Growth of heat-treated enterotoxin-positive Clostridium perfringens and the implications for safe cooling rates

Clostridium perfringens 790-94 and 44071.C05 carrying a chromosomal and a plasmid cpe gene, respectively, were used to determine differences in heat resistance and growth characteristics between the genotypes. Heat inactivation experiments were conducted using an immersed coil apparatus. Spore germination, outgrowth, and lag phase, together named GOL time, as well as generation times were determined during constant temperatures in fluid thioglycollate (FTG) medium as well as in vacuum-packed, heat-treated minced turkey. GOL time and growth were also monitored during cooling scenarios from 65 to 10 degrees C for 3, 4, 5, 6, and 7 h in vacuum-packed, heat-treated minced turkey. Spores of strain 790-94 were approximately 10-fold more heat resistant at 85 degrees C than those of strain 44071.C05, and strain 790-94 also had a higher temperature growth range in FTG. The higher growth range for a chromosomal enterotoxin-producing CPE+ strain was confirmed using two other strains carrying a chromosomal (NCTC8239) and plasmid (945P) cpe gene. Moreover, strain 790-94 had shorter GOL times at 50 degrees C in turkey and approximately half the generation time compared with strain 44071.C05 at temperatures > or = 45 degrees C in both FTG and turkey. Strain 790-94 increased with 0.3, 1.0, 1.7, and 2.0 logs, respectively, during cooling from 65 to 10 degrees C in 4, 5, 6, and 7 h, which was significantly higher than for strain 44071.C05. A maximum acceptable cooling time of 5 h between 65 and 10 degrees C is suggested.     

Survival and growth of Clostridium perfringens in commercial no-nitrate-or-nitrite-added (natural and organic) frankfurters, hams, and bacon

The popularity of "preservative-free" foods among consumers has stimulated rapid growth of processed meats manufactured without sodium nitrite. The objective of this study was to quantify the potential for Clostridium perfringens growth in commercially available processed meats manufactured without the direct addition of nitrite or nitrate. Commercial brands of naturally cured, no-nitrate-or-nitrite-added frankfurters (10 samples), hams (7 samples), and bacon (9 samples) were obtained from retail stores and challenged with a three-strain inoculation (5 log CFU/g) of C. perfringens. Reduced inhibition (P < 0.05) was observed in seven brands of frankfurters, six brands of hams, and four brands of bacon when compared with each respective sodium nitrite-added control. In naturally cured and truly uncured commercial frankfurters, growth over time was approximately 4.7 log, while conventionally cured frankfurters exhibited growth at 1.7 log. Naturally cured ham and bacon products exhibited growth at 4.8 and 3.4 log, respectively, while their conventionally cured counterparts exhibited growth at 2.6 and 2.3 log, respectively. These products also demonstrated variation in growth response. The results indicate that commercially available natural/organic naturally cured meats have more potential for growth of this pathogen than do conventionally cured products. Natural and organic processed meats may require additional protective measures in order to consistently provide the level of safety from bacterial pathogens achieved by conventionally cured meat products, and which is expected by consumers.     

Survival and germination of Clostridium perfringens spores during heating and cooling of ground pork

The effect of heating rate on the heat resistance, germination, and outgrowth of Clostridium perfringens spores during cooking of cured ground pork was investigated. Inoculated cured ground pork portions were heated from 20 to 75°C at a rate of 4, 8, or 12°C/h and then held at 75°C for 48 h. No significant differences (P > 0.05) in the heat resistance of C. perfringens spores were observed in cured ground pork heated at 4, 8, or 12°C/h. At heating rates of 8 and 12°C/h, no significant differences in the germination and outgrowth of spores were observed (P > 0.05). However, when pork was heated at 4°C/h, growth of C. perfringens occurred when the temperature of the product was between 44 and 56°C. In another set of experiments, the behavior of C. perfringens spores under temperature abuse conditions was studied in cured and noncured ground pork heated at 4°C/h and then cooled from 54.4 to 7.2°C within 20 h. Temperature abuse during cooling of noncured ground pork resulted in a 2.8-log CFU/g increase in C. perfringens. In cured ground pork, C. perfringens decreased by 1.1 log CFU/g during cooling from 54.4 to 36.3°C and then increased by 0.9 log CFU/g until the product reached 7.2°C. Even when the initial level of C. perfringens spores in cured ground pork was 5 log CFU/g, the final counts after abusive cooling did not exceed 3.4 log CFU/g. These results suggest that there is no risk associated with C. perfringens in cured pork products under the tested conditions.     

