Growth of Clostridium perfringens in Cooked Chili during Cooling

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1988, p. 114-118 99-224/88/5114-5$2./ Copyright C) 1988, American Society for Microbiology Vol. 54, No. 5 Growth of Clostridium perfringens in Cooked Chili during Cooling L. C. BLANKENSHIP,'* S. E. CRAVEN,' R. G. LEFFLER,' AND C. CUSTER2 Richard B. Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia 3613,1 and Food Safety and Inspection Service, U.S. Department of Agriculture, Washington, D.C. 2252 Received 4 May 1987/Accepted 1 February 1988 U.S. Department of Agriculture regulations require that brick chili be cooled from 48.9TC to 4.4AC within 2 h of cooking, but processors may not always be able to comply. Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures. Inoculated samples were heated (75 C for 2 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h. No growth was observed for C. perfringens in samples held at 26.7 C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 C and after 2 h in samples held at temperatures between 37.8 C and 48.9 C. Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions. The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase. It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C. perfringens in chili. Actual data agreed closely with predicted results. The results should be useful for evaluating the hazard potential for growth of C. perfringens in chili. Clostridium perfringens food poisoning outbreaks occur primarily as a result of consumption of foods that are improperly handled after cooking. Spores of C. perfringens may survive the cooking process used for products such as brick chili. U.S. Department of Agriculture regulations require that heat-processed meat products of this type be cooled from 48.9 to 4.4 C within 2 h (8a). Some chili producers may not be able to comply with this specification. Consequently, the question has been raised of whether this cooling period can be extended beyond 2 h without posing a potential health hazard due to growth from surviving clostridial spores. This report describes the characteristics of growth from spores of C. perfringens which were heat activated in chili and either incubated at various constant temperatures or cooled to simulate processing conditions. A model was developed for predicting growth during cooling by using isothermal data. The model was tested by using two temperature programs, both of which are exponentially decreasing functions, in which the starting temperature was 5 C and the final temperature approached C. In one, the time to reach 25 C was 4 h, and in the other it was 6 h. Five sets of isothermal bacterial growth curves were used which covered the range of interest between 5 and 25 C. The model is an empirical one and consists of fitting an equation developed by Ratkowsky et al. (5) to the isothermal growth rate data. The growth rate equation is then integrated numerically to give bacterial numbers as a function of time. In this study we also developed a computerized model for two such temperature programs, and we believe that this approach is sufficiently general to be applied to any physically realizable temperature program of practical interest. MATERIALS AND METHODS Clostridium strains and spore production. Strains of C. perfringens used in this study included NCTC 8679, NCTC * Corresponding author. 114 8238, NCTC 8239, R42, and PS44, which were supplied by S. E. Craven. Spores of C. perfringens were produced by the procedure described by Craven and Blankenship (3). After spore crops of each strain had been washed several times in sterile distilled water, the individual strains were divided into 2-ml volumes and stored at -2 C until needed. Composites of spore strains were prepared immediately prior to experiments by combining equivalent numbers of spores from suspensions of individual strains. Systems for heating and cooling inoculated chili. Portions (2 g) of commercially prepared chili were packed into sterile 5-ml disposable polyethylene centrifuge tubes. We inoculated spore suspensions into chili by injecting.2 ml of the inoculum into each tube near the bottom. These conditions were found by preliminary experimentation to allow optimum outgrowth of the spore inoculum in the chili mass while allowing accurate control of temperature. All chili samples were heated at 75 C for 2 min to activate spores. For isothermal experiments, samples were quickly chilled in an ice bath to the desired temperature before being incubated in a constant-temperature water bath. Samples for exponential cooling experiments were cooled to 5 C at room temperature (about 22 C) after the heat activation step and then placed in a refrigerated water bath initially set at 5 C. Computer-generated time-temperature tables were used to manually adjust the water bath temperature to simulate exponential cooling rates. A thermocouple placed in an uninoculated chili sample at a location equivalent to that of the spore inoculum was used to monitor the temperature decline. Comparison of the water bath temperature and the thermocouple temperature used to monitor the chili temperature during cooling indicated little or no difference. Growth measurements. At appropriate times, each 2-g chili sample was mixed with 8 ml of.1% sterile peptone water and macerated for 1 min in a Colworth 4 stomacher. Dilutions for pour plating were also made in.1% peptone. Samples containing C. perfringens were plated in tryptosesulfite-cycloserine agar (TSC) (4) containing 1.,ug of lyso- Downloaded from http://aem.asm.org/ on January 27, 219 by guest

