Nonproteolytic Clostridium Botulinum Research Articles

Selection and Development of Nontoxic Nonproteolytic Clostridium botulinum Surrogate Strains for Food Challenge Testing.

Clostridium botulinum causes severe foodborne intoxications by producing a potent neurotoxin. Challenge studies with this pathogen are an important tool to ensure the safety of new processing techniques and newly designed or modified foods, but they are hazardous and complicated by the lack of an effective selective counting medium. Therefore, this study aimed to develop selectable nontoxic surrogate strains for group II, or nonproteolytic, C. botulinum, which are psychotropic and hence of particular concern in mildly treated, refrigerated foods. Thirty-one natural nontoxic nonproteolytic strains, 16 of which were isolated in this work, were characterized in detail, revealing that 28 strains were genomically and phenotypically indistinguishable from toxic strains. Five strains, representing the genomic and phenotypic diversity of group II C. botulinum, were selected and successfully equipped with an erythromycin (Em) resistance marker in a defective structural phage gene without altering phenotypic features. Finally, a selective medium containing Em, cycloserine (Cs), gentamicin (Gm), and lysozyme (Ly) was developed, which inhibited the background microbiota of commercial cooked ham, chicken filet, and salami, but supported spore germination and growth of the Em-resistant surrogate strains. The surrogates developed in this work are expected to facilitate food challenge studies with nonproteolytic C. botulinum for the food industry and can also provide a safe alternative for basic C. botulinum research.

Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum – Evaluation and validation with MAP and smoked foods

The growth inhibiting effect of lactic acid bacteria (LAB) on non-proteolytic Clostridium botulinum was studied. LAB had no significant effect on growth of C. botulinum and their effect was not included in the model to be evaluated. An available cardinal parameter growth and growth boundary model for non-proteolytic C. botulinum (Koukou et al., 2021; https://doi.org/10.1016/j.ijfoodmicro.2021.109162) was evaluated using a total of 822 time-to-toxin (TTT) formation data extracted from the scientific literature for seafood, poultry, vegetables and meat products. These data included smoked products and food stored in air, vacuum or modified atmosphere packaging (MAP) with added CO2. The available extensive model predicted TTT formation without bias (Bf-TTT value = 0.99) and with a reasonable accuracy (Af-TTT value = 1.76). The model was successfully validated for seafood and poultry products. This study substantially increased the range of applicability of the available growth and growth boundary model for non-proteolytic C. botulinum. The performed evaluation showed this model can be used to predict environmental conditions to prevent growth in seafood and poultry products including smoked fish and MAP foods. It is expected that this validated model will contribute to product development and innovation including new sodium reduced foods.

Cardinal parameter growth and growth boundary model for non-proteolytic Clostridium botulinum – Effect of eight environmental factors

A new cardinal parameter growth and growth boundary model for non-proteolytic C. botulinum was developed and validated for fresh and lightly preserved seafood and poultry products. 523 growth rates in broth were used to determine cardinal parameter values and terms for temperature, pH, NaCl/water activity, acetic, benzoic, citric, lactic and sorbic acids. The new growth and growth boundary model included the inhibiting interactive effect between these factors and it was calibrated using growth curves from 10 challenge tests with unprocessed seafood. For model evaluation, 40 challenge tests with well characterized fresh and lightly preserved seafood were performed. Comparison of these observed growth curves and growth rates (μmax-values) predicted by the new model resulted in a bias factor (Bf) of 1.12 and an accuracy factor (Af) of 1.40. Furthermore, the new model was evaluated with 94 growth rates and 432 time to toxin formation data extracted from the scientific literature for seafood, poultry, meat, pasta and prepared meals. These data included responses for 36 different toxigenic strains of non-proteolytic C. botulinum. The obtained Bf-/Af-values were 0.97/2.04 for μmax-values and 0.96/1.80 for time to toxin formation. The model correctly predicted 93.8% of the growth responses with 5.6% being fail-safe and <1% fail-dangerous. A cocktail of four non-toxin producing Clostridium spp. isolates was used to develop the new model and these isolates had more than 99.8% 16S rRNA gene similarity to non-proteolytic C. botulinum (Group II). The high number of environmental factors included in the new model makes it a flexible tool to facilitate development or reformulation of seafood and poultry products that do not support the growth of non-proteolytic C. botulinum. Further, evaluation of the new model with well characterized products is desirable particularly for meat, vegetables, pasta and prepared meals as well as for dairy products that was not included in the present study.