Dynamic computer simulation of Clostridium perfringens growth in cooked ground beef

The objective of this study was to develop a computer simulation algorithm to dynamically estimate and predict the growth of Clostridium perfringens in cooked ground beef. The computational algorithm was based on the implicit form of the Gompertz model, the growth kinetics of C. perfringens in cooked ground beef, and the fourth-order Runge-Kutta numerical method. This algorithm was validated using a cocktail of three strains of C. perfringens spores grown under isothermal, square-waved, linear cooling, and exponential cooling temperature profiles. In general, the results of computer simulation matched closely with the experimental data with the absolute errors less than 0.5 log(10) CFU/g. This method may be a useful tool for the food industry, regulatory agencies, distributors, and retailers to predict the effect of temperature abuse on the microbial safety of C. perfringens and other foodborne pathogens in processed meat products.     

Predictive model for growth of Clostridium perfringens in cooked cured pork

Mathematical models have been developed and used for predicting growth of foodborne pathogens in various food matrices. However, these early models either used microbiological media or other model systems to develop the predictive models. Some of these models have been shown to be inaccurate for applications in meat and specific food matrices, especially under dynamic conditions, such as constantly changing temperatures that are encountered during food processing. The objective of this investigation was to develop a model for predicting growth of Clostridium perfringens from spore inocula in cured pork ham. Isothermal growth of C. perfringens at various temperatures from 10 to 48.9 degrees C were evaluated using a methodology that employed a numerical technique to solve a set of differential equations. The estimated theoretical minimum and maximum growth temperatures of C. perfringens in cooked cured pork were 13.5 and 50.6 degrees C, respectively. The kinetic and growth parameters obtained from this study can be used in evaluating growth of C. perfringens from spore populations during dynamically changing temperature conditions such as those encountered in meat processing. Further, this model can be successfully used to design microbiologically "safe" cooling regimes for cured pork hams and similar products.     

A probabilistic analysis of Clostridium perfringens growth during food service operations

The purpose of this study was threefold: first, the study was designed to illustrate the use of data and information collected in food safety surveys in a quantitative risk assessment. In this case, the focus was on the food service industry; however, similar data from other parts of the food chain could be similarly incorporated. The second objective was to quantitatively describe and better understand the role that the food service industry plays in the safety of food. The third objective was to illustrate the additional decision-making information that is available when uncertainty and variability are incorporated into the modelling of systems.     

Evaluation of a predictive model for Clostridium perfringens growth during cooling

Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production. The U.S. Department of Agriculture Food Safety and Inspection Service offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimes result in no more than a 1-log CFU/g increase of C. perfringens and no growth of Clostridium botulinum. The Juneja 1999 model for C. perfringens growth during cooling may be helpful in determining whether the C. perfringens performance standard has been achieved, but this model has not been extensively validated. The objective of this study was to validate the Juneja 1999 model under a variety of temperature situations. The Juneja 1999 model for C. perfringens growth during cooling is fail safe when low (<1 log CFU/ml) or high (>3 log CFU/ml) observed increases occur during exponential cooling. The Juneja 1999 model consistently underpredicted growth at intermediate observed increases (1 to 3 log CFU/ml). The Juneja 1999 model also underpredicted growth whenever exponential cooling took place at two different rates in the first and second portions of the cooling process. This error may be due to faster than predicted growth of C. perfringens cells during cooling or to an inaccuracy in the Juneja 1999 model.     

Clostridium perfringens in Dehydrated Soups and Sauces

SUMMARY– Fifty-five samples of nationally advertised dehydrated sauce and gravy mixes, soup mixes, spaghetti sauce mixes, and cheese sauce mixes were examined for the presence of Clostridium perfringens. The organism was found in 18.2% of the samples. Spaghetti sauce mixes had the highest incidence of C. perfringens and the soup mixes had the lowest incidence. One strain possessed heat-resistant spores that were able to withstand boiling at 97.4°C for one hour prior to isolation. The presence of preservatives in the food products did not influence the presence of C. perfringens in these food preparations. No common ingredient was detected as the source of contamination. The general presence of this organism in dehydrated soups and sauces may have epidemiological significance in C. perfringens food poisoning, especially since these products are exposed to short besting periods.     