VOL. 54, 1988 zyme per ml and incubated anaerobically at 37 C for 24 h. All experiments were carried out in duplicate. Preliminary experiments with strain NTCT 8679 indicated very similar lag phases in four commercial chili brands during a 6-h incubation at 4 C (data not shown). All the remaining experiments were conducted with a single brand of chili. Model development. Equation 1 gives the form of the temperature-time relationship in which the parameter a is the cooling-rate constant. T = Toe-a (1) To is the starting temperature and is numerically equal to 5 C. In the two temperature programs, the chili temperature reached 25 C in 4 and 6 h. This corresponds to values for the cooling rate constant of.2888/min and.1925/min, respectively. These temperature programs were chosen to simulate a commercial cooling process in which the chili was first cooled to 5 C and then placed in a C environment. Material at the center of the container would cool more slowly than that near the container boundary, but both regions would cool in an approximately exponential fashion. Bacterial growth from spores can be characterized by an initial lag phase during which spores germinate but no multiplication occurs, an exponential growth phase characterized by a growth rate constant (k), and a stationary phase in which the growth rate declines (6). This model assumes that the stationary phase is never reached. The model consists of two parts: the prediction of the lag phase duration, to', and the prediction of bacterial number as a function of time, B(t). These are treated as independent processes occurring in series. The typical knee-shaped transition region between the two is not modeled, but is accounted for qualitatively by the assumption that there is a spread in the values of t' for the individual spores. The process occurring during the lag phase consists of a complex combination of chemical and physical steps during which heat-activated spores undergo initiation and germination followed by outgrowth and, finally, division. The assumption is made that the rate for the development of events throughout this process may be characterized by a single rate-limiting step for a given temperature and that this rate is equal to lito, where to is the lag phase time for a given temperature. A plot of lito versus temperature, with the data from the five constant-temperature data sets, gives the general shape of a declining exponential, and the following equation was fitted to the data lito = a2(1 - e -ao(t - al)) (2) where ao, a1, and a2 are adjustable parameters. Since T (the temperature) is a known function of t (the time) (equation 1), Ilto can also be expressed as a function of time. Let S be the degree of completion of the lag phase process. Then dsidt = lito and fol ds = 1 = fof' lltodt (3) to is the time necessary for completing the lag phase process when the temperature is changing. A numerical integration of the right-hand side of equation 3 gives the value for to'. This completes the lag phase part of the model. In the second part of the model, it is assumed that to' has been determined from the first part. The exponential growth phase response to the condition of changing temperature is then constructed. To do this, the growth rate constant, k, C. PERFRINGENS GROWTH IN COOKED CHILI 115 must be found as a function of the temperature, and hence of the time, again through equation 1. For constant temperature the growth rate is given by the following equations: db(t)ldt = kb(t) B(t) = Boek, where B(t) is the number of bacteria as a function of time starting at to'. Bo is the number of bacteria initially present, and k is the growth rate constant. However, when k is a function of the temperature and it is recognized that new bacteria appearing at time t were incubated during a previous time interval during which the temperature was changing, the explicit time and temperature dependence of B must be expressed. This suggests that the equations take the following form: db(t)ldt = kb (t) = kboekt B(t)IBo = 1 + f kek'dt log1o [B(t)iBO1 = log1o [1 + f'k' In (1) lok' dt] (4) Here, k' = k/ln (1), which enables a decade scale to be used throughout the enusing development. Ratkowsky et al. (5) have shown that for a large number of bacterial types, the temperature dependence of the rate constant k' is given by the following equation: k' = [F(T - Tmin)(l -ec(t - Tmax))]2 (5) The values of k' derived from the five isothermal determinations are used, and a fit of equation 5 to these data gives the values of F, C, Tmin, and Tmax. This enables k' to be found as a function of temperature. The use of equation 1 then enables k' to be found as a function of time. Replacing the integration with a summation in equation 4 gives the following equation: log1o [B(t)iBO] = log1o [1 + t/at I k'ln (1) 1k ia At (6) i=o One final factor remains to be accounted for in the model development. The mean generation time for bacterial growth is the time needed to double the bacteria number. For a constant temperature, this is given by (log1o 2)ik'. It is seen that bacteria newly formed at time t were germinated during a preceding time interval roughly equal to the mean generation time for that value of k'. For the constant-temperature case this process is taken into account