Evidence for Bacillus cereus Spores as the Target Pathogen in Thermally Processed Extended Shelf Life Refrigerated Foods

The microbial safety concern associated with thermally processed extended shelf life (ESL) refrigerated foods is based on adequate elimination of spore-forming pathogens such as nonproteolytic Clostridium botulinum types B, E, and F. These pathogens are traditionally regarded as targets for validation of thermally processed ESL foods. However, their use for research is restricted due to their designation as select agents. In this study, the thermal resistances of spores of 10 nonproteolytic C. botulinum types B and F and seven psychrotrophic Bacillus cereus strains were evaluated in ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer (0.05 M, pH 7.00) and compared to determine whether any of the B. cereus strains could serve as a nonselect agent for establishing thermal processes for ESL refrigerated foods. Thermal decimal reduction times (DT-values) of both nonproteolytic C. botulinum types B and F and psychrotrophic B. cereus strains decreased as process temperature increased from 80 to 91°C, and the highest values were obtained at 80°C. All psychrotrophic B. cereus strains tested were more thermally resistant than nonproteolytic C. botulinum types B and F. DT-values of nonproteolytic C. botulinum types B and F decreased to <1.0 min at 87°C, whereas all psychrotrophic B. cereus strains had higher DT-values (i.e., 52.35 to 133.69 min) at the same temperature. Among all psychrotrophic B. cereus strains tested, BC-6A16 had the highest DT-values at any given temperature. The DT-values indicated that the psychrotrophic B. cereus strains were more thermally resistant than the nonproteolytic C. botulinum strains and therefore may be potential target pathogens for thermal process validation of ESL refrigerated foods. However, further comparative challenge studies are needed with a model food system or an ESL refrigerated food to confirm these results.

Extensive Growth and Growth Boundary Model for Non-Proteolytic &lt;i&gt;Clostridium Botulinum&lt;/i&gt; – New Term for Smoke Components and Validation with Map Foods

The growth inhibiting effect of smoke components (phenol) and lactic acid bacteria on non-proteolytic Clostridium botulinum were studied. An available cardinal parameter growth and growth boundary model for non-proteolytic C. botulinum (Koukou et al., 2021; https://doi.org/10.1016/j.ijfoodmicro.2021.109162) was expanded with a term for phenol to include the combined inhibiting effect of nine environmental factors. The expanded model was then evaluated using 810 time-to-toxin (TTT) formation data extracted from the scientific literature for seafood, poultry, vegetables and meat products in different atmospheres including air, vacuum or modified atmosphere packaging (MAP) with added CO2. Cold-smoking inhibited growth of non-proteolytic C. botulinum whereas hot-smoking and lactic acid bacteria had no significant inhibiting effect on growth rates (P > 0.2). The expanded model predicted TTT formation without bias (Bf-TTT value = 1.00) and with a reasonable accuracy (Af-TTT value = 1.77). This study substantially increased the range of applicability of the available growth and growth boundary model for non-proteolytic C. botulinum as the expanded model allowed prediction of environmental conditions to prevent growth in seafood (including smoked fish), poultry and vegetable products. It is expected that the expanded model will contribute to product development including development of new sodium reduced foods.

