to reduce the spore population by 90% with heat treatment alone. Incorporation of a 1% NaOH solution into the heating menstruum effected the same degree of destruction of the spores within a 2 min period at the same temperature. From a series of thermal destruction and alkaline destruction curves, an empirical equation expressing the relationship between the death rate of bacterial spores, and the intensity of temperature and the concentration of alkali was established. The equation reveals that the death rate of bacterial spores is affected in an exponential manner by temperature and in a direct relationship by alkali concentration. Using the equation, sterilization time for various combinations of temperature and alkali concentration was determined and the overall correlation index between the experimental data and computed value was 0.877.     

Effect of environmental parameters on growth kinetics of Bacillus cereus (ATCC 7004) after mild heat treatment

The influence of temperature (10 to 50 degrees C), initial pH (4.0 to 6.0) and sodium chloride concentration (0.5 to 3.0%) on the growth in nutrient broth and in meat extract of Bacillus cereus after mild heat treatment (90 degrees C--10 min) was determined. B. cereus spores survived after heating and they were able to germinate and grow in both media when post-treatment conditions were favourable. Heated B. cereus did not grow at 10 and 50 degrees C or in a medium with pH 4.0. Decreasing pH values and increasing levels of sodium chloride decreased growth rate and increased the lag phase of B. cereus. pH 4.5 was unable to prevent the growth of heated spores in a meat substrate with 0.5% NaCl at 12 degrees C. The combination of pH /=1.0% and temperatures /=60 degrees C could control heated B. cereus ATCC 7004 growth.     

Modeling the influence of electron beam irradiation on the heat resistance of Bacillus cereus spores

The effect of electron beam irradiation (EBI) on Bacillus cereus spore heat resistance was investigated. Irradiation with accelerated electrons had an important heat-sensitizing effect on distilled-water spore suspensions. After irradiation doses of 1.3, 3.1, or 5.7 kGy followed by heating at 90 degrees C, calculated D(90)-values for strains Escuela Politécnica Superior de Orihuela (EPSO)-41WR and EPSO-50UR were reduced more than 1.3, 2.4, and 4.6 times, respectively. Plots of calculated log D(T)-values versus irradiation doses (1.3, 3.1, and 5.7 kGy) yielded straight parallel lines for the 85-100 degrees C heating temperature range, which made it possible to develop an equation to predict the changes in heat sensitivity of B. cereus spores that occurred with changing irradiation dose. Radiation-induced heat-sensitivity was characterized by a z(EBI)-value which was determined as the irradiation dose that should be required to reduce the decimal reduction time (D(T)) by one log(10) cycle when log(10)D(T) was plotted against irradiation treatment. A model is proposed to describe the influence of a pre-irradiation treatment with electron beams followed by heating on the heat resistance of B. cereus spores. This study also suggests the potential use of EBI followed by heating for food preservation.     

Quantifying the effects of heating temperature, and combined effects of heating medium pH and recovery medium pH on the heat resistance of Salmonella typhimurium

The influence of heating treatment temperature, pH of heating and recovery medium on the survival kinetics of Salmonella typhimurium ATCC 13311 is studied and quantified. From each non-log linear survival curve, Weibull model parameters were estimated. An average shape parameter value of 1.67 was found, which is characteristic of downward concavity curves and is in agreement with values estimated from other S. typhimurium strains. Bigelow type models quantifying the heating temperature, heating and recovery medium pH influences are fitted on scale parameter delta data (time of first decimal reduction), which reflects the bacterial heat resistance. The estimate of z(T) (4.64 degrees C) is in the range of values given in the literature for this species. The influence of pH of the heating medium on the scale parameter (z(pH): 8.25) is lower than that of the recovery pH medium influence (z(')(pH): 3.65).     

Heat resistance of Alicyclobacillus acidocaldarius in water, various buffers, and orange juice

The effect of the pH or the composition of the heating medium and of the sporulation temperature on the heat resistance of spores of a thermoacidophilic spore-forming microorganism isolated from a dairy beverage containing orange fruit concentrate was investigated. The species was identified as Alicyclobacillus acidocaldarius. The spores showed the same heat resistance in citrate-phosphate buffers of pH 4 and 7, in distilled water, and in orange juice at any of the temperatures tested (D120 degrees C = 0.1 min and z = 7 degrees C). A raise in 20 degrees C in the sporulation temperature (from 45 to 65 degrees C) increased the heat resistance eightfold (from D110 degrees C = 0.48 min when sporulated at 45 degrees C to 3.9 min when sporulated at 65 degrees C). The z-values remained constant for all sporulation temperatures. The spores of this strain of A. acidocaldarius were very heat resistant and could easily survive any heat treatment currently applied to pasteurize fruit juices.     

