INTRODUCTION

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Predictive microbiology combines mathematical modeling with experimental data on combinations of factors that influence the growth of food spoilage and/or food-borne pathogenic microorganisms. The models developed are intended to predict the fate of microorganisms in foods. Since the experimental data are usually derived from studies using laboratory media, the models must be validated with data collected under conditions under which food products are customarily stored.

Predictive microbiology models can be divided into kinetic models and probability models. With the former type, one calculates the microbiological life of food products, i.e., the period of time during which the number of microorganisms in the food is less than a specified value. With the latter type, one determines whether a microorganism can grow and identifies storage conditions with a low or nil probability of growth.

Kinetic and probability models may be closely related, because the probability of detectable growth within a specified time period depends on germination, lag, and generation times, i.e., on kinetic parameters. In some cases, a probability model may be derived from a kinetic model by some simple mathematical transformations. For example, in references 33, 35 and 41, a kinetic model was transformed into a probability model by taking the natural logarithm of both sides of the original equation and then replacing one side with the "logit" of a probability, i.e., ln [P/(1  P)], where P is the probability that growth occurs. Low levels of Listeria monocytogenes, i.e., levels not exceeding 10² to 10³ CFU per gram of food at the time of consumption, are considered by many authorities to pose a low risk for most consumers (7, 15). Consequently, some national regulatory authorities, including Germany, The Netherlands, France, and Canada, advocate or have adopted a food safety risk management strategy that involves tolerance of low levels of L. monocytogenes in foods, provided that the organism cannot grow to unacceptable levels during the shelf life of the product (13). Thus, there is a need for a methodology for evaluating rapidly the potential for growth of L. monocytogenes in particular products. Mathematical models to describe the probability of growth of L. monocytogenes in foods can fulfill that need.

Our objective in this study was to develop methods to identify combinations of environmental variables that just permit or just prevent growth. This set of combinations also defines the growth rate of an organism in multidimensional space, the so-called "hyperspace cloud" (4). We also wanted to determine the potential for L. monocytogenes to grow in cold-smoked salmon. We modified the model-fitting procedure described in reference 35 and used nonlinear logistic regression techniques to estimate as many of the cardinal parameters (e.g., T_min, a_wmin, etc.) as possible. We tested this model on experimental data from two strains and evaluated the resulting predictive equations by using previously published data from several laboratories (8, 14, 26).

	MATERIALS AND METHODS

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Culture and inoculum preparation. Two strains of L. monocytogenes, Scott A and L5, the latter a wild-type strain isolated from cold-smoked salmon, were used. We inoculated two 250-ml Erlenmeyer flasks containing 50 ml of TSB-YE (tryptone soya broth [CM 129; Oxoid] with 0.6% yeast extract [L21; Oxoid]) each with one of the above strains and incubated them with shaking (50 ± 2 rpm) for 18 h at 30°C. Fifty microliters of that culture was transferred to 50 ml of fresh TSB-YE in a 250-ml Erlenmeyer flask, and the incubation was repeated for 18 h at 30°C. Cultures were grown until the late exponential phase of growth, when the optical density (at 540 nm) of the culture was 0.8 (45).

Inoculation procedures. TSB-YE was used as the basal medium for all experiments but was modified by the addition of NaCl or lactic acid or of HCl or NaOH to adjust the pH. Under aseptic conditions, 100 µl of inoculum was added to 50 ml of culture medium and mixed well, and the pH was measured immediately. Two milliliters of each broth was pipetted into four wells of each of four 24-well plates (4 wells by 6 wells). This inoculum produced turbidity just visible to the unaided eye and was used so that growth would immediately increase the turbidity of the broth without a lag in detection time. Two wells served as negative controls (sterile TSB-YE [pH 7.2]), and another two served as positive controls (TSB-YE [pH 7.2] containing 100 µl of the inoculum), in each well plate. We used two well plates to test 10 different pH levels for each lactic acid concentration in quadruplicate. Replicates were incubated at 4, 10, and 20°C in constant-temperature rooms, at 6 and 8°C in waterbaths, and at 30°C in a laboratory incubator. We also studied water activity, pH, and lactic acid effects in duplicate cultures incubated at 20 and 30°C. Details of the experimental design for each strain are given in Table 1.

