The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history.

While bacterial spores are mostly produced in a continuous process, this study reports a two-step sporulation methodology. Even though spore heat resistance of numerous spore-forming bacteria is known to be dependent on sporulation conditions, this approach enables the distinction between the vegetative cell growth phase in nutrient broth and the sporulation phase in specific buffer. This study aims at investigating whether the conditions of growth of the vegetative cells, prior to sporulation, could affect spore heat resistance. For that purpose, wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores, produced via a two-step sporulation process, was determined from vegetative cells harvested at four different stages of the growth kinetics, i.e. early exponential phase, late exponential phase, transition phase or early stationary phase. To assess the impact of the temperature on spore heat resistance, sporulation was performed at 10 °C, 20 °C and 30 °C from cells grown during a continuous or a discontinuous temperature process, differentiating or not the growth and sporulation temperatures. Induction of sporulation seems possible for a large range of growth stages. Final spore concentration was not significantly affected by the vegetative cell growth stage while it was by the temperature during growing and sporulation steps. The sporulation temperature influences the heat resistance of B. weihenstephanensis KBAB4 spores much more than growth temperature prior to sporulation. Spores produced at 10 °C were up to 3 times less heat resistant than spores produced at 30 °C.

Quantification of spore resistance for assessment and optimization of heating processes: a never-ending story.

The assessment and optimization of food heating processes require knowledge of the thermal resistance of target spores. Although the concept of spore resistance may seem simple, the establishment of a reliable quantification system for characterizing the heat resistance of spores has proven far more complex than imagined by early researchers. This paper points out the main difficulties encountered by reviewing the historical works on the subject. During an early period, the concept of individual spore resistance had not yet been considered and the resistance of a strain of spore-forming bacterium was related to a global population regarded as alive or dead. A second period was opened by the introduction of the well-known D parameter (decimal reduction time) associated with the previously introduced z-concept. The present period has introduced three new sources of complexity: consideration of non log-linear survival curves, consideration of environmental factors other than temperature, and awareness of the variability of resistance parameters. The occurrence of non log-linear survival curves makes spore resistance dependent on heating time. Consequently, spore resistance characterisation requires at least two parameters. While early resistance models took only heating temperature into account, new models consider other environmental factors such as pH and water activity ("horizontal extension"). Similarly the new generation of models also considers certain environmental factors of the recovery medium for quantifying "apparent heat resistance" ("vertical extension"). Because the conventional F-value is no longer additive in cases of non log-linear survival curves, the decimal reduction ratio should be preferred for assessing the efficiency of a heating process.

A new predictive dynamic model describing the effect of the ambient temperature and the convective heat transfer coefficient on bacterial growth.

In this study, predictive microbiology and food engineering were combined in order to develop a new analytical model predicting the bacterial growth under dynamic temperature conditions. The proposed model associates a simplified primary bacterial growth model without lag, the secondary Ratkowsky "square root" model and a simplified two-parameter heat transfer model regarding an infinite slab. The model takes into consideration the product thickness, its thermal properties, the ambient air temperature, the convective heat transfer coefficient and the growth parameters of the micro organism of concern. For the validation of the overall model, five different combinations of ambient air temperature (ranging from 8 degrees C to 12 degrees C), product thickness (ranging from 1 cm to 6 cm) and convective heat transfer coefficient (ranging from 8 W/(m(2) K) to 60 W/(m(2) K)) were tested during a cooling procedure. Moreover, three different ambient air temperature scenarios assuming alternated cooling and heating stages, drawn from real refrigerated food processes, were tested. General agreement between predicted and observed bacterial growth was obtained and less than 5% of the experimental data fell outside the 95% confidence bands estimated by the bootstrap percentile method, at all the tested conditions. Accordingly, the overall model was successfully validated for isothermal and dynamic refrigeration cycles allowing for temperature dynamic changes at the centre and at the surface of the product. The major impact of the convective heat transfer coefficient and the product thickness on bacterial growth during the product cooling was demonstrated. For instance, the time needed for the same level of bacterial growth to be reached at the product's half thickness was estimated to be 5 and 16.5 h at low and high convection level, respectively. Moreover, simulation results demonstrated that the predicted bacterial growth at the air ambient temperature cannot be assumed to be equivalent to the bacterial growth occurring at the product's surface or centre when convection heat transfer is taken into account. Our results indicate that combining food engineering and predictive microbiology models is an interesting approach providing very useful tools for food safety and process optimisation.