Modeling the combined effects of pH, temperature and ascorbic acid concentration on the heat resistance of Alicyclobacillus acidoterrestis

In this study, thermal inactivation parameters (D- and z-values) of Alicyclobacillus acidoterrestris spores in McIlvaine buffers at different pH, apple juice and apple nectar produced with and without ascorbic acid addition were determined. The effects of pH, temperature and ascorbic acid concentration on D-values of A. acidoterrestris spores were also investigated using response surface methodology. A second order polynomial equation was used to describe the relationship between pH, temperature, ascorbic acid concentration and the D-values of A. acidoterrestris spores. Temperature was the most important factor on D-values, and its effect was three times higher than those of pH. Although the statistically significant, heat resistance of A. acidoterrestris spores was not so influenced from the ascorbic acid within the concentration studied. D-values in apple juice and apple nectars were higher than those in buffers as heating medium at similar pH. The D-values ranged from 11.1 (90 °C) to 0.7 min (100 °C) in apple juice, 14.1 (90 °C) to 1.0 min (100 °C) in apple nectar produced with ascorbic acid addition, and 14.4 (90 °C) to 1.2 min (100 °C) in apple nectar produced without ascorbic acid addition. However, no significant difference in z-values was observed among spores in the juices and buffers at different pH, and it was between 8.2 and 9.2 °C. The results indicated that the spores of A. acidoterrestris may survive in fruit juices and nectars after pasteurization treatment commonly applied in the food industry.     

Measurement of optical absorbance of Clostridium sporogenes PA 3679 spores as a method for determining their thermal inactivation

 The loss of optical density of Clostridium sporogenes PA 3679 spores after heating at temperatures of 121, 126, 130 and 135°C was studied, together with the relationship between this parameter and the recovery capacity of the heated spores. The results show that the spores suffered a greater loss of optical density when they were subjected to more severe heating. A linear relationship was observed between the loss of viability and the optical absorbance of the spores. A certain parallel was detected between the heat resistance parameters of the spores D_T (decimal reduction time) and z (thermal inactivation coefficient), and the kinetic parameters D_T ^A and z^A, which describe the reduction in absorbance. Received: 14 April 1997     

Predicting thermal inactivation in media of different pH of Salmonella grown at different temperatures

The influence of the growth temperature and the pH of the heating medium on the heat resistance at different temperatures of Salmonella typhimurium ATCC 13311 was studied and described mathematically. The shift of the growth temperature from 10 to 37 degrees C increased heat resistance of S. typhimurium fourfold. The pH of the heating medium at which heat resistance was maximum was pH 6 for cells grown at 37 degrees C, but changed with growth temperature. The alkalinization of the heating medium from pH 6 to pH 7.7 decreased the heat resistance of cells grown at 37 degrees C by a factor of 3. Neither the growth temperature nor the pH modified the z values significantly (4.9 degrees C). The decimal reduction times at different treatment temperatures, in buffers of different pH of cells of S. typhimurium grown at different temperatures, were accurately described by a mathematical equation (correlation coefficient of 0.97). This equation was also tested for Salmonella senftenberg 775W (ATCC 43845) and Salmonella enteritidis ATCC 13076, strains in which the correlation coefficients between the observed and the theoretically calculated values were 0.91 and 0.98, respectively.     

Measurement of optical absorbance of Clostridium sporogenes PA 3679 spores as a method for determining their thermal inactivation

 The loss of optical density of Clostridium sporogenes PA 3679 spores after heating at temperatures of 121, 126, 130 and 135°C was studied, together with the relationship between this parameter and the recovery capacity of the heated spores. The results show that the spores suffered a greater loss of optical density when they were subjected to more severe heating. A linear relationship was observed between the loss of viability and the optical absorbance of the spores. A certain parallel was detected between the heat resistance parameters of the spores D_T (decimal reduction time) and z (thermal inactivation coefficient), and the kinetic parameters D_T ^A and z^A, which describe the reduction in absorbance. Received: 14 April 1997     

Effect of a previous heat shock on the thermal resistance of Listeria monocytogenes and Pseudomonas aeruginosa at different pHs

