POPULATION MODEL of BACTERIAL SPORES FOR VALIDATION of DYNAMIC THERMAL PROCESSES1

Data are presented supporting a new model of spore populations during isothermal and dynamic lethal heat treatments. the model incorporates activation, injury, and preliminary inactivation of less-resistant fractions as well as the usual predominant inactivation. Rate constants of those transformations were determined experimentally for Bacillus subtilis strain A and were found to vary with temperature according to Arrhenius equations. Model generated and experimental isothermal survivor curves compared well. Comparison of model and experimental survivor curves for this species in a time-varying temperature regime showed the model to be a potentially good predictor of survivors during dynamic lethanl heat treatment. the new model could be particularly important in simulating sterilization and pasteurization processes, especially short duration UHT treatments, and microbiological validation of arbitrary, dynamic thermal processes.     

Comparison of Predictive Models for Bacterial Spore Population Resources to Sterilization Temperatures

Comparisons of characteristics of recent models and the conventional model of bacterial spore populations during thermal sterilization showed the conventional model was inadequate for general representation because it lacks activation of dormant spores. New models accounting for activation differed in other assumptions but obviated heat shock of indicator spores required when using the conventional model in validations of thermal sterilization. Comparisons of rate constants and simulated and experimental responses of models of B. stearother-mophilus spores in constant and dynamic temperatures showed one new model was more general, more accurate and preferred. Arrhenius equations accurately described temperature dependencies of all rate constants of that model.     

Thermal inactivation of <Emphasis Type="Italic">Alicyclobacillus acidoterrestris</Emphasis> spores under conditions simulating industrial heating processes of tangerine vesicles and its use in time temperature integrators

The aim of the present study was to characterize the thermal inactivation of Alicyclobacillus acidoterrestris spores under isothermal and non-isothermal conditions simulating industrial heating processes applied to tangerine vesicles. A microbiological time temperature integrator (TTI) suitable for estimating the severity of thermal processes applied to acid foods was also developed. The heat resistance of A. acidoterrestris was characterized by D _105 °C = 0.63 min and z = 10.8 °C in tangerine juice, showing linear survival curves, without shoulders and tails. Under non-isothermal conditions, the use of isothermal data allowed for an accurate prediction of the inactivation. The spores were included in alginate TTIs and they were used to estimate the severity of thermal treatments applied both in a thermoresistometer Mastia and in a food pilot plant scale system, which allows fast heating of the product to 93 °C and then a short holding time (2 min). In the thermoresistometer, tangerine juice was used as heating medium. In the food pilot plant scale system, thermal treatments were applied in batch to unpackaged tangerine vesicles. In both equipments, the TTIs accurately predicted the lethality of the thermal treatments applied. The percent coefficients of variation for survivor counting in TTIs showed that distribution of heat is homogeneous both in the thermoresistometer and in the reactor, being lower than 10% in all cases. The logistic and normal distributions were found to be the best for fitting the different survivor datasets.     

Comparative effect of different test methodologies on Bacillus coagulans spores inactivation kinetics in tomato pulp under isothermal conditions

Heat resistant micro‐organisms are an ongoing challenge to the food industry. Various factors may influence the heat resistance of micro‐organisms including type and strain; the environmental influences during cell and spore formations and during heat exposure; and the equipment and test tools used to perform the experimental process. In an attempt to analyse the influence of different test tools used on the heat inactivation processes, this study aimed to define the isothermal inactivation kinetics of Bacillus coagulans spores in tomato pulp at different temperatures and compare the inactivation of this bacterium when thermal death time (TDT) and capillary tube methods were used. Temperature ranges from 95 °C to 120 °C were studied, and inactivation kinetic parameters were estimated through the application of primary models. TDT inactivation curves consisted of shoulder and linear decline, while capillary method inactivation curves consisted of shoulder, linear decline and long tail. A secondary model was used to describe the influence of the temperature on spore inactivation parameters. The results showed test methods are at least as important in determining thermal processes as the micro‐organisms and media used.     