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).

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.

General model, based on two mixed weibull distributions of bacterial resistance, for describing various shapes of inactivation curves.

Cells of Listeria monocytogenes or Salmonella enterica serovar Typhimurium taken from six characteristic stages of growth were subjected to an acidic stress (pH 3.3). As expected, the bacterial resistance increased from the end of the exponential phase to the late stationary phase. Moreover, the shapes of the survival curves gradually evolved as the physiological states of the cells changed. A new primary model, based on two mixed Weibull distributions of cell resistance, is proposed to describe the survival curves and the change in the pattern with the modifications of resistance of two assumed subpopulations. This model resulted from simplification of the first model proposed. These models were compared to the Whiting's model. The parameters of the proposed model were stable and showed consistent evolution according to the initial physiological state of the bacterial population. Compared to the Whiting's model, the proposed model allowed a better fit and more accurate estimation of the parameters. Finally, the parameters of the simplified model had biological significance, which facilitated their interpretation.

Modelling the influence of the incubation temperature upon the estimated heat resistance of heated bacillus spores.

AIMS: In this study, the influence of the incubation temperature on the D-values was described according to a simple Bigelow-like model. METHODS: Model parameters were estimated from different sets of data from the literature and from our own data. For different Bacillus species and heat-treatment conditions, the influence of the recovery temperature was quantified and the optimal recovery temperature was determined. RESULTS: The impacts of species, bacterial strains belonging to the same species, heat-treatment temperature and composition of recovery media on the model parameters were analysed. The optimum recovery temperatures differ greatly from one species to another; however, no difference appears clearly between strains belonging to the same species. D values were significantly affected by recovery temperature. This influence of recovery temperature was dependent on the species, and affected by the composition of recovery media but not by the heating temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed model could be useful for determining the optimal incubation temperature and quantifying the weight of the recovery temperature influence for safe security control in the canning industry.

Quantifying the combined effects of the heating time, the temperature and the recovery medium pH on the regrowth lag time of Bacillus cereus spores after a heat treatment.

The purpose of this study was to quantify the lag time of re-growth of heated spores of Bacillus cereus as a function of the conditions of the heat treatment: temperature, duration and pH of the recovery medium. For a given heating temperature, curves plotting lag times versus time of heating show more or less complex patterns. However, under a heating time corresponding to a decrease of 2 decimal logarithms of the surviving populations of spores, a linear relationship between the lag time of growth and the time of the previous heat treatment can be observed. The slope of this linear relationship followed itself a Bigelow type linear relationship, the slope of which yielded a zeta-value very close to the observed conventional z-value. It was then concluded that the slope of the regrowth lag time versus the heating time followed a linear relationship with the sterilisation value reached in the course of the previous heat treatment. A sharp effect of the pH of the medium which could be described by a simple "secondary" model was observed. As expected, the observed intercept of the linear relationship between lag time and heating time (lag without previous heating) was dependent on only the pH of the medium and not on the heating temperature.

A modified Weibull model for bacterial inactivation.

In this paper, a modified Weibull model is proposed to fit microbial survival curves. This model can incorporate shoulder and/or tailing phenomena if they are encountered. We aim to obtain an accurate fit of the "primary" modelling of the bacterial inactivation and to provide a useful and meaningful model for biologists and food industry. A delta parameter close to the classical concept of the D value, established for sterilisation processes, is used in the model. The specific parameterisation of the Weibull model is evaluated for the parameter of interest delta. The goodness-of-fit of the model is compared to the one produced by the model proposed by Geeraerd et al., [Geeraerd, A.H., Herremans, C.H., Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. Int. J. Food Microbiol. 59, 185-209.] on experimental data. As our model provides good fits for the different types of survival curves analysed, further research can focus on the development of suitable secondary model types. In this respect, it is interesting to note that the delta parameter is close to the D concept.