Assessment of growth. Well plate cultures were examined daily for 90 days. "Presumptive growth" was recorded if there was a visible increase in the turbidity of the broths. The day on which growth was first observed was recorded, although that information played no role in the subsequent fitting of the probability model. Growth-positive cultures were mixed, and 0.3 ml was withdrawn for pH measurement in a small capule by using a flat-tip pH probe (model 250A with calomel-sealed flat-tip probe, AEP433; Orion Research Inc., Boston, Mass.). A 0.1-ml aliquot of the culture was streaked onto TSA-YE (tryptone soya agar [CM 131; Oxoid] with 0.6% yeast extract [L21; Oxoid]) and incubated at 30°C for 24 to 48 h. Typical L. monocytogenes colonies were subcultured onto Oxford Formulation Listeria Selective Agar (Oxoid CM856 including selective supplement Oxoid SR140) and incubated at 37°C for 24 to 48 h. If we saw only one colony type on TSA-YE and if colonies typical of Listeria were seen on Oxford Formulation Listeria selective agar, then growth of L. monocytogenes was presumed confirmed. When turbidity did not increase, or if only a deposit formed in the bottom of the well by the end of the incubation period, we used a standardized ecometric technique (29, 30, 33) calibrated to viable counts to identify culture conditions that were lethal to L. monocytogenes, i.e., in which cell numbers declined during incubation. These determinations could not be made using turbidimetric methods alone.

Experimental design. We conducted three sets of experiments to test the effect of combinations of temperature, pH, and the concentration of lactic acid on the growth of L. monocytogenes. The experimental design covered more than 500 different sets of conditions for each strain tested (see Table 1). The structure of each data set was such that it varied from sparse for certain combinations of factors to more complete in other regions of "factor space." There were usually four replicates when the experiments were conducted in a water bath, but there was only a single observation for each condition when a temperature gradient incubator was used. Two forms of acidulant, HCl and lactic acid, were used in combination so that inhibition due to pH or to lactic acid could be distinguished. Filter-sterilized 5 M HCl or 4 N NaOH was used to adjust the pH of media containing various levels of lactic acid. Water activity was adjusted with NaCl. The contribution of sodium lactate to the water activity of the media was included in calculations, and the final a_w of each medium was measured using a dew point water activity meter (Aqualab CX-2; Decagon Devices, Pullman, Wash.).

Effects of temperature, pH, and lactic acid concentration on growth limits. Filter-sterilized lactic acid (Univar, analytical-grade reagent; minimum, 88% [wt/wt] Ajax Chemicals) was added to sterile over-strength TSB-YE, prepared in a 1-liter volumetric flask. The broths were made up to final volume with sterile distilled water to yield final lactic acid concentrations of 10, 20, 30, or 50 mM. TSB-YE with no lactic acid was prepared in the same manner. Each medium was adjusted to 10 different pH levels, and each pH-lactic acid combination was dispensed into 25-ml McArtney bottles.

Effects of water activity, pH, and concentration of lactic acid on growth limits. Three levels of lactic acid, 0, 20, and 50 mM, and four levels of water activity, 0.929, 0.940, 0.954, and 0.965, were selected for additional studies. We prepared a series of over-strength TSB-YE broths of different a_ws that included NaCl to the desired level. These media were autoclaved at 105°C for 30 min. We added 50 mM sterile lactic acid to the sterile a_w-adjusted media in a volumetric flask, and sterile distilled water was added to achieve the required final volume and concentration. At each water activity level, the pH was adjusted to ca. 5.4, 5.7, and 6.1, and 15 ml of the broth was then dispensed into duplicate L-shaped glass tubes (L-tubes) (diameter, 15 mm) designed for use with a temperature gradient incubator (model TN3; Advantec, Toyo Roshi International, Pleasanton, Calif.) L-tubes were incubated at 20°C overnight prior to inoculation.

	THEORY

Model derivation. Suitable kinetic models (32, 38) can be converted to a probability model. The most general form of kinetic model employed in those studies is given by equation 1 and has four variables that affect growth, namely, temperature (T [in degrees Celsius]), water activity (a_w), pH, and lactic acid concentration ([LAC] [in millimolar units]).

(1)

where µ_max is the maximum specific growth rate, c, d, and k are scale parameters, T_min is the theoretical minimum temperature for growth, T_max is the theoretical maximum temperature for growth, a_wmin is the theoretical minimum water activity for growth, a_wmax is the theoretical maximum water activity for growth, pH has its usual meaning, pH_min is the theoretical minimum pH for growth, pH_max is the theoretical maximum pH for growth, U_MIC is the minimum concentration of undissociated lactic acid which prevents growth, D_MIC is the minimum concentration of dissociated lactic acid which prevents growth, pK_a is the pH at which levels of undissociated and dissociated acid are equal (3.86 for lactic acid [6]), and e is the error term.