Optimizing sporulation of Clostridium perfringens

Many sporulation media have been developed for Clostridium perfringens, but none stimulates sporulation for all strains. The aim of our experiments was to develop a sporulation method using Duncan and Strong (DS) medium, which supports sporulation of a wide variety of strains. Different inoculation levels were tested, and the effects of sporulation-promoting substances and acid shock were evaluated. Furthermore, DS medium was compared with other sporulation media. Highest spore numbers in DS medium were obtained with a 10% 24-h fluid thioglycollate broth inoculum (5.0 x 10(5)/ml). Addition of theophylline and replacement of starch by raffinose increased spore yields for some strains, but most strains were not affected (average increases in log N/ml of 0.2 and 0.3, respectively). One strain was enhanced by the addition of bile, but other strains were strongly inhibited (average decrease in log N/ml of 2.5); agar did not influence sporulation. Neither short-time acid exposure nor addition of culture supernatant fluids of well-sporulating strains resulted in higher spore numbers in DS medium. None of the tested methods enhanced sporulation in general; only strain-dependent effects were obtained. Peptone bile theophylline medium was the most promising sporulation medium tested; peptone bile theophylline starch medium yielded highest spore numbers (2.5 x 10(5)/ml), but some strains failed to sporulate. In conclusion, adding theophylline to DS medium may optimize sporulation of C. perfringens, but peptone bile theophylline medium with or without starch is most suitable.     

Feasibility of using food-grade additives to control the growth of Clostridium perfringens

Previously, it was demonstrated that the combination of sucrose laurate (SL) ethylenediaminetetraacetate (E) and butylated hydroxyl anisole (B) (SLEB) was an effective antimicrobial agent against both gram-negative (aerobes) and gram-positive (facultative anaerobes) foodborne bacteria. This investigation examines the sensitivity of Clostridium perfringens to SLEB relative to: (1) the minimum inhibitory concentration (MIC) of SLEB required to inhibit the growth of C. perfringens and (2) the antibacterial effectiveness of different combination ratios of SLEB in fluid thioglycollate medium (FTM). Results indicated that the MIC of SLEB (1:1:1, v/v/v) against C. perfringens on tryptose sulfite cycloserine (TSC) agar was > 150 ppm at 37 degrees C. However, in FTM, a SLEB (1:1:1, v/v/v) concentration of > 100 ppm inhibited C. perfringens during an incubation (anaerobic) period of 196 h at 37 degrees C. The sensitivity of C. perfringens to different combination ratios was also investigated in FTM. The results showed that, when the concentrations of SL and E were held at 75 ppm in the SLEB combination, and the concentration of B increased from 0 to 75 ppm, C. perfringens growth increased initially during the first 24 h of incubation (37 degrees C) but remained constant during the next 48 h. Similarly, when concentrations of SL and E were held constant at 150 ppm in the SLEB combination and the B ratio increased from 50 to 150 ppm in FTM, C. perfringens viability decreased in all of the treated samples during 72-h incubation at 37 degrees C. The results indicated that SLEB was an effective inhibitor of C. perfringens growth activities, and the ratios of the components of SLEB can be adjusted to meet specific preservation needs.     

Inhibitory effects of polyphosphates on Clostridium perfringens growth, sporulation and spore outgrowth

This study evaluated the effects of various polyphosphates (SPP, STPP, SAPP and TSPP) on growth, sporulation and spore germination of Clostridium perfringens, and germination and outgrowth of C. perfrinegns spores in poultry meat. We have found that the requirements of polyP (0.8–1.0%) to inhibit C. perfringens bacterial growth were higher than those reported for other bacteria. Sub-lethal concentrations of polyP significantly (p<0.01) inhibited sporulation of C. perfringens by reducing sporulating cells (heat-resistant cells) 5–6 log₁₀. While C. perfringens spores were able to germinate in the presence of 1% STPP, their outgrowth was significantly (p<0.01) inhibited. Finally, a significant (p<0.01) reduction of survival of C. perfringens was observed when meat samples contaminated with a cocktail of spores of C. perfringens isolates carrying enterotoxin gene on the chromosome were treated with 1% STPP. Collectively, this study demonstrated the inhibitory effects of polyP on growth, sporulation and spore outgrowth of C. perfringens, and suggests that polyP can be used not only as an enhancer of the functional properties of meat products, but also as a promising C. perfringens antimicrobial agent.     

Predictive model for growth of Clostridium perfringens during cooling of cooked ground chicken

Traditional methodologies for development of microbial growth models under dynamic temperature conditions do not take into account the organism's history. Such models have been shown to be inadequate in predicting growth of the organisms under dynamic conditions commonly encountered in the food industry. The objective of the current research was to develop a predictive model for Clostridium perfringens spore germination and outgrowth in cooked chicken products during cooling by incorporating a function to describe the prior history of the microbial cell in the secondary model. Incorporating an assumption that growth kinetics depends in an explicit way on the cells' history could provide accurate estimates of growth or inactivation.Cooked, ground uncured chicken was inoculated with C. perfringens spores, and from this chicken, samples were formed and vacuum packaged. For the isothermal experiments, all samples were incubated in a constant temperature water baths stabilized at selected temperatures between 10 and 51 °C and sampled periodically. The samples were cooled from 54.4 to 27 °C and subsequently from 27 to 4 °C at different time periods (cooling rates) for dynamic cooling experiments. The standard model provided predictions that varied from the observed mean log₁₀ growth values by magnitudes up to about 0.65 log₁₀. However, for a selected memory model, estimates of log₁₀ relative growth provided predictions within 0.3 log₁₀ of the mean observed log₁₀ growth values. These findings point to an improvement of predictions obtained by memory models over those obtained by the standard model. More study though is needed to validate the selected model.Industrial relevanceMention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Inhibition of Clostridium perfringens by Naphthoquinones