Validation of an overall model describing the effect of three environmental factors on the apparent D-value of Bacillus cereus spores.

Several factorial models extending the famous Bigelow model to describe the influence of the heating and recovery pH and a(w) conditions on bacterial heat resistance have been developed. These models can be associated in an overall multifactorial model describing the influences of heating and recovery conditions on D values. For Bacillus cereus strain ADQP 407 the model parameters characterising the environmental factor influences (pH, Temperature, a(w)) were evaluated. Determination of bacterial heat resistance in cream chocolate have been realised to validate these parameter values and to evaluate the level of the influence of food texture or different compounds not taken account of in the model.

Food engineering and predictive microbiology: on the necessity to combine biological and physical kinetics.

Predictive microbiology is mainly applied in the area of risk assessment, but unusually regarded as a tool for the optimisation of processes, which needs the implementation of food engineering. Combination of predictive microbiology and food engineering allows both the assessment of a process in relation to risk and its optimisation. Intrinsic comparison between processes in relation to risk, on one hand, and the development of process optimisation tools on the other hand, necessitates the implementation of new concepts and the involvement of simplified and standard calculations based upon reference target strains and environmental conditions. Some conventional concepts related to heat treatments are extended, while some new ones related to bacterial growth are derived from the gamma concept of Marcel Zwietering.

Modelling the growth kinetics of Listeria as a function of temperature, pH and organic acid concentration.

The combined effects of temperature, pH and organic acids (lactic, acetic and propionic) on the growth kinetics of Listeria innocua ATCC 33090 were studied. First, a multiplicative model was built assuming independent effects of all environmental factors. Thus, the model was expanded by the inclusion of a novel term describing the effects of interactions on the growth/no growth limits. The proposed approach allows an accurate description of the boundary between growth and no growth of Listeria.

On calculating sterility in thermal preservation methods: application of the Weibull frequency distribution model.

A simple and parsimonious model which originated from the Weibull frequency distribution was proposed to describe nonlinear survival curves of spores. This model was suitable for downward concavity curves (Bacillus cereus and Bacillus pumilus), as well as for upward concavity curves (Clostridium botulinum). It was shown that traditional F values calculated from this new model were no longer additive, to such an extent that a heat treatment should be better characterized by the obtained decimal reduction of spores. A modified Bigelow method was then proposed to assess this decade reduction or to optimize the heat treatment for a target reduction ratio.

Effect of sodium chloride concentration on the heat resistance and recovery of Salmonella typhimurium.

The survival of Salmonella typhimurium (ATCC 13311) heated and recovered in media with 0, 1, 2, 3, 4 or 5% (w/w) added sodium chloride was investigated. A protective effect in the heating medium and an inhibitory effect in the recovery medium were observed. The results showed an interaction between the effect on, D(58 degrees C) values, of sodium chloride concentration in both media. Lower concentration in the heating media led to a greater effect of the sodium chloride concentration in the recovery media. When the sodium chloride concentration was the same in both media, the protective effect exerted in the heating media dominated over its inhibitory effect in the recovery media.

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.

Effect of pH on the heat resistance of spores comparison of two models.

All published models describing the effect of pH on the heat resistance of spores can be regarded either as a linear first degree equation or a linear second degree equation. This work aimed to compare both models from three sets of published data for, Clostridium sporogenes and Bacillus stearothermophilus, respectively. The relative quality of fit of each model with respect to the other depends on the species, the strain and the heating temperature. Parameter estimation was more reliable for the second degree model than for of the simple first degree equation. However, in the case of acidic foodstuffs, predictions obtained from the second degree model are more sensitive toward errors of parameter values. The second degree model is better from the point of view of safety at most frequent ranges of pH of foods. Moreover, for Clostridium botulinum the goodness of fit of this model is clearly higher than that of the first degree equation. If this observation is confirmed by further work, the second degree model in application of standard calculations of heat processes of foods would be preferred.

Effect of water activities of heating and recovery media on apparent heat resistance of Bacillus cereus spores.