Kinetic studies on high-pressure inactivation of Bacillus stearothermophilus spores suspended in food matrices

Bacillus stearothermophilus spores ATCC 7953 can effectively be inactivated by high-pressure treatment, but only if it is applied at elevated temperatures; however, these temperatures are much lower compared to the temperature level used in heat inactivation under atmospheric pressure. Temperature and pressure in a range between 60 and 120°C and 50–600 MPa were applied to inactivate spores suspended in mashed broccoli and in cocoa mass. Utilizing an empirical mathematical model, derived from nth order kinetics, the survival curves of the spore strain investigated could be described accurately. The model can predict the impact of combined action of pressure and temperature on spore reduction. It was demonstrated that the inactivation of B. stearothermophilus spores ATCC 7953 improved with increasing treatment intensity. Beside intrinsic microbial inactivation mechanisms, the role of the pressure-induced shift in crystallization temperature of fat on spore inactivation in cocoa mass is discussed.     

High pressure thermal processing for the inactivation of Clostridium perfringens spores in beef slurry

The spores of Clostridium perfringens can survive and grow in cooked/pasteurized meat, especially during the cooling of large portions. In this study, 600 MPa high pressure thermal processing (HPTP) at 75 °C for the inactivation of C. perfringens spores was compared with 75 °C thermal processing alone. The HPTP enhanced the inactivation of C. perfringens spores in beef slurry, resulting in 2.2 log reductions for HPTP vs. no reductions for thermal processing after 20 min. Then, the HPTP resistance of two C. perfringens spore strains in beef slurry at 600 MPa was compared and modeled, and the effect of temperature investigated. The NZRM 898 and NZRM 2621 exhibited similar resistance, and Weibull modeled well the log spore survivor curves. The spore inactivation increased when HPTP temperature was raised from 38 to 75 °C. The results confirm the advantage of high pressure technology to increase the thermal inactivation of C. perfringens spores in beef slurry.Industrial relevanceC. perfringens spores may cause food/meat poisoning as a result of improperly handled and prepared foods in industrial kitchens. Thermal processes at 100 °C or higher are generally carried out to ensure the elimination of these pathogenic spores. High pressure processing (HPP) is a food pasteurization technique which would help to maintain the sensorial and nutritional properties of food. Preservation of foods with HPP in conjunction with mild heat (HPTP) would enhance the spore inactivation compared to thermal processing alone at the same temperature, due to a known germination–inactivation mechanism. This technology, together with the application of Good Manufacturing Practices, including rapid cooling, is a good alternative to the traditional methods for producing safe processed meat and poultry products with enhanced sensory and nutritional quality.     

Accurate quantification of thermophilic spores in dairy powders

Internationally, there are no official guidelines for the quantification of thermophilic spores in dairy products, which leads to variations in applied methodology. In this study, we assess the heat sensitivity of thermophilic spores, vegetative cells grown under laboratory conditions and spores in German dairy powders to determine appropriate heating conditions for accurate quantification of total thermophilic spores. The heat inactivation effect (80–95 °C) is limited for spores of Anoxybacillus flavithermus and Geobacillus stearothermophilus grown under laboratory conditions. However, for spores originating from whey, whey powder and skimmed milk powder (mostly identified as A. flavithermus), a different trend was observed; spore counts continuously reduced when heating time and temperature increased (80–98 °C, 10–30 min). The results indicate that data obtained using laboratory cultures cannot be extrapolated to commercial powders, and in this case, applying temperatures above 80 °C leads to an underestimation of spore counts in dairy powders.     

Combined thermal and high pressure inactivation kinetics of tomato lipoxygenase

The combined isothermal (10–60 °C) and isobaric (0.1–650 MPa) inactivation kinetics of lipoxygenase (LOX) extracted from tomatoes and reconstituted in a tomato purée were studied. Thermal inactivation of LOX at atmospheric pressure proceeded in the temperature range of 45–65 °C. LOX inactivation did not follow first order kinetics; the data could be fitted assuming that the two isoforms of LOX with different thermostability were present. Combined thermal and high pressure inactivation occurs at pressures in the range of 100–650 MPa combined with temperatures from 10–60 °C, and followed first-order kinetics. In the high-temperature/low-pressure range, (T≥50 °C and P≤300 MPa) an antagonistic effect is observed, therefore, the Arrhenius and Eyring equation cannot be used over the entire temperature and pressure range. Small temperature dependence is found in the low-temperature/high pressure range. A third degree polynomial model was successfully applied to describe the temperature–pressure dependence of the inactivation rate constants, which can be useful to predict inactivation rate constants of tomato LOX reconstituted in tomato purée in the temperature–pressure range studied.     

