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Analysis and Validation of a Predictive Model for Growth and Death of Aeromonas hydrophila under Modified Atmospheres at Refrigeration

Temperatures
Carmen Pin, Raquel Velasco de Diego, Susan George, Gonzalo D. Garca de Fernando and Jzsef Baranyi Appl. Environ. Microbiol. 2004, 70(7):3925. DOI: 10.1128/AEM.70.7.3925-3932.2004.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2004, p. 39253932 0099-2240/04/$08.000 DOI: 10.1128/AEM.70.7.39253932.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved.

Vol. 70, No. 7

Analysis and Validation of a Predictive Model for Growth and Death of Aeromonas hydrophila under Modied Atmospheres at Refrigeration Temperatures
Carmen Pin,1* Raquel Velasco de Diego,2 Susan George,1 Gonzalo D. Garca de Fernando,2 and Jo zsef Baranyi1
Institute of Food Research, Norwich NR4 7UA, United Kingdom,1 and Departamento de Higiene y Tecnologa de los Alimentos (Nutricio n y Bromatologa III), Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid 28040, Spain2
Received 29 October 2003/Accepted 1 April 2004

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Specic growth and death rates of Aeromonas hydrophila were measured in laboratory media under various combinations of temperature, pH, and percent CO2 and O2 in the atmosphere. Predictive models were developed from the data and validated by means of observations obtained from (i) seafood experiments set up for this purpose and (ii) the ComBase database (http://www.combase.cc; http://wyndmoor.arserrc.gov/combase/). Two main reasons were identied for the differences between the predicted and observed growth in food: they were the variability of the growth rates in food and the bias of the model predictions when applied to food environments. A statistical method is presented to quantitatively analyze these differences. The method was also used to extend the interpolation region of the model. In this extension, the concept of generalized Z values (C. Pin, G. Garca de Fernando, J. A. Ordo n ez, and J. Baranyi, Food Microbiol. 18:539-545, 2001) played an important role. The extension depended partly on the density of the model-generating observations and partly on the accuracy of extrapolated predictions close to the boundary of the interpolation region. The boundary of the growth region of the organism was also estimated by means of experimental results for growth and death rates.

Most predictive models in food microbiology focus on the specic growth and/or death rate (or the doubling time [D value]) of a microorganism as a function of the main environmental factors, such as temperature, pH, and others. These models are commonly based on observations made in a welldened and controlled laboratory environment, using microbiological media. It is also vital to validate the predictions in food environments, which can be highly complex and sometimes difcult to characterize. The overall error of a model is dened by means of the mean square error (MSE) between predictions and observations made in food (19). If extrapolations are omitted from the predictions, as they should be, then the overall error refers only to the interpolation region. Sometimes, depending on the experimental design and available data, it is difcult to determine the interpolation region of a multivariate empirical model based purely on observations. Baranyi et al. (3) dened it as a minimum convex polyhedron (MCP), or convex hull, in the space of environmental factors. As Fig. 1 shows, the MCP encompasses those combinations of the environmental conditions for which observations were made to generate the model. Its vertices can be calculated as described previously (3). Model predictions outside the MCP are extrapolations. Often, conditions observed in food fall outside of the interpolation region but are close enough to it that they can be

* Corresponding author. Mailing address: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom. Phone: 44 1603 255021. Fax: 44 1603 255288. E-mail: carmen.pin @bbsrc.ac.uk. 3925