Spores of Bacillus cereus were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores. The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors. Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity. As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose. In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values. This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose. When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the efficiency of depressors was also less pronounced. It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.

Taking injuries of surviving bacteria into account for optimising heat treatments.

The main assets of early conventional models applied in the field of canned food industries are their simplicity and their robustness. Moreover, a certain standardisation of these models allows the intrinsic quantification of a food process like sterilisation, regardless of the nature of concerned microbial populations. However, a first drawback of conventional models is their monofactorial nature: only temperature is considered for the evaluation of microbial heat resistance. A second limit of early survival models is that conventional estimates of heat resistance are made by recovering heated surviving cells at optimal incubation conditions. However, many investigators observed that culture medium and incubation temperature influence both the ratio of injured cell recovery and estimated heat resistance values. Mafart and Leguérinel [Mafart, P., Leguérinel, I., 1998. Modeling combined effects of temperature and pH on heat resistance of spores by a linear-Bigelow equation. J. Food Sci. 63, 6-8] developed a model describing the heat resistance of spores as a function of temperature and pH which is an extension of the Bigelow equation. A short while later, they added a further term to their model in order to consider the water activity of the heating medium (Gaillard, S., Leguérinel, I., Mafart, P., 1998. Model for combined effects of temperature, pH and water activity on thermal inactivation of Bacillus cereus spores. J. Food Sci. 63, 887-889). From their model, the authors proposed an extension of the concept of biological destruction value (BDV) noted L(T, pH, a(w)), that they called primary BDV. More recently, we developed a second multifactorial model describing the effect of the temperature, the pH and the water activity of the recovery medium on the estimated D-value of heated spores (unpublished). From this new model we propose the concept of secondary BDV noted L'(T, pH, a(w)). We show that, for calculations of heat processes, the effective BDV, M, to be considered is the overall function M = LL'. From another model [Mafart, P., Leguerinel, I., 1997. Modelling the heat stress and the recovery of bacterial spores. Int. J. Food Microbiol. 37, 131-135], it can be shown that the secondary BDV is also the corrective factor to be considered to assess the overall decimal reduction ratio q: q = L'n where n is the conventional decimal reduction ratio without taking into account the effect of environmental factors of the recovery medium on the effective heat resistance of injured spores.

Relationship between the apparent heat resistance of Bacillus cereus spores and the pH and NaCl concentration of the recovery medium.

Conventional heat resistance data, D values, were previously established by other workers at optimal condition for spores outgrowth. However, in canned food conditions of outgrowth are generally suboptimal in term of pH, salt concentration, water activity. The combined effects of pH and NaCl level of the recovery medium for the D value and z(pH) value were studied. Spores of Bacillus cereus were heated at 95 degrees C in phosphate-citrate buffer media at pH 7. Cells were recovered at 25 degrees C in Nutrient Agar with pH ranging from 5 to 7 and 1% to 4% (w/w) NaCl concentration. For each condition D' values (decimal reduction time associated with the recovery media characteristics) were determined. The results show a major influence of the recovery pH on the D' values. This effect is characterised by the z'(pH) values, distance of recovery medium pH from optimum recovery pH* medium (6.7) which leads to a tenfold reduction time of D value. The increase of the salt concentration leads to a slight decrease of D' value. However z'(pH) values are not significantly affected by the salt concentration. A simple three parameter model describing the effects of pH and NaCl concentration of the recovery medium upon the heat resistance of spores is proposed. The interaction between pH and salt concentration is sufficiently low to be neglected by the model.

Modelling the overall effect of pH on the apparent heat resistance of Bacillus cereus spores.

A simple overall model is proposed to describe the effect of both the pH of the heating menstruum and the pH of the recovery medium on the apparent spore heat resistance of Bacillus cereus. Applied to foods making up both heating and recovery media, the model can be reduced to only two parameters. Its goodness of fit and its robustness enable it to be applied to the optimisation of heat treatments. However. further experiments should be undertaken to validate the model for other species and to determine the parameters related to reference species such as Clostridium botulinum.