Assessment of the risk of botulism from chilled, vacuum/modified atmosphere packed fresh beef, lamb and pork held at 3 °C–8 °C

The safety of current UK industry practice (including shelf-life) for chilled, vacuum/modified atmosphere-packed fresh red meat (beef, lamb and pork) held at 3°C–8°C has been evaluated with respect to non-proteolytic Clostridium botulinum. UK industry typically applies a retail pack shelf-life at 3°C–8°C to 13 days for fresh red meat, with a maximum of 23 days for beef, 27 days for lamb, and 18 days for pork. An exposure assessment established that current commercial practice for fresh red meat provided strong protection with more than 1010 person servings marketed in the UK without association with foodborne botulism. A challenge test demonstrated that spores of non-proteolytic C. botulinum inoculated on chilled vacuum-packed fresh red meat did not lead to detectable neurotoxin at day 50 for beef, day 35 for lamb, or day 25 for pork (i.e. <40 pg type B toxin and type E toxin g−1 of meat). The products were visually spoiled many days before these end points. The exposure assessment and challenge test demonstrated the safety of current UK industry practices for the shelf-life of fresh, vacuum-packed beef, lamb and pork held at 3°C–8°C with respect to C. botulinum, and that botulinum neurotoxin was not detected within their organoleptic shelf-life.

The use of the so-called 'tubs' for transporting and storing fresh fishery products.

On‐land transport/storage of fresh fishery products (FFP) for up to 3 days in ‘tubs’ of three‐layered poly‐ethylene filled with freshwater and ice was compared to the currently authorised practice (fish boxes of high‐density poly‐ethylene filled with ice). The impact on the survival and growth of biological hazards in fish and the histamine production in fish species associated with a high amount of histidine was assessed. In different modelling scenarios, the FFP are stored on‐board in freshwater or seawater/ice (in tubs) and once on‐land they are ‘handled’ (i.e. sorted or gutted and/or filleted) and transferred to either tubs or boxes. The temperature of the FFP was assumed to be the most influential factor affecting relevant hazards. Under reasonably foreseeable ‘abusive’ scenarios and using a conservative modelling approach, the growth of the relevant hazards (i.e. Listeria monocytogenes, Aeromonas spp. and non‐proteolytic Clostridium botulinum), is expected to be < 0.2 log10 units higher in tubs than in boxes after 3 days when the initial temperature of the fish is 0°C (‘keeping’ process). Starting at 7°C (‘cooling‐keeping’ process), the expected difference in the growth potential is higher (< 1 log10 for A. hydrophila and < 0.5 log10 for the other two hazards) due to the poorer cooling capacity of water and ice (tub) compared with ice (box). The survival of relevant hazards is not or is negligibly impacted. Histamine formation due to growth of Morganella psychrotolerans under the ‘keeping’ or ‘cooling‐keeping’ process can be up to 0.4 ppm and 1.5 ppm higher, respectively, in tubs as compared to boxes after 3 days, without reaching the legal limit of 100 ppm. The water uptake associated with the storage of the FFP in tubs (which may be up to 6%) does not make a relevant contribution to the differences in microbial growth potential compared to boxes.

Thermal gelation of Pacific whiting surimi in microwave assisted pasteurization

This research invetigated the potential of microwave assisted pasteurization systems (MAPS) for production of molded or thick sheeted products from Pacific whiting (PW) surimi. Prior to MAPS processing, a systematic study was conducted to evaluate the effect of heating rate on textural properties of PW gels by heating surimi paste from 3 °C/min to 24 °C/min. No gel was formed at a slow heating rate (3 °C/min). Gels with greater strength were achieved with higher heating rates. The heating pattern for the surimi pastes in microwave heating was predicted using a gellan gel model food in which 2% salt and 40% sucrose were added to adjust the dielectric properties. Prepackaged PW surimi paste in pouches were pasteurized to a similiar leathal lethalty for non-proteolytic Clostridium botulinum spores with MAPS or water bath as control. The breaking force and penetration distance of the PW surimi gels were significantly (P < 0.05) higher when processed in MAPS as compared to water bath.

Inactivation of non-proteolytic Clostridium botulinum type E in low-acid foods and phosphate buffer by heat and pressure.