useful for model validation. These observations can also help to extend the interpolation region of the model. In this paper, we report new experimental data about the growth and death rates of Aeromonas hydrophila which vary with temperature, pH, and percent CO2 and O2 in the atmosphere. Both death and growth data were used to estimate the growth-no growth boundary of the organism. The growth data were used to generate a predictive growth model, which was then extensively validated by comparing its predictions with various observations in food. Some observations were outside of but close enough to the interpolation region of the growth model to be useful for the validation procedure. We developed an algorithm to extend the interpolation region of the model in order to utilize those originally extrapolated values. The line of thought leading to the method can be summarized as follows. Predictive models are usually based on observations of the response parameter (in this case, the logarithm of the specic growth rate) in broth. Standard tting methods assume that the bias is 0 and that the variance is constant throughout the interpolation region when predictions and observations are compared in broth. A partition of the MSE between predictions and observations in food is illustrated in Fig. 2. Such partitioning is commonly used in statistics to estimate bias and variance. Here we use this technique to analyze the error between broth-generated model predictions and food observations. The bias is due to the fact that the data for the model were generated in laboratory media, generally producing higher growth rates than in food. The other component of the error expresses the variability of the predicted parameter in food. It is due to factors that are not taken into account in the

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PIN ET AL.

APPL. ENVIRON. MICROBIOL.

of vacuum-packaged products stored between 2 and 10C, such as smoked cod (4), cooked craysh (12), beef (9), roast beef (11), and pork (24), as well as for modied atmospherepackaged foods (5, 13, 14). Some results were occasionally contradictory (24). Apart from extending the interpolation region of a predictive model, another aim of the present work was to study the behavior of this organism in culture medium under modied atmospheres by means of mathematical models and to test how these results can be applied to modied atmosphere-packaged meat and sh.
MATERIALS AND METHODS Bacterial strain. A. hydrophila CECT 398 (Spanish Type Culture Collection) was maintained at 20C. Immediately before the experiments, it was subcultured three times consecutively in tryptic soy broth (CM129; Oxoid) incubated at 25C for 24 h. Broth experiments. Broth experiments were performed as previously described (16). Bottles containing 200 ml of tryptic soy broth, with the pH adjusted to the target value and with different atmospheric compositions, were prepared and stored at the required experimental temperature. Each bottle was inoculated with 1 ml of the appropriate dilution of the bacterial culture to give a nal concentration of ca. 103 CFU/ml. At each sampling time, bacterial counts were estimated by plating samples onto tryptic soy agar (CM131; Oxoid). In this way, bacterial kinetic curves were generated for 110 combinations of environmental conditions (Table 1). The conditions were intended to be uniformly distributed in the environmental region between 1.5 and 11C and pHs 5.2 and 7.2 and in atmospheres containing 0 to 80% CO2 combined with 0 to 80% O2, with balanced N2. Seafood experiments. Seafood was purchased from a local shmonger in Madrid. Striped tunny (Sarda sarda), hake (Merluccius merluccius), and salmon (Salmo salar) were lleted under aseptic conditions. Whole Kuruma prawns (Penaeus japonicus) and sole (Solea solea) and the llets were inoculated with A. hydrophila by immersion in a bacterial suspension of ca. 104 CFU/ml. For each storage condition, 12 samples of each type of seafood (whole sh or llet) were individually packaged under modied atmospheres as previously described (16). Table 2 shows the pH values of the seafood samples and the conditions in which they were stored. The storage temperatures and atmospheric compositions were chosen according to the most frequently used commercial conditions to store that type of seafood. At suitable time intervals, one sample was removed, homogenized, diluted, and plated onto both tryptic soy agar, for total counts, and Aeromonas medium (RYAN agar, Oxoid CM 833, SR136E), for A. hydrophila counts. Predictive modeling of specic growth and death rates. The bacterial growth and death curves, generated either with broth or seafood, were tted by the model of Baranyi and Roberts (2). This estimated the main parameter, the maximum specic growth or death rate (measured as the maximum slope of the curve of the natural logarithms of cell concentration versus time), for combinations of environmental factors. The natural logarithms of the maximum specic growth and death rates obtained [ln(g) (growth) and ln(d) (death)] were then modeled separately by two multivariate quadratic polynomials of temperature, pH, and percent CO2 and O2 in the atmosphere. A stepwise procedure (22) was used to remove those coefcients that did not contribute signicantly to the model from both polynomials (P 0.10). The effects of the environmental variables on the growth and death rates were quantied by the averages of the generalized Z values, calculated by a Monte Carlo simulation for both models (17). The most important feature of the generalized Z value can be summarized as follows: if xi and Zi denote the ith environmental variable and its generalized Z value, respectively, then the effect of one unit change in the xi/Zi normalized variable on the modeled parameter is about the same, regardless of which environmental variable was considered. Analysis of error of predictions. In what follows, bold, lowercase letters will denote vectors that are commonly used in mathematical texts. Let x (x1. . .xk) denote the vector of the studied environmental factors (in this paper, these are temperature, pH, CO2, and O2; thus, k 4). Let fx be the natural logarithm of the maximum specic rate observed at x (so fx is a random variable and Efx is its expected value). When tting the secondary model, Efx is described by the quadratic polynomial model g(x): g(x) Efx. The core assumption of the least squares method when tting broth data is that the g(x) fx error has a zero mean and a constant variance, independent of