Thermal resistance of aerobic spore formers isolated from food products

The heat resistance of aerobic spore formers isolated from dairy products and cocoa powder was examined to give an overview of occurring highly heat‐resistant spores. Experiments were conducted in phosphate buffer at different temperatures for 30 min. Two Geobacillus pallidus strains survived the heat treatment at 125 °C with log reductions of 6.68 and 6.73. Furthermore, the inactivation kinetics of one of these strains (Bacillus amyloliquefaciens) were determined using a modified Arrhenius model. The inactivation followed a reaction order of 1 with a reaction rate constant (k_ref) of 0.085/s at 394 K and an activation energy (E_a) of 209 kJ/mol.     

A model of non‐isothermal degradation of nutrients,pigments and enzymes

Published isothermal degradation curves for chlorophyll A and thiamine in the range 100–150 °C and the inactivation curves of polyphenol oxidase (PPO) in the range 50–80 °C could be described by the model C(t)/C₀ = exp[?b(T)tⁿ] where C(t) and C₀ are the momentary and initial concentrations, respectively, b(T) a temperature dependent ‘rate parameter’ and n, a constant. This suggested that the temporal degradation/inactivation events of all three had a Weibull distribution with a practically constant shape factor. The temperature dependence of the ‘rate parameter’ could be described by the log logistic model, b(T) = log_e[1 + exp[k(T ? T_c)], where T_c is a marker of the temperature level where the degradation/inactivation occurs at a significant rate and k the steepness of the b(T) increase once this temperature range has been exceeded. These two models were combined to produce a non‐isothermal degradation/inactivation model, similar to one recently developed for microbial inactivation. It is based on the assumption that the local slope of the non‐isothermal decay curve, ie the momentary decay rate, is the slope of the isothermal curve at the momentary temperature at a time that corresponds to the momentary concentration of the still intact or active molecules. This model, in the form of a differential equation, was solved numerically to produce degradation/inactivation curves under temperature profiles that included heating and cooling and oscillating temperatures. Such simulations can be used to assess the impact of planned commercial heat processes on the stability of compounds of nutritional and quality concerns and the efficacy of methods to inactivate enzymes. Simulated decay curves on which a random noise was superimposed were used to demonstrate that the degradation/inactivation parameters, k and T_c, can be calculated directly from non‐isothermal decay curves, provided that the validity of the Weibullian and log logistic models and the constancy of the shape factor n could be assumed. Copyright © 2004 Society of Chemical Industry     

A study on degradation kinetics of ascorbic acid in drumstick (Moringa olifera) leaves during cooking

The kinetics of ascorbic acid degradation in drumstick (Moringa olifera) leaves as well as in pure ascorbic acid solutions at the initial concentrations present in drumstick leaves over a temperature range of 50–120 °C (isothermal temperature process) has been studied. The degradation kinetics of ascorbic acid was also evaluated in normal open‐pan cooking, pressure‐cooking and a newly developed and patented fuel‐efficient eco cooker (non‐isothermal heating process). The ascorbic acid degradation followed first‐order reaction kinetics where the rate constant increased with an increase in the temperature. The temperature dependence of degradation was adequately modelled by the Arrhenius equation. A mathematical model was developed using the isothermal kinetic parameters obtained to predict the losses of ascorbic acid from the time–temperature data of the non‐isothermal heating/processing method. The results obtained indicate the ascorbic acid degradation is of similar order of magnitude in all the methods of cooking. Copyright © 2005 Society of Chemical Industry     

Mechanism of Bacillus subtilis spore inactivation induced by moderate electric fields