The minimum inhibitory concentrations (MICs) of several naphthoquinones (NQ) were determined in the presence or absence of nitrite (NO₂^?) against various strains of Clostridium perfringens. In fluid thioglycollate medium, MICs ranged from 70–100 ppm for 2-methyl-1,4-NQ (menadione), 200–280 ppm for 1,4-NQ, 180–250 ppm for 1,2-NQ, >500 ppm for several water-soluble derivatives and 100–300 ppm for NO₂^?. Using a type B strain in homogenized meat medium, MICs were 670 ppm for menadione, 620 ppm for 1,4-NQ and 770 ppm for NO₂^?. Nitrite, menadione and 1,4-NQ exhibited comparable and additive rather than synergistic inhibition. Some NQ compounds may have potential as partial nitrite substitutes subject to safety evaluation.     

Predictive model for growth of Clostridium perfringens during cooling of cooked uncured beef

This paper considers growth models including one based on Baranyi's equations for growth and the other based on the logistic function. Using a common approach for constructing dynamic models for predicting Clostridium perfringens growth in ready-to-eat uncured beef during cooling, there was no appreciable difference between the models' predictions when the population of cells was within the lag or exponential phases of growth. The developed models can be used for designing safe cooling processes; however, the discrepancies between predicted and observed growths obtained in this study, together with discrepancies reported in other papers using the same, or similar methodology as used in this paper, point to a possible inadequacy of the derived models. In particular, the appropriateness of the methodology depends on the appropriateness of using estimated growth kinetics obtained from experiments conducted in isothermal environments for determining coefficients of differential equations that are used for predicting growth in constantly changing (dynamic) environments. The coefficients are interpreted as instantaneous specific rates of change that are independent of prior history. However, there is no known scientific reason that would imply the truth of this assumption. Incorporating a different, less restrictive assumption, allowing for a dependency on the prior history of cells for these kinetic parameters, might lead to models that provide more accurate estimates of growth. For example, a cooling scenario of 54.4-27 degrees C in 1.5h, the average predicted and observed log(10) relative growths were 1.1log(10) and 0.66log(10), respectively, a difference of 0.44log(10,) whereas, when assuming a particular dependency of history, the predicted value was 0.8log(10). More research is needed to characterize the behavior of growth kinetic parameters relative to prior history in dynamic environments.     

Predictive model for growth of Clostridium perfringens during cooling of cooked ground pork

A predictive dynamic model for Clostridium perfringens spore germination and outgrowth in cooked pork products during cooling is presented. Cooked, ground pork was inoculated with C. perfringens spores and vacuum packaged. For the isothermal experiments, all samples were incubated in a water bath stabilized at selected temperatures between 10 and 51 °C and sampled periodically. For dynamic experiments, the samples were cooled from 54.4 to 27 °C and subsequently from 27 to 4 °C for different time periods, designated as x and y hours, respectively. The growth models used were based on a model developed by Baranyi and Roberts (1994), which incorporates a constant, referred to as the physiological state constant, q₀. The value of this constant captures the cells' history before the cooling begins. To estimate specific growth rates, data from isothermal experiments were used, from which a secondary model was developed, based on a particular form of Ratkowsky's 4-parameter equation. Using the data from dynamic experiments and the Ratkowsky model, an optimal value of q₀ (=0.01375) was derived minimizing the mean square error of predictions. However, using this estimate, the model had a tendency to over-predict relative growth when there was observed small amounts of relative growth, and under-predict relative growth when there was observed large relative growth. To provide more fail-safe estimates, rather than using the derived value of q₀, a value of 0.04 is recommended. The predictive model with this value of q₀ would provide more fail-safe estimates of relative growth and could aid producers and regulatory agencies with determining disposition of products that were subjected to cooling deviations.Industrial relevanceSafe time/temperature for cooling of cooked pork is very important to guard against the pathogen in cooked products. Predictive model will assist industry to determine compliance with regulatory performance standards and to ensure microbiological safety of cooked products.