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.     

The effect of pH,sodium chloride,sodium nitrite and storage temperature on the growth of Clostridium perfringens and faecal streptococci in laboratory media

The effects of salt concentration (1–12% w/v) in combination with unheated sodium nitrite (0–400 μg/ml) on growth of mixed strains of Clostridium perfringens and of faecal streptococci at three pH values (5.6, 6.2, 6.8) and storage temperatures ranging from 10°C to 35°C is reported. At pH 6.2, following storage at 15°C, 1% salt and 50 μg/ml nitrite inhibited growth of C. perfringens. At 20°C and pH 6.2, 200 μg/ml nitrite plus 3% salt, or 50 μg/ml plus 4% salt were required to inhibit growth. Growth of C. perfringens was prevented by levels of curing salts used commercially providing the pH was 6.2 or below. At pH 6.8 or above at least 4% salt and 50 μg/ml nitrite was required to prevent growth at 20°C. The faecal streptococci grew in medium containing 6% salt and 400 μg/ml nitrite irrespective of pH or storage temperature. In 8% salt growth was prevented by storing at or below 17.5°C or, if pH was 6.2 or lower, by adding 200 μg/ml nitrite irrespective of storage temperature. Growth of faecal streptococci was not controlled by concentrations of curing salts which would be acceptable in meat products.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Variability in growth and fermentation products of Clostridium sporogenes inoculated into canned foods and grown in laboratory media for various time intervals

Previous studies on the use of gas-liquid chromatography (GLC) in solving food spoilage problems have proven the technique to be rapid, simple and inexpensive. However, the limitation of this approach is the effect of food composition on metabolic activity as reflected in the GLC profile of the organism involved. In this study, the influence of the food composition and incubation time upon the fermentation end-product patterns of Clostridium sporogenes was investigated. The results illustrated the fact that each of these environmental variables had a profound effect on the GLC profile. When the test organism was grown in peptone yeast (PY) medium with or without 1% glucose, the characteristic profile was detected only after 24 h which corresponds to the stationary phase of growth. An initial lag (12 h) of glucose utilization was observed. Glucose supplementation of the PY medium resulted in certain metabolic activities which are explained by reference to known physiological facts. Examples include the production of higher amounts of alcohol, especially ethanol, the production of iso-amyl alcohol and the suppression of certain branched chain amino acids. A proposal is made to show how the interaction of bacteria with environmental variables could be studied systematically.     

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 cured chicken

Estimates of the growth kinetics of Clostridium perfringens from spores at temperatures applicable to the cooling of cooked cured chicken products are presented. A model for predicting relative growth of C. perfringens from spores during cooling of cured chicken is derived using a nonlinear mixed effects analysis of the data. This statistical procedure has not been used in the predictive microbiology literature that has been written for microbiologists. However, recently software systems have been including this statistical procedure. The primary growth curves, based on the stages of cell development, identify two parameters: (1) germination, outgrowth, and lag (GOL) time, or lag phase time; and (2) exponential growth rate, egr. The mixed effects model does not consider GOL and egr as constants, but as random variables that would, in all likelihood, differ for different cooling events with the same temperature. As such, it is estimated that the egr, for a given temperature, has a CV of approximately 19%. The model obtained by the mixed effects model is compared to the one obtained by the more traditional two-stage approach. The estimated parameters from the derived models are virtually the same. The model predicts, for example, a geometric mean relative growth of about 9·4 with an upper 95% confidence limit of 21·3 when cooling the product from 51°C to 12°C in 8 h, assuming log linear decline in temperature with time. C. perfringens growth from spores was not observed at a temperature of 12°C for up to 3 weeks.