Bacterial endospores are the key safety targets for inactivation within low-acid foods. Herein, we investigated the inactivation of Bacillus subtilis CGMCC 1.1087 spores (10⁷ CFU/mL) in sterile distilled water using moderate electric fields (MEF, 300 V/cm) under various temperatures (<30, 55, 65 and 75 °C). MEF treatment at below 30 °C resulted in 0.6-log reduction of spores, while treatments for 60 min without electric fields showed no inactivation. Inactivation induced by MEF in the same treatment time increased to 1.8-, 2.0- and 2.5-log as temperature increased to 55, 65 and 75 °C. Spores treated with MEF at <30, 55, 65 and 75 °C or mild heat (55, 65 and 75 °C) scarcely lost heat resistance, suggesting that spores did not germinate during MEF or mild heat treatment. The viability of MEF-treated spores did not increase by addition of lysozyme (3 μg/mL) in recovery plates, preincubation for 1 h in a 1:1 mixture of 60 mM Ca²⁺ and DPA, or lysozyme treatment in hypertonic medium. Confocal laser scanning microscopy photomicrographs showed that exposure to MEF induced a marked increase in the permeability of inner membrane and cortex. These findings suggested that damage of the cortex and inner membrane, rather than spore nutrient germinant receptors or cortex lytic enzymes, are possible reasons contributing to inactivation of B. subtilis spores by MEF. This study indicates that MEF at mild temperatures (55 to 75 °C) have the potential for spore inactivation.Industrial relevanceLiterature in the past few years has shown that moderate electric fields (MEF), typically associated with ohmic heating, have nonthermal effects on bacterial spores, leading to accelerated inactivation. The current work extends the range of temperatures to those well below thermally lethal conditions, and shows that some spore inactivation occurs under MEF, even when temperatures are sublethal. Little or no germination is observed, and spore inner membranes are increasingly compromised over time. The elucidation of such nonthermal effects would be significant to the food industry as it seeks increasingly nonthermal methods for inactivation of spores.     

Kinetic Parameters for Inactivation of Bacillus stearothermophilus at High Temperatures

High temperature thermal death parameters for Bacillus stearothermophilus TH24 (NCDO 1096) spores in water were determined using a computer-controlled reactor. The equipment produced and recorded accurate, reproducible square-wave temperature transients during very short heating times (?0.1 sec). Survivor curves and the phantom thermal death time (TDT) curve between 130°C and 155°C from reactor data were compared to results from oilbath-heated capillary tubes between 115°C and 135°C. The TDT curve was nonlinear in the high temperature range but could be described by two lines with z = 9.3C° for temperatures between 115°C and 145°C and z = 16.9C° for temperatures above 145°C. An Arrhenius plot did not represent the data better.     

Thermal Inactivation Kinetics of Peroxidase in Coriander Leaves

Design of efficient blanching treatments requires knowledge of critical factors such as enzyme inactivation kinetic parameters and relative proportions of heat-labile and heat-resistant fractions, which is unique in each vegetable. Thermal inactivation curves for peroxidase in coriander leaves were determined in the temperature range of 70 to 100 °C and in steam. The isothermal data were statistically treated using both linear and nonlinear regression. Applicability of various enzyme inactivation models available in the literature was critically evaluated. The two-fraction first-order model was found to be the best model to describe the peroxidase inactivation kinetics in coriander leaves (R ² > 0.97). Kinetic parameters were determined for heat-labile and heat-resistant isoenzyme fractions. The temperature dependence of the rate parameters in the present study did not follow the Arrhenius relationship.     

Effect of small, acid-soluble proteins on spore resistance and germination under a combination of pressure and heat treatment

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of alpha/beta-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65 degrees C and 100 to 500 MPa) and applied to wild-type and SASP-alpha-/beta- Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65 degrees C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65 degrees C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65 degrees C inactivated wild-type SASP-alpha+/beta+ spores more than mutant SASP-alpha-/beta- spores, and this was attributed to less pressure-induced germination in SASP-alpha-/beta- spores than in wild-type SASP-alpha+/beta+ spores. However, there was no difference in the pressure resistance between SASP-alpha+/beta+ and SASP-alpha-/beta- spores at 100 MPa and ambient temperature (approximately 22 degrees C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that alpha/beta-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.     