The effect of high pressure thermal (HPT) treatments on the inactivation of spores of non-proteolytic type E Clostridium botulinum TMW 2.990 was investigated at high pressures (300 to 600 MPa) and elevated temperatures (80 to 100 °C) in four low-acid foods (steamed sole, green peas with ham, vegetable soup, braised veal) and imidazole phosphate buffer (IPB). In addition, corresponding conventional thermal treatments at ambient pressure were performed to expose possible synergisms of pressure and temperature on spore inactivation. In general, spore count reduction was more efficient by combining pressure and temperatures < 100 °C and the overall process duration could be shortened due to accelerated heating rates (adiabatic effect). Processing at 90 °C and 600 MPa resulted in inactivation below the detection limit after 5 min in all foods except steamed sole. Traditional thermal processing of spores at 90 °C for 10 min, on the other hand, did not result in an estimated 6-log reduction. Additional HPT treatments in steamed sole and IPB did not reveal pronounced food matrix dependent protective effects. Here, varying pressure levels did not appear to be the driving force for spore count reduction in steamed sole at any temperature. By applying a Weibull distribution on destruction kinetics of isobaric/isothermal holding times, 6D-values were calculated. Compression and decompression phase (1 s pressure holding time) had a considerable impact on spore count reduction (max. -2.9 log units) in both, foods and buffer. Hence, compression and decompression phases should directly be included into the total lethal effect of HPT treatments to avoid prolonged holding times and overprocessing.

Effect of High Pressures in Combination with Temperature on the Inactivation of Spores of Nonproteolytic Clostridium botulinum Types B and F

The impact of high pressure processing on the inactivation of spores of nonproteolytic Clostridium botulinum is important in extended shelf life chilled low-acid foods. The three most resistant C. botulinum strains (Ham-B, Kap 9-B, and 610-F) were selected for comparison of their thermal and pressure-assisted thermal resistance after screening 17 nonproteolytic C. botulinum strains (8 type B, 7 type E, and 2 type F). Spores of strains Ham-B, Kap 9-B, and 610-F were prepared using a biphasic media method, diluted in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.00) to 105 to 106 CFU/mL, placed into a modified sterile transfer pipette, heat sealed, and subjected to a combination of high pressures (600 to 750 MPa) and high temperatures (80 to 91°C) using laboratory and pilot-scale pressure test systems. Diluted spores from the same crops were placed in nuclear magnetic resonance tubes, which were heat sealed, and subjected to 80 to 91°C in a Fluke 7321 high precision bath with Duratheram S oil as the heat transfer fluid. After incubation for 3 months, survivors in both studies were determined by the five-tube most-probable-number method using Trypticase-peptone-glucose-yeast extract broth. The highest (>5.0) log reductions in spore counts for Ham-B, Kap 9-B, and 610-F occurred at the highest temperature and pressure combination tested (91°C and 750 MPa). Thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 87°C, decreasing to <1.0 min at 87°C for these strains. Pressure-assisted thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 91°C with any pressure combination and decreased to <1.0 min as the pressure increased from 600 to 750 MPa at 91°C. Based on the pressure-assisted thermal D-values, pressure exerted a more protective effect on spores of Ham-B, Kap 9-B, and 610-F when processed at 83 to 91°C combined with pressures of 600 to 700 MPa when compared with thermal treatment only. No protective effect was observed when the spores of Ham-B, Kap9-B, and 610-F were treated at lower temperatures (80 to 83°C) in combination with 750 MPa. However, at higher temperatures (87 to 91°C) in combination with 750 MPa, a protective effect was seen for Ham-B, Kap9-B, and 610-F spores based on the calculated pressure-assisted thermal D-values.

Food Quality Evaluation using Model Foods: a Comparison Study between Microwave-Assisted and Conventional Thermal Pasteurization Processes

Thermal process optimization has focused on traditional sterilization, with limited research on pasteurization or microwave-assisted thermal processing. Model foods have been developed as quality evaluation tools for thermal pasteurization processes, but there are no comprehensive studies demonstrating how these model foods could be used to evaluate and compare the resulting food quality after different pasteurization processes. The aim of this study was to develop a methodology using image and quantitative analyses for quality evaluation of pre-packaged food pasteurized using a microwave-assisted pasteurization system (MAPS) and traditional hot water method. Four pasteurization processes (MAPS and hot water method at 90 and 95 °C) were designed to have an equivalent accumulated thermal lethality at the cold spot of at least 90 °C for 10 min to control nonproteolytic Clostridium botulinum spores. Color-based time-temperature indicators in mashed potato and green pea model foods were quantified using image analysis. Results showed that median color values were useful in assessing overall color change, and interquartile range was an indicator of burnt areas. MAPS 95 °C was the best process because it had the smallest hot spot cook values and the least color change, while the 90 °C hot water process was the worst. Model foods and image analysis techniques were useful pasteurization process quality evaluation tools and made it possible to visualize the potential food quality change volumetrically, throughout a food package. In the future, these tools could be combined with computer simulations to optimize the quality of pilot-scale and industrial MAPS or conventional pasteurization processes.