by using the measured value of k'. When the temperature is changing, an additional correction is needed. It was found to be conceptually simple and empirically successful to do this by replacing T in equation 5 by T + T,, where T, is a temperature lag. Thus, equation 5 becomes: k'={f(t - Tmin + T,) [1 -ec(t - Tmax + T)]12 (7) It is this value of k' which must be substituted into equation 6 to permit the calculation of log1o[b(t)ibo) as a function of time. This completes the model development. RESULTS The growth curves for the five-strain composite of C. perfringens in commercial chili at various constant temperatures between 48.9 and 21.1 C during a 6-h incubation are shown in Fig. 1. No growth was observed at 26.7 or 21.1 C. Initiation of growth was observed after 5 h of incubation at 32.2 C after 4 h of incubation at 35 C, and after 2 to 3 h of incubation at temperatures between 37.8 and 48.9 C. Similar Downloaded from http://aem.asm.org/ on January 27, 219 by guest

116 BLANKENSHIP ET AL. 1 (. 5. 4. - 3. 1 2 3 4 5 26.7C/SOF Hours FIG. 1. Isothermal growth curves of a five-strain composite of C. perfringens in chili. Points are means of duplicate experiments. results were obtained in initial experiments when a single strain (NCTC 8679) of C. perfringens was tested at the same temperatures (data not shown). No C. perfringens organisms were detected in uninoculated chili. To simulate commercial chili cooling procedures, we conducted experiments with two exponential cooling curves based on computer-generated time-temperature tables to measure growth from the five-strain composite of C. perfringens. Additionally, data from isothermal growth experiments were used to develop a mathematical equation to predict the extent of growth that might occur under various mm Ico 1 -J 4. 3.o 4 2. 1. TABLE 1. Values of lag phase time (to) and rate constant (k') for isothermal data Temp ( C) t (h) Observed k' (1/h) Predicted 32.2 3.33.8.79 35. 2.63 1. 1. 37.8 2.22 1.25 1.23 43.3 2. 1.77 1.74 48.9 2. 1.33 1.33 cooling conditions. The results presented in Fig. 2 show good agreement between the predicted counts and actual counts in chili during the two exponential cooling trials. The model. The basic data consist of discrete pairs of log1ob(t) versus time. Values of to were determined by inspection of plots of the data. The fit of equation 2 to the to-versus-time data gave values for its constants of ao =.1989, a1 = 27.92, and a2 =.5159. Values of k' were also determined from plots of the isothermal data pairs and are summarized in Table 1. The least-squares fit of equation 5 to these data gave F = 5.11, C =.6566, Tmin = 9.78, and Tmax = 5.96. The only remaining step in implementing equation 6 is the determination of the parameter T, in equation 7. T, was determined initially for the two temperature programs by manually inserting values into the computer program which performed the calculation of equation 6. The best values of T, were determined by comparing the data generated by the program with the experimental data. Inspection of these values, along with the observation that T, must approach zero as the cooling-rate constant, a, approaches zero (constant-temperature case), suggested that the value of T, varied as the product of a and the cooling rate, at. Thus, it was found that T, = p Ta2 = 45, Ta2 (8) A single empirical parameter, p = 45,, gives a successful prediction of the exponential cooling data for the given starting and asymptotic temperatures. The form of equation 8 is suggested from the following argument. A time lag is ~~ 5 6 h Eh _ 4 o 4h 3-6hE E I APPL. ENVIRON. MICROBIOL. 2 Io2 Downloaded from http://aem.asm.org/ on January 27, 219 by guest I 1 1. 2. 3. 4. 5. 6. Time (h) FIG. 2. Plot of log1o (C. perfringens count/initial count) versus time for the 4- and 6-h cooling times. Symbols: -, model prediction;, experimental data; -----, temperature changes with time. Abbreviations: Bo, Initial count; B, count after incubation.

VOL. 54, 1988 C. PERFRINGENS GROWTH IN COOKED CHILI 117 m m 2. cm 1. found that the counts increased during the 4 to 7 h that the 6 gravy temperature was in a range within which growth could occur, but the experimental design did not allow an assess- 5 ment of the individual contributions of spores or vegetative 4 cells. Shigehisa et al. (7) determined the germination and growth profiles of C. perfringens spores inoculated into 3 ground beef at 6 C and cooled to 15 C with the temperature constantly falling at a rate of 5 to 25 C/h. They found that no I growth occurred during the first 15 min regardless of the cooling rate. We observed similar results with no growth occurring within 12 min in isothermal experiments at 37.8, 43.3, and 48.9 C and even longer periods being required at lower temperatures before growth was initiated. Shigehisa et al. (7) further reported that growth was observed only at constant linear temperature decline rates of 1, 7.5, and I I I I I 5 C/h over the range of 6 to 15 C. Cooling periods were 4.5, 1. 2. 3. 4. 5. 6. 