In this work we study the effect of heat shocks of various durations up to 60 min, at different temperatures between 35 and 45 degrees C, in media of pH 4.0, 5.5 and 7.4 on the heat resistance of Listeria monocytogenes and Pseudomonas aeruginosa. The pattern of survival curves after heat treatment did not change with the application of a previous heat shock. However, the kinetics of inactivation was different for the two microorganisms studied. Whereas the inactivation of L. monocytogenes was similar to an exponential function of heating time and therefore straight survival curves were obtained, survival curves corresponding to P. aeruginosa showed convex profiles. All survival curves obtained in this investigation were fitted to Weibull-based Mafart equation: log(10)S(t)=-(t / delta)(p). The magnitude of the heat shock induced thermotolerance increased with treatment medium pH. At pH 7.4 the increase in heat tolerance depended on the duration and temperature of the heat shock. On the contrary, at pH 5.5 and pH 4.0, the heat-shock temperature did not exert any effect. The observed maximum delta values increased 2.3, 4.0 and 9.3 fold for L. monocytogenes, and 1.3, 2.1 and 8.4 fold for P. aeruginosa, at pH 4.0, 5.5 and 7.4, respectively. This research has proven that Mafart equation allows studying and quantifying the effect of heat shocks on bacterial heat resistance.     

Use of sensitivity analysis to aid interpretation of a probabilistic Bacillus cereus spore lag time model applied to heat-treated chilled foods (REPFEDs)

The microbiological safety and quality of REfrigerated Processed Foods of Extended Durability (REPFEDs) relies on a combination of mild heat treatment and refrigeration, sometimes in combination with other inhibitory agents that are not effective when used alone. In this context, the output of a probabilistic model predicting the lag time of heat-treated Bacillus cereus spores under realistic heat-treatment profile and chilled supply-chain conditions, has been investigated using a sensitivity analysis technique. Indeed, knowing that there was uncertainty in the model (e.g. due to lack of data to build the model input probability density function), the objective of the analysis was to evaluate if the variability associated with some inputs (e.g. the consumers' refrigerator temperature values reported in Europe and US markets were different) had a significant impact on the model output, i.e. on the expected lag time of heat-treated B. cereus spores in REPFEDs. To do so, the uncertainty and variability associated with the various model inputs have been identified and then separated using a second order Monte Carlo decomposition. Concerning the variability, there was a significant difference between the chilled supply-chains (Europe, US) and between the raw material groups (low, medium or high contamination levels). For example, in the European market, after a heat treatment of 90 degrees C for 10 min, with a high raw material contamination level, the predicted 5th percentile of the lag time was 17 days, while it was 35 days with a low raw material contamination level. This was confirmed with an ANOVA. The impact of the uncertainty on the lag time has been illustrated graphically by building confidence intervals around its 5th percentile. A sensitivity analysis based upon uncertainty and variability decomposition is clearly a complex and time consuming exercise; however, it provides a greater confidence (greater transparency and better understanding) in the model output when making food safety decisions (e.g. determining the safe shelf-life of REPFEDs).     

Modeling heat resistance of Bacillus weihenstephanensis and Bacillus licheniformis spores as function of sporulation temperature and pH

Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T(min(R)), T(opt(R)), T(max(R)), pH(min(R)) and pH(opt(R))) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry.     

Altered ability of Bacillus cereus spores to grow under unfavorable conditions (presence of nisin,low temperature,acidic pH,presence of NaCl) following heat treatment during sporulation

The effect of thermal treatment on the heat resistance of Bacillus cereus spores and their ability to germinate and grow under more or less adverse conditions during sporulation was investigated. Spores produced by sporulating cells subjected to a mild heat treatment (at a temperature 15 degrees C higher than the growth temperature) were more resistant to heat than were spores produced by untreated cells. Spore germination and growth (the lag time, the maximal growth rate, and the occurrence of a decrease in population) may be greatly affected by adverse environmental conditions brought about by the addition of nisin, low temperatures, acidic pHs, and, to a lesser extent, the addition of NaCl. Furthermore, heat treatments applied to sporulating cells or to mature spores induced a modification of the lag time (interaction of both treatments). Therefore, mild heat treatments applied during sporulation may affect the heat resistance of spores and the ability of these spores to germinate under adverse conditions and may thus increase the risk associated with the presence of spores in lightly processed foods.