Spores Of Non-proteolytic Clostridium Botulinum Research Articles

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.

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.

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.

Germination and growth from spores: variability and uncertainty in the assessment of food borne hazards

We have developed a model for the variability of spore lag times and shown that variability has an important role in the quantitative assessment of risks associated with spore forming bacteria in food. The model includes two sequential independent delay times that contribute to the lag time for a single spore. We have shown that a population of variable spores also has a variable lag time, and we have emphasised the significance of this variability in quantitative representations of population dynamics for small populations. We have made a Bayesian estimate for the extent of the variability in spore lag times and made a comparison with direct microscopic observations of individual spores of nonproteolytic Clostridium botulinum. We conclude that Bayesian inference is a practical method for quantifying variability and hence a significant element in the development of quantitative risk assessments for hazards associated with spore forming bacteria.

A predictive model of growth from spores of non-proteolytic Clostridium botulinum in the presence of different CO2concentrations as influenced by chill temperature, pH and NaCl

In the present study, the effect of modified atmospheres ranging from 0–90% CO2(balance 10% H2and N2) was evaluated on growth from spores of non-proteolyticClostridium botulinum (types B, E and F), at different pH and NaCl concentrations, over the temperature range 5–12°C. Growth tests were carried out using a model broth system, PYGS (peptone, yeast, glucose, starch). In general terms, a CO2concentration of 5% enhanced growth, 50% CO2showed a moderate inhibition and 90% had a considerable inhibitory effect. A predictive model has been developed that describes the effect of CO2, in combination with other mild preservative factors, on growth from spores of non-proteolytic C. botulinum. The model provides a good description of the experimental data.

Combinations of Heat Treatment and Sodium Chloride That Prevent Growth from Spores of Nonproteolytic Clostridium botulinum

The ability of spores of nonproteolytic Clostridium botulinum to survive heat treatment and subsequently produce turbidity at 10°C in the presence of NaCl was quantified for different incubation times. Spores of nonproteolytic C. botulinum strain Eklund 17B were heated at 75°C for up to 4 min or 90°C for up to 30 min and subsequently incubated at l0°C in PYGS broth containing 1.5%. 3.0%, or 4.0% NaCl (wt/vol) with or without 10 μg hen egg white lysozyme ml−1. Heat treatment at 90°C for 30 min or incubation at 10°C in the presence of 4.0% NaCl did not prevent growth from up to 1.4 × l05 spores. Heat treatment at 90°C for 15 min and incubation in the presence of 4.0% NaCl did prevent growth in the same conditions.

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum.

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90 degrees C for between 0 and 60 min and subsequently incubated at 5 degrees, 10 degrees or 30 degrees C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90 degrees C for 1 or more min ensured a 10(6) reduction when spores were subsequently incubated at 5 degrees C for up to 23 weeks. Heating at 90 degrees C for 60 min ensured a 10(6) reduction over 23 weeks when subsequent incubation was at 10 degrees C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10(6) when subsequent incubation was at 30 degrees C.

Vegetable juice aids the recovery of heated spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic Clostridium botulinum at 85d̀C for 2 min followed by plating on a standard laboratory medium reduced the count of viable spores by a factor of greater than 104. A similar result was obtained when the plating medium was supplemented with juice from courgette, carrot or mung bean sprout. When plating was on media supplemented with hen egg white lysozyme or juice from turnip, swede, flat bean, cabbage or potato, heating at 85d̀C for 10 min did not reduce the viable count by a factor of 104. Thus these vegetable juices increased the measured heat resistance of spores of non-proteolytic Cl. botulinum. These findings are relevant to the safety of minimally processed (e.g. sous-vide) foods.