6., and 9 h, respectively. In our exponential cooling experiments in which the cooling time was 4 and 6 h for the Time ( h ) temperature decline from 5 to 25 C, a declining rate of Plot of log1o (C. perfringens count/initial count) versus bacterial multiplication was detected. FIG. 3. time for siix cooling rates as predicted by the model. Curves 1 to 6 Hence, cooling brick chili in compliance with U.S. De- to decline times of 3.25, 3.5, 4., 5., 6., and 8. h, partment of Agriculture regulations, i.e., from 48.9 to 4.4 C correspond respective ly. Abbreviations: Bo = initial count; B, count after within 2 h, would appear to provide safe conditions under incubation which the spores of C. perfringens which do germinate are unlikely to multiply. However, if the cooling period is associated with the changing temperature, which for a extended beyond 2 h, there is a probability that growth will slowly alnd monotonically changing temperature could be occur. expectedl to change linearly with the temperature cooling It is further concluded that the prediction of bacterial rate constant, a, i.e., At = pa. Any time interval will have a growth under varying temperature conditions is possible temperatiure interval associated with it: AT = Toea(r - At) - using only constant-temperature data covering the tempera- Toeat= T(eaAt - 1) and AT TaAt = pta2, which has the ture range of interest in which the temperature declines are form of equation 8. The values for to' predicted by equation relatively slow compared with the mean generation times. 2 for the two temperature profiles are 127 min for 4 h of (The maximum rate used in this experiment was 25 C in 4 h.) cooling and 121 min for 6 h of cooling. These are comparable A single adjustable parameter suffices to characterize a class with the observed values, determined by inspection of the of temperature profiles of the exponential cooling type. The plotted data, of to' 2 12 min. predictability found here is not an ab initio predictability, but The finial model predictions are shown in Fig. 2 for the 4 relies on the use of variable-temperature data to characterize and 6-h cooling data along with the datum points being the behavior of a class of phenomena. Now that the class modeled. As a check on the calculations for equation 6, behavior pattern has been established, new members of that isotherm; al conditions were simulated by entering the value class should show predictable behavior within the range of of each cf the five temperatures for which data were avail- applicability of the model development. able as the starting temperature and entering a very large value for the cooling time, giving a cooling-rate constant ACKNOWLEDGMENT close to.. The resulting data were characterized by k' We gratefully acknowledge the excellent technical assistance of values, and these are listed in Table 1 as the predicted k' George J. Magner III. values. Figure 3 shows the prediction of the model for six different values of the cooling-rate constant. Integrations were implemented as simple sums with At intervals of 1 min. Curve fits were done by using a grid search method based on the approach of Bevington (1). In the application of these results to bacterial growth in bulk material cooled by a constant ambient temperature, it is understood that cooling rates in bulk material do not follow a strictly exponential pattern (2), but it is believed that a time delay followed by an exponential temperature decline may well approximate such temperature profiles. DISCUSSION The growth of C. perfringens from vegetative cells in various beef-containing media at static and continuously rising temperatures has been reported by Willardson et al. (9); however, growth during cooling has received less attention. Tuomi et al. (8) examined the effect of refrigerator cooling of ground-beef gravy that was experimentally inoculated with C. perfringens vegetative cells and spores. They LITERATURE CITED 1. Bevington, P. R. 1969. Data reduction and error analysis for the physical sciences, p. 24-215. McGraw Hill Book Co., New York. 2. Charm, S. 1961. A method for calculating the distribution and mass average temperature in conduction-heated canned foods during water cooling. Food Technol. 15:248-253. 3. Craven, S. E., and L. C. Blankenship. 1985. Activation and injury of Clostridium perfringens spores by alcohols. Appl. Environ. Microbiol. 5:249-276. 4. Harmon, S. M. 1976. Collaborative study of an improved method for the enumeration and confirmation of Clostridium perfringens in foods. J. Assoc. Off. Anal. Chem. 59:66-612. 5. Ratkowsky, D. A., R. K. Lowry, T. A. McMeekin, A. N. Stokes, and R. E. Chandler. 1983. Model for bacterial culture growth rate throughout the enteric biokinetic temperature range. J. Bacteriol. 154:1222-1226. 6. Setlow, R. B., and E. C. Pollard. 1962. Molecular biophysics, p. 28-3, Addison-Wesley Publishing Co., Inc., Reading, Mass. 7. Shigehisa, T., T. Nakagami, and S. Taji. 1985. Influence of heating and cooling rates on spore germination and growth of Clostridium perfringens in media and in roast beef. 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118 BLANKENSHIP ET AL. Sci. 47(2):259-267. 8. Tuomi, S., M. E. Matthews, and E. H. Marth. 1974. Behavior of Clostridium perfringens in precooked chilled ground beef gravy during cooling, holding, and reheating. J. Milk Food Technol. 37:494-498. 8a.U.S. Department of Agriculture. 1973. Meat and poultry inspec- APPL. ENVIRON. MICROBIOL. tion manual, section 8.55e. Food Safety and Inspection Service, U.S. Department of agriculture, Washington, D.C. 9. Willardson, R. R., F. F. Busta, and C. E. Allen. 1979. Growth of Clostridium perfringens in three different beef media and fluid thiogylcollate medium at static and constantly rising temperatures. J. Food Prot. 42:144-148. Downloaded from http://aem.asm.org/ on January 27, 219 by guest