T  T

pH  pH_max

pK_a  pH

Interface model. Using an approach proposed previously (35), equation 1 or modifications of it were converted to an interface model for growth/no growth by taking the natural logarithm of both sides and replacing the left-hand side with the logit of the probability, P, that the organism will grow, where logit (P) is a mathematical shorthand for ln[P/(1  P)]. This operation and substitution result in the following model:

(2)

Equation 2 contains nine `linear-appearing' parameters to be estimated, namely b₀, b₁, b₂, b₃, b₄, b₅, b₆, b₇, and b₈. Had the analogy that equation 2 was obtained by log-transformation of equation 1 been strictly followed, then b₁ = b₂ = 1, and b₃ = b₄ = b₅ = b₆ = b₇ = b₈ = 0.5. However, the analogy is incomplete, and these parameters are treated as free parameters and are estimated with the same maximum likelihood or weighted least-squares procedure that simultaneously produces estimates of T_min, d, T_max, a_wmin, k, a_wmax, pH_min, pH_max, U_MIC, and D_MIC. In this respect, the growth/no growth model takes its form from that of equation 1, even though it is not a literal translation of it. Forcing b₁ = b₂ = 1.0 and b₃ = b₄ = b₅ = b₆ = b₇ = b₈ = 0.5, so that b₀ is the only b parameter estimated, resulted in a much poorer fit (results not shown).

Statistical software. Equation 2 is an example of a nonlinear logistic regression model. It is nonlinear because the expression on the right-hand side contains both linear and nonlinear parameters. It is a logistic regression because the right-hand side of the expression is linked to the response variable P, the probability of growth, by a logit link function. Linear logistic regression is a well-established statistical procedure which can be fitted by a wide variety of available statistical packages, utilizing built-in procedures, e.g., PROC LOGISTIC and PROC GENMOD of SAS (SAS/STAT user's guide, version 6, 4th ed., vol. 2; SAS/STAT software: changes and enhancements, through release 6.11; SAS Institute Inc., Cary, N.C.) or directives, such as in GENSTAT (Genstat S, release 3, manual; Genstat, Downer's Grove, Ill.). Nonlinear logistic regression, however, requires more than a standard procedure or directive. Equation 2 was fitted by adapting code in an example in PROC NLIN of SAS (SAS/STAT user's guide, version 6, p. 1168); see the Appendix for a partial listing of the modified code. The critical change is to use a weight function, which for a binomial probability distribution is nP(1  P), where P is the probability of growth and n is the number of replicates. The mean value of the nonlinear predictor, the expression involving the growth regulating factors, is linked to the probability P by the logit link function. Convergence to an optimum solution was improved by use of a "loss function" that is described in the same example, although its use is not obligatory.

3.35  pH

pH  3.86

3.35  pH

pH  3.86

3.35  pH

pH  9.5

Assessment of model performance. The receiver operating characteristic (ROC) curve (e.g., see reference 25), the Hosmer-Lemeshow goodness-of-fit statistic (18), and the maximum rescaled R-square statistic (31) were used as measures of goodness of fit of the model developed. The area under the ROC curve, c, is a measure of discrimination, obtained from a plot of sensitivity, i.e., the proportion of observed events that were correctly predicted to be events, against the complement of specificity, i.e., the proportion of nonevents that were correctly predicted to be nonevents. The closer the value of c is to 1, the greater is the discrimination. In epidemiological studies, a c value of >0.7 is considered acceptable discrimination, a c value of >0.8 as excellent discrimination, and a c value of >0.9 as outstanding discrimination (25); however, in epidemiology, usually not all of the factors that influence the response variable are known. For our model, a high degree of discrimination is expected, since the identity and approximate range of values of the important factors that prevent the growth of the organism in the experimental system are well known. The Hosmer-Lemeshow goodness-of-fit statistic, which involves grouping objects into a contingency table and calculating a Pearson chi-square statistic, was proposed (18) as a means of estimating goodness of fit when there is no replication or insufficient replication in any of the subpopulations. Small values of the statistic (large P values) indicate a good fit of the model to the data.

	RESULTS

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Since we did not have sufficient data to fit all the terms in equation 2, we used a simpler model instead. For example, we collected no data for an a_w of >0.997, precluding estimation of a_wmax. Also, the term representing the effect of dissociated lactic acid (i.e., the one with the parameter D_MIC) was not needed to model the present data sets. The parameters T_max and pH_max of equation 2 had to be fixed to obtain convergence, because insufficient data were available in the high-temperature and high-pH regions. We set these values to T_max = 48.0°C and pH_max = 9.5 based on work summarized in references 20 and 38. The coefficient of the high-temperature term, d, was fixed at 0.536, the converged value it attained with the kinetic model. This led to the following simplified model.

(3)

which was tested on the data from both strains.