ULTRA-HIGH TEMPERATURE EFFECT ON B. STEAROTHERMOPHILUS DURING PUFFING OF RICE1

Destruction, damage and activation of thermophilic bacterial spores of Bacillus stearothermophilus at ultra-high heating temperatures (170°C to 210°C) were studied using a rice cake machine as a model system. Activation of spores at ultra-high temperatures was observed after 99.9% of the original spores were killed (P < 0.05). Significant difference (P < 0.01) in spore counts was found when a rich protein medium and a minimal nutritious medium were used simultaneously to recover spores after heating. This indicated that heat damage did occur and that amino acids were required to repair the damage.     

A time temperature integrator for particulated foods: thermal process evaluation

A time temperature integrator (TTI) was developed by immobilizing Bacillus stearothermophilus spores in a cylindrical particle consisting of an alginate-starch-mushroom purée. The particle showed homogeneous spore distribution, and when heated over a temperature range of 121?–?130°?C negligible spore leakage was observed after the thermal process. The experimental data on spore survivor levels obtained for each temperature-time combination were compared with theoretical predictions using a mathematical model. The results showed a good correlation between the experimental and theoretical data. All these results provide evidence that this artificial particle could be a very reliable TTI for monitoring the thermal impact on micro-organisms during validating sterilization processes in continuous aseptic systems.     

Mathematical modeling of Saccharomyces cerevisiae inactivation under high‐pressure carbon dioxide

High‐pressure carbon dioxide inactivation curves of Saccharomyces cerevisiae at different temperatures were analysed using the modified Gompertz model. Comparable λ and μ values were obtained under pressure treatment as function of temperature. The phase of disappearance (λ) and the inactivation rate (μ) of S. cerevisiae were inversely related. Higher μ values were obtained at 50°C than at 40, 30, and 20°C under 10.0 MPa CO₂ pressure. Increased pressure and temperature had significant effects on the survival of S. cerevisiae. Arrhenius, linear and square‐root models were used to analyse the temperature dependence of the inactivation rate constant. For the Arrhenius model the activation energy (E_μ) was 56.49 kJ/mol at 10.0 MPa, and 55.70, 53.83, and 52.20 kJ/mol at 7.5, 5.0, and 2.5 MPa, respectively. Results of this study enable the prediction of yeast inactivation exposed to different CO₂ pressures and temperatures.     

A time temperature integrator for particulated foods: thermal process evaluation

 A time temperature integrator (TTI) was developed by immobilizing Bacillus stearothermophilus spores in a cylindrical particle consisting of an alginate-starch-mushroom purée. The particle showed homogeneous spore distribution, and when heated over a temperature range of 121 – 130° C negligible spore leakage was observed after the thermal process. The experimental data on spore survivor levels obtained for each temperature-time combination were compared with theoretical predictions using a mathematical model. The results showed a good correlation between the experimental and theoretical data. All these results provide evidence that this artificial particle could be a very reliable TTI for monitoring the thermal impact on micro-organisms during validating sterilization processes in continuous aseptic systems. Received: 25 February 1997     

Inactivation Kinetics of Pectin Methylesterase,Polyphenol Oxidase,and Peroxidase in Cloudy Apple Juice under Microwave and Conventional Heating to Evaluate Non-Thermal Microwave Effects

Continuous-flow microwave pasteurization provides important advantages over conventional heat exchangers such as fast volumetric heating, lower tube surface temperature, and possible non-thermal effects that enhance enzymatic and bacterial inactivation. Conventional and microwave-assisted inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) in cloudy apple juice were investigated to evaluate non-thermal effects. Experiments were conducted to provide uniform heating with accurate temperature acquisition and similar temperature profiles for conventional and microwave treatments. A two-fraction first-order kinetic model was successfully fitted to the data in a procedure that took into account the whole time-temperature profile instead of assuming isothermal conditions. Predicted inactivation curves for pasteurization at 70 and 80 °C of the cloudy apple juice showed that PME has the highest thermal resistance (residual activity of 30% after 250 s at 80 °C) and that there was no evidence of non-thermal microwave effects on the inactivation of these enzymes.