Predictive model of the effect of temperature, pH and sodium chloride on growth from spores of non-proteolytic Clostridium botulinum

Non-proteolytic strains of Clostridium botulinum are capable of growth at chill temperatures and thus pose a potential hazard in minimally-processed chilled foods. The combined effect of pH (5.0–7.3), NaCl concentration (0.1–5.0%) and temperature (4–30 °C) on growth of non-proteolytic C. botulinum in laboratory media was studied. Growth curves at various combinations of pH, NaCl concentration and temperature were fitted by the Gompertz and Baranyi models, and parameters derived from the curve-fit were modelled. Predictions of growth from the models were compared with data in the literature and this showed them to be suitable for use with fish, meat and poultry products. This model should contribute to ensuring the safety of minimally-processed foods with respect to non-proteolytic C. botulinum.

Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature.

Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods. Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium. The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined. No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.

Effect of sporulation temperature on some properties of spores of non-proteolytic Clostridium botulinum

Effect of sporulation temperature on some properties of spores of non-proteolytic Clostridium botulinum

Effect of lysozyme concentration, heating at 90 degrees C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum.

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10(6) spores per tube. Hen egg white lysozyme (0-50 micrograms ml-1 was added, and the tubes were given a heat treatment equivalent to 19.8 min at 90 degrees C, cooled, and incubated at 8 degrees, 12 degrees, 16 degrees and 25 degrees C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6-D inactivation was therefore achieved. In tubes to which lysozyme (5-50 micrograms ml-1 had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 micrograms ml-1, growth was first observed after 68 d at 8 degrees C, 31 d at 12 degrees C, 24 d at 16 degrees C, and 9 d at 25 degrees C. Thus, in these circumstances, a heat treatment equivalent to 19.8 min at 90 degrees C was not sufficient, on its own, to give a 6-D inactivation. A combination of the heat treatment, maintenance at less than 12 degrees C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10(6).

Heat resistance and recovery of spores of non-proteolytic Clostridium botulinum in relation to refrigerated, processed foods with an extended shelf-life.

Journal of Applied BacteriologyVolume 76, Issue S23 p. 115S-128S Heat resistance and recovery of spores of non-proteolytic Clostridium botulinum in relation to refrigerated, processed foods with an extended shelf-life Barbara M. Lund, Corresponding Author Barbara M. Lund 8 The Walnuts, Branksome Road, Norwich8 The Walnuts, Branksome Road, Norwich NR4 6SR, UK.Search for more papers by this authorM.W. Peck, M.W. Peck Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich, UKSearch for more papers by this author Barbara M. Lund, Corresponding Author Barbara M. Lund 8 The Walnuts, Branksome Road, Norwich8 The Walnuts, Branksome Road, Norwich NR4 6SR, UK.Search for more papers by this authorM.W. Peck, M.W. Peck Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich, UKSearch for more papers by this author First published: June 1994 https://doi.org/10.1111/j.1365-2672.1994.tb04363.xCitations: 50AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume76, IssueS23June 1994Pages 115S-128S RelatedInformation

Heat-resistance of spores of non-proteolytic Clostridium botulinum estimated on medium containing lysozyme

Heat treatment of spores of non‐proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat‐resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat‐resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat‐inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D‐values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D85°C, 100 min; D90°C, 18.7 min; D95°C, 4.4 min; for strain Beluga, D85°C, 46 min; D90°C, 11.8 min; D95°C, 2.8 min. The z‐values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum.

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.

Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum.

Heat treatment of spores of non-proteolytic Clostridium botulinum at 85°C for 120 min followed by enumeration of survivors on a medium containing lysozyme resulted in a 4.1 and 4.8 decimal reduction in numbers of spores of strains 17B (type B) and Beluga (type E), respectively. Only a small proportion of heated spores formed colonies on medium containing lysozyme; this proportion could be increased by treatments designed to increase the permeability of heated spores. The results indicate that the germination system in spores of non-proteolytic Cl. botulinum was destroyed by heating, that lysozyme could replace this germination system, and that treatments that increased the permeability of the spore coat could increase the proportion of heated spores that germinated on medium containing lysozyme. These results are important in relation to the assessment of heat-treatments required to reduce the risk of survival and growth of non-proteolytic Clostridium botulinum in processed (pasteurized) refrigerated foods for extended storage.