Using PROC LOGISTIC of SAS (SAS/STAT user's guide, version 6), we examined the effect of the six model terms in equation 3, along with terms for the square of ln(T  T_min), the square of ln(a_w  a_wmin), the square of ln(1  10^pH_min  pH), the square of ln(1  {[LAC]/[U_MIC (1 + 10^pH  pK_a)]}), and the cross-product term ln(T  T_min) × ln(1  10^pH_min  pH). The results from the two strains were the same, with the terms ln²(T  T_min) and ln²(1  10^pH_min  pH) explaining a statistically significant proportion of the total variation in logit (P), whereas the other three additional terms and the superoptimal pH term ln(1  10^pH  9.5) were not significant. This resulted in the following final model, which was used for both data sets:

(4)

Parameter estimates and their standard errors, obtained with nonlinear logistic regression using PROC NLIN of SAS (SAS/STAT user's guide, version 6) are given in Table 2 for the data from the two strains.

	MODEL PERFORMANCE

Goodness of fit. We compared the modeled growth/no growth interface to the data upon which the model was based (Fig. 1). Since four factors affect growth, it is difficult to display the model in all dimensions, but Fig. 1 is representative of the performance of the models for both strains for all pairs of variables. Objective measures of how well the model describes the data set are summarized in Table 3.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Selected growth/no growth interfaces (P = 0.5) predicted by equation 4 for L. monocytogenes, compared to the data used to generate the model. (a) Temperature/pH interface predicted by equation 4 fitted to data for L. monocytogenes Scott A compared to observed growth responses of that strain in TSB-YE with added salt (aw = 0.993 ± 0.001) and without added lactate. Growth () or no growth () within 90 days is shown. (b) pH/water activity interface predicted by equation 4 fitted to data for L. monocytogenes Scott A compared to observed growth responses of that strain in TSB-YE in the presence of 50 mM lactate at 20°C. Growth () or no growth () within 90 days from probabilistic and kinetic experiments, respectively, is shown.

Comparison to independent data. We also compared the predictions of the model to other data collected under laboratory conditions (Fig. 2 and 3) and under diverse conditions reported in the literature (data not shown). We collated ~500 data for growth in laboratory broth and ~500 data for growth in foods derived from 28 and 60 sources, respectively. Unlike the results shown in Fig. 1 to 3, a comparison to all literature data revealed some cases in which the model predicts growth is not possible where growth has been reported. Of particular note were poor predictions at high (i.e., >35°C) and low (<3°C) temperatures, discussed below.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Evaluation of the probability models for strains Scott A and L5 fitted to equation 4. The model predictions were compared to the data of George et al. (14) for the effect of temperature and pH on the potential for growth of L. monocytogenes NCTC 10357 (a and c) and Scott A (b and d) in TSB plus 1% glucose plus 0.3% yeast extract (aw, ~0.995). , growth observed; , growth not observed. In each panel, the growth/no growth interfaces predicted at P values of 0.1 (lower curve), 0.5 (middle curve), and 0.9 (upper curve) are shown. (a) George et al. data for strain NCTC 10357 compared to data from model for Scott A. (b) George et al. data for strain Scott A compared to data from model for Scott A. (c) George et al. data for strain NCTC 10357 compared to data from model for strain L5. (d) George et al. data for strain Scott A compared to data from model for strain L5.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Evaluation of the probability models for strains Scott A and L5 fitted to equation 4. The model predictions were compared to the data of McClure et al. (26) for the effect of water activity and pH on the potential for growth of L. monocytogenes NCTC 9863 in TSB at 25°C at three levels of probability of growth: P = 0.1 (lower curve), P = 0.5 (middle curve), and P = 0.9 (upper curve). (a) (Upper) equation 4 fitted to L. monocytogenes Scott A data; (b) (Lower) equation 4 fitted to L. monocytogenes L5 data. Conditions under which growth was reported are indicated by , those under which growth was not observed are indicated by , and those under which growth was observed in some, but not all, trials are indicated by . The abruptness of the predicted transition from a high (P = 0.9) to a low (P = 0.1) probability of growth is illustrated by the closeness of the three predicted boundaries. In both panels, three observations lie within the 10 to 90% probability-of-growth limits.

	DISCUSSION

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Probability or growth/no growth interface models are of particular interest in managing the safety of foods which occasionally may be contaminated with pathogens and for which subsequent growth of the pathogen would increase the risk of food-borne illness. Definition of all combinations of environmental factors that prevent growth of L. monocytogenes in foods will, in effect, quantify the Hurdle Concept (23, 24). The data underlying the growth/no growth models presented here are based on an observation period of 90 days to ensure that if growth were possible it would have been detected.

