Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time.

Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH.IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.

Validation of a predictive model coupling gas transfer and microbial growth in fresh food packed under modified atmosphere.

Predicting microbial safety of fresh products in modified atmosphere packaging implies to take into account the dynamic of O2, CO2 and N2 exchanges in the system and its effect on microbial growth. In this paper a mechanistic model coupling gas transfer and predictive microbiology was validated using dedicated challenge-tests performed on poultry meat, fresh salmon and processed cheese, inoculated with either Listeria monocytogenes or Pseudomonas fluorescens and packed in commercially used packaging materials (tray + lid films). The model succeeded in predicting the relative variation of O2, CO2 and N2 partial pressure in headspace and the growth of the studied microorganisms without any parameter identification. This work highlighted that the respiration of the targeted microorganism itself and/or that of the naturally present microflora could not be neglected in most of the cases, and could, in the particular case of aerobic microbes contribute to limit the growth by removing all residual O2 in the package. This work also confirmed the low sensitivity of L. monocytogenes toward CO2 while that of P. fluorescens permitted to efficiently prevent its growth by choosing the right combination of packaging gas permeability value and initial % of CO2 initially flushed in the pack.

Modeling the recovery of heat-treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal temperature and pH using growth limits.

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

Mechanistic model coupling gas exchange dynamics and Listeria monocytogenes growth in modified atmosphere packaging of non respiring food.

A mechanistic model coupling O2 and CO2 mass transfer (namely diffusion and solubilisation in the food itself and permeation through the packaging material) to microbial growth models was developed aiming at predicting the shelf life of modified atmosphere packaging (MAP) systems. It was experimentally validated on a non-respiring food by investigating concomitantly the O2/CO2 partial pressure in packaging headspace and the growth of Listeria monocytogenes (average microbial count) within the food sample. A sensitivity analysis has revealed that the reliability of the prediction by this "super-parametrized" model (no less than 47 parameters were required for running one simulation) was strongly dependent on the accuracy of the microbial input parameters. Once validated, this model was used to decipher the role of O2/CO2 mass transfer on microbial growth and as a MAP design tool: an example of MAP dimensioning was provided in this paper as a proof of concept.

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.

Predicting heat process efficiency in thermal processes when bacterial inactivation is not log-linear.

The food industry widely uses the F-value which considers microbial log-linear inactivation, while microbial heat inactivation may result in a non-log-linear inactivation pattern due to genetic or phenotypical heterogeneity. This may yield discrepancies in predicting microbial heat inactivation under dynamic conditions of heat treatment. In this paper, we suggest the calculation of the equivalent time of heat treatment at a given temperature to overcome these constraints. To validate our proposal, the heat inactivation of Bacillus pumilus, showing non-log-linear behavior, was predicted for 4 different heat inactivation profiles and bacterial enumeration was performed to determine whether prediction errors were acceptable. When the proportion of residuals in an acceptable zone from 1 log (fail safe) to 0.5 log (fail dangerous) was greater or equal to 70%, the model was considered as acceptable for predictions of the tested data. The new approach gave four different temperature profiles, with 96, 85, 85 and 100% of the residuals in the acceptable zone, indicating satisfactory prediction. Thus the proposed practical alternative to simulate microbial heat inactivation kinetics is able to extend the F-value to non-log-linear inactivation patterns.

Modelling pH evolution and lactic acid production in the growth medium of a lactic acid bacterium: application to set a biological TTI.

Time temperature integrators or indicators (TTIs) are effective tools making the continuous monitoring of the time temperature history of chilled products possible throughout the cold chain. Their correct setting is of critical importance to ensure food quality. The objective of this study was to develop a model to facilitate accurate settings of the CRYOLOG biological TTI, TRACEO. Experimental designs were used to investigate and model the effects of the temperature, the TTI inoculum size, pH, and water activity on its response time. The modelling process went through several steps addressing growth, acidification and inhibition phenomena in dynamic conditions. The model showed satisfactory results and validations in industrial conditions gave clear evidence that such a model is a valuable tool, not only to predict accurate response times of TRACEO, but also to propose precise settings to manufacture the appropriate TTI to trace a particular food according to a given time temperature scenario.

Effect of incubation temperature and pH on the recovery of Bacillus weihenstephanensis spores after exposure to a peracetic acid-based disinfectant or to pulsed light.

The recovery at a range of incubation temperatures and pH of spores of Bacillus weihenstephanensis KBAB4 exposed to a peracetic acid-based disinfectant (PABD) or to pulsed light was estimated. Spores of B. weihenstephanensis were produced at 30 °C and pH 7.00, at 30 °C and pH 5.50, or at 12 °C and pH 7.00. The spores were treated with a commercial peracetic acid-based disinfectant at 80 mg·mL-1 for 0 to 200 min at 18 °C or by pulsed light at fluences ranging between 0.4 and 2.3 J·cm-2 for pulsed light treatment. After each treatment, the spores were incubated on nutrient agar at 12 °C, 30 °C or 37 °C, or at pH 5.10, 6.00 or 7.40. Incubation temperature during recovery had a significant impact only near the recovery limits, beyond which surviving spores previously exposed to a PABD or to pulsed light were not able to form colonies. In contrast, a decrease in pH of the recovery nutrient agar had a progressive impact on the ability of spores to form colonies. The time to first log reduction after PABD treatment was 29.5 ±â€¯0.7 min with recovery at pH 7.40, and was tremendously shortened 5.1 ±â€¯0.2 min with recovery at pH 5.10. Concerning the fluence necessary for the first log reduction, it was 1.5 times higher when the spores were recovered at pH 6.00 compared to a recovery at pH 5.10. The impact of recovery temperature and pH can be described with a mathematical model using cardinal temperature and pH as parameters. These effects of temperature and pH on recovery of Bacillus weihenstephanensis spores exposed to a disinfectant combining peracetic acid and hydrogen peroxide, or pulsed light are similar, although these treatments are of different natures. Sporulation temperature or pH did not impact resistance to the peracetic acid-based disinfectant or pulsed light.

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.

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).

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.

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.

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.

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.

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.