Mathematical modeling and Monte Carlo simulation of thermal inactivation of non-proteolytic Clostridium botulinum spores during continuous microwave-assisted pasteurization

The objective of this study was to develop a mathematical method to predict the internal temperature history of products exposed to a microwave-assisted pasteurization system and use Monte Carlo simulation to analyze the inactivation of the spores of Clostridium botulinum Types B in beef meatball trays and Type E in salmon fillet trays. With a target of 6 log-reduction in the spores, the simulation showed >98.8% and 99.1% of the processes will achieve a minimum of 5-log reductions of C. botulinum Type B in beef meatball trays and Type E in salmon fillet trays, respectively. Sensitivity analysis showed that the heating temperature, time, and product heating rate in the Microwave-Assisted Heating section and the heating temperature in the Pre-Heating section are four most critical factors affecting the accumulation of lethality. The results of this study may be used to guide the development of more effective thermal processes in microwave-assisted pasteurization systems.

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement.

Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials.

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg−1. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments.

Characterisation of non-toxigenic Clostridium spp. strains, to use as surrogates for non-proteolytic Clostridium botulinum in chilled food challenge testing

Under many of the conditions studied, a two-strain cocktail of non-toxigenic Clostridium spp. was found to be suitable as a surrogate for non-proteolytic Clostridium botulinum, and has the potential for use in chilled food challenge tests measuring growth. Non-toxigenic surrogates could also be used in thermal process screening studies.

Inability of non-proteolytic Clostridium botulinum to grow in mussels inoculated via immersion and packaged in high oxygen atmospheres

A series of botulism challenge studies were conducted to determine if botulinum toxin would be produced in mussels (Mytilus edulis) inoculated with non-proteolytic Clostridium botulinum spores and held under modified atmosphere (MA) packaging conditions at normal (4 °C) and abusive (12 °C) temperatures. Spore mixtures of six strains of non-proteolytic C. botulinum were introduced into live mussels through immersion in a seawater solution with cultured algae. Mussels were packed in a commercial high-oxygen (60–65% O2) MA-package with a buffer, and also packed under a vacuum. Feeding live mussels cultured algae (104 cells/ml) with a C. botulinum spore suspension (103 spores/ml) in seawater at 4 °C for 6 h resulted in the uptake of spores into mussel tissue (500/g) and the mussel GI tract (100/g). Under all of the experimental conditions evaluated, none of the fresh mussels became toxic, even after spoilage and in the absence of oxygen. However, control samples using tuna or cooked mussel meats became toxic in the absence of oxygen. Botulinum toxin was not produced in fresh mussels packaged under the MA-packaging conditions evaluated, even at an abusive storage temperature (12 °C) for at least 12 days or at normal storage temperate (4 °C) for at least 21 days, which is beyond their shelf life.

Botulism challenge studies of a modified atmosphere package for fresh mussels: inoculated pack studies.

A series of botulism challenge studies were performed to determine the possibility of production of botulinum toxin in mussels (Mytilus edulis) held under a commercial high-oxygen (60 to 65% O(2)), modified atmosphere packaging (MAP) condition. Spore mixtures of six strains of nonproteolytic Clostridium botulinum were introduced into mussel MAP packages receiving different packaging buffers with or without the addition of lactic acid bacteria. Dye studies and package flipping trials were conducted to ensure internalization of spores by packed mussels. Inoculated mussel packages were stored at normal (4°C) and abusive (12°C) temperatures for 21 and 13 days, respectively, which were beyond the packaged mussels' intended shelf life. Microbiological and chemical analyses were conducted at predetermined intervals (a total of five sampling times at each temperature), including total aerobic plate counts, C. botulinum counts, lactic acid bacterial counts, package headspace gas composition, pH of packaging buffer and mussel meat, and botulinum toxin assays of packaging buffer and mussel meat. Results revealed that C. botulinum inoculated in fresh mussels packed under MAP packaging did not produce toxin, even at an abusive storage temperature and when held beyond their shelf life. No evidence was found that packaging buffers or gas composition influenced the lack of botulinum toxin production in packed mussels.