Model stability and convergence. There was a strong similarity between the estimates of the cardinal model parameters T_min, a_wmin, pH_min, and U_MIC for the two strains (see Table 2). Although it is tempting to interpret these parameters as, respectively, the "true" minimum levels of temperature, pH, water activity (controlled by NaCl), and undissociated lactic acid that permit growth under otherwise optimal conditions, they should be seen as notional. The estimate of one of these parameters, pH_min, converged to 3.35, the lower bound that was specified for that parameter (using the "bounds" directive of SAS PROC NLIN). Relaxing the boundary condition and allowing pH_min to take on lower values resulted in the estimate converging to the new lower bound. In addition, allowing parameter estimates to deviate too far from realistic values caused other parameters to hit bounds. This behavior reflects the fact that nonlinear logistic regression, like its counterpart nonlinear regression with normally distributed error, does not necessarily guarantee convergence to a global optimum. Even when modeling with normally distributed error, a subject for which there is an immense literature (see, for example, references 34 and 42), the behavior of the parameter estimators can be very erratic, with the estimators exhibiting considerable bias and having a highly non-normal distribution. Nonlinear logistic regression is a new field with few published applications, and problems in estimation and interpretation of results should be expected.

Transition between growth and no growth. The transition from "likely to grow" conditions (P = 0.9, or 90% likelihood of growth) to "unlikely to grow" conditions (P = 0.1, or 10% likelihood of growth), as predicted from the fitted model, was abrupt as can be seen graphically for combinations of pH and temperatures (Fig. 1a) and pH and water activity (Fig. 1b). The abruptness of the transition between growth or no-growth conditions influenced by pH can be as little as 0.1 to 0.2, which is close to the limit of reproducibility for pH measurements. For temperature and water activity, the transition is much less abrupt, occurring over increments of temperature and water activity that exceed that of measurement or experimental error. Most of the raw data were of the form of all n replicates being observed either to grow or not to grow. For the Scott A strain (521 factor combinations), only four of these combinations gave a response different from "all grew" or "none grew," while for the L5 strain (541 data points), only seven of these outcomes were not in the "all" or "none" categories. Thus, the experimental data showed an abrupt transition between growth and no growth that is not closely reflected by the results of the mathematical modeling. This abruptness does not indicate inadequate modeling but rather a microbiological reality in which small changes in environmental factors within an experiment may have a strong influence on the position of the interface. These differences are reflected in the predictions of the model at different levels of P.

Validation using other published data. Both models perform well when their data are compared to the data of George et al. (14), which cover a wide range of pH and temperature (up to 30°C; see Fig. 2) at near-optimal water activity, and of McClure et al. (26), which cover a wide range of pH and water activity (see Fig. 3) at 25°C, which is near optimal for the tolerance of L. monocytogenes to environmental stresses (see, e.g., reference 12). In these comparisons, sufficient details were known about the experimental conditions to use the model for prediction. In contrast, the comparison of model predictions to a collation of growth data from the literature revealed poorer concordance between predictions and observations, with the model generally predicting the growth/no growth interface to lie beyond the region where growth has been reported. This was expected because a worst-case strategy was adopted. For example, inhibitory factors other than those included in the model, such as other acidulants or humectants, preservatives, or other microorganisms, were present in many of the foods tested but could not be included in the model predictions. This led to conservative model predictions. The limitations of using literature data to evaluate model performance have been discussed previously (37, 44).

Cold-smoked salmon. In cold-smoked salmon, water activity, pH, and lactic acid present in the muscle tissue interact to retard microbial growth, while low-temperature storage further extends the shelf life of the product. Typical levels are water activity of 0.96 to 0.98 (with NaCl as a humectant), pH 6, and lactic acid levels of 80 to 100 mM (9), with the recommended storage temperature being typically 5°C. Equation 4 enables prediction of the growth potential of L. monocytogenes under these conditions and, for the most stringent conditions of the ranges indicated above, predicts a probability of growth of 0.58, whereas for the least stringent of that range, P equals 0.69. Results (10) demonstrate that when L. monocytogenes is inoculated onto smoked salmon, growth is possible, but in naturally contaminated salmon, growth was more inhibited, suggesting that other factors, such as smoke components, microbial injury, or the presence of other microorganisms, are important inhibitors of the growth potential of L. monocytogenes.

Conclusions. The growth or no-growth boundary was previously successfully defined and modeled using only kinetic data (35). In the present study, the good fit to the kinetic data by the probability model is evident (Fig. 1). This may represent an integration of the two extremes, kinetic and probabilistic aspects, of predictive microbiology.