Does proximity to neighbours affect germination of spores of non-proteolytic Clostridium botulinum?

It is recognised that inoculum size affects the rate and extent of bacterial spore germination. It has been proposed that this is due to spores interacting: molecules released from germinated spores trigger germination of dormant neighbours. This study investigated whether changes to the total number of spores in a system or proximity to other spores (local spore density) had a more significant effect on interaction between spores of non-proteolytic Clostridium botulinum strain Eklund 17B attached to defined areas of microscope slides. Both the number of spores attached to the slides and local spore density (number of spores per mm2) were varied by a factor of nine. Germination was observed microscopically at 15 °C for 8 h and the probability of, and time to, germination calculated from image analysis measurements. Statistical analysis revealed that the effect of total spore number on the probability of germination within 8 h was more significant than that of proximity to neighbours (local spore density); its influence on germination probability was approximately four-times greater. Total spore number had an even more significant affect on time to germination; it had a nine-fold greater influence than proximity to neighbours. The applied models provide a means to characterise, quantitatively, the effect of the total spore number on spore germination relative to the effect of proximity to neighbouring spores.

Thermal inactivation of foodborne pathogens and the USDA pathogen modeling program

The use of heat to inactivate foodborne pathogens is a critical control point and the most common means for assuring the microbiological safety of processed foods. A key to optimization of the heating step is defining the target pathogens’ heat resistance. Sufficient evidence exists to document that insufficient cooking, reheating, and/or subsequent cooling are often contributing factors in food-poisoning outbreaks. Accordingly, the objective of thermal processing is to design sufficient heating regiments to achieve a specific lethality for foodborne pathogens in foods. The effects and interactions of temperature, pH, sodium chloride content, sodium pyrophosphate, and sodium lactate concentration are among the variables that were considered when attempting to assess the heat inactivation kinetics of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and spores of non-proteolytic Clostridium botulinum. Incorporation of these multiple barriers usually increases the sensitivity of pathogens to heat, thereby reducing heat requirements and ensuring the safety of ready-to-eat food products. Complex multifactorial experiments and analysis to quantify the effects and interactions of additional intrinsic and extrinsic factors and development of “enhanced” predictive models are underway to ensure the microbiological safety of thermally processed foods. Predictive inactivation kinetics (thermal death) models for foodborne pathogens have been converted into an easy-to-use computer program that is available on the USDA–Eastern Regional Research Center website. These models should aid in evaluating the safety of cooked products and are being used as building blocks for microbial risk assessment.

Quantitative risk assessment for hazards that arise from non-proteolytic Clostridium botulinum in minimally processed chilled dairy-based foods

A modular process risk model has been constructed that describes the manufacture of dairy dessert products and hazards that arise from non-proteolytic Clostridium botulinum. The model describes batch manufacture and consumer storage of a family size generic dairy dessert but includes a realistic quantification that could apply to a specific food product. The dairy dessert sector is an expanding part of the UK market. The model includes modules that describe spore loads in raw materials, spore inactivation during thermal processing, volume partition and the population kinetics for non-proteolytic C. botulinum during sequential isothermal storage regimes. Where possible elements of uncertainty and variability are identified explicitly. The model is constructed as a belief network from published data and expert opinions. The model provides marginal probabilities, and associated sensitivities, for a range of endpoint measures centred on the toxicity of a single retail unit after an extended period of storage. The decimal reduction time for non-proteolytic C. botulinum spore populations at the highest (hold) temperature of the primary thermal process and the highest temperature experienced during poorly controlled (consumer) storage are dominant factors determining risks. Priorities for additional information to support risk assessments have been identified.