FIG. 1. The interpolation region of the model is described by the MCP, which encompasses all of the observations used to t the model. Those environmental conditions outside the MCP are extrapolations.

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model, such as food structure or microbial interactions. Assuming that the bias and variance in food are constant, they can be estimated inside the interpolation region and then extrapolated to those regions for which only food data exist. In another words, we determine how far the model can be extended so that the constant bias and variance, estimated inside the interpolation region, still hold. The extended region was constructed from data close to the boundary of the original MCP. The extension depends partly on the density of model-generating data and partly on the accuracy of extrapolated predictions. To do this objectively, we dened a distance concept in the space of the environmental variables in such a way that one unit change had a similar effect on the modeled response, whichever variable had changed. This is, in a sense, equivalent to the normalization of the variables. In our algorithm, we rescaled the environmental variables according to this technique (C. Pin and J. Baranyi, Abstr. 4th Int. Conf. Predictive Modelling in Foods, p. 147, 2003). Some strains of Aeromonas spp., especially those of A. hydrophila, are enteropathogens with virulence properties, such as the ability to produce enterotoxins, cytotoxins, and hemolysins and/or the ability to invade epithelial cells (15, 18). Infection may produce localized illnesses, mainly in the gastrointestinal tract, and exceptionally may affect systemic processes and require hospitalization. The growth of A. hydrophila has been reported for a variety

FIG. 2. Analysis of the error. The MSE between food observations and predictions can be broken down as the sum of two components, Bias2(g,ffood) Var(ffood), where Bias(g,ffood) denotes the bias of the g model when applied to food and Var(ffood) denotes the variance of the observations in food.

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AEROMONAS HYDROPHILA UNDER MODIFIED ATMOSPHERES TABLE 1. Death and growth rates of A. hydrophila under modied atmospheres in tryptic soy broth

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Temp (C)

pH

CO2 (%)

O2 (%)

N2 (%)

SE

Temp (C)

pH

CO2 (%)

O2 (%)

N2 (%)

SE

1.5 1.5 1.5 1.5 1.5 1.5 1.5 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 1.5 1.5 1.5 1.5 4.0 4.0 4.0 4.0 4.0 4.0 4.0
a

6.64 7.20 7.20 7.20 7.20 7.20 7.20 7.20 5.33 5.33 5.82 5.82 5.82 5.82 5.82 5.82 5.82 5.82 6.06 6.06 6.06 6.06 6.06 6.06 6.06 6.30 6.30 6.30 5.05 5.11 5.11 5.12 5.12 5.52 5.55 5.55 5.55 5.57 5.61 6.08 6.08 6.13 6.13 6.13 6.61 6.61 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20

19.0 0.00 0.00 25.0 38.0 55.0 80.0 0.00 0.00 0.00 0.00 0.00 0.00 9.50 10.5 48.0 62.0 81.5 0.00 20.5 24.0 28.0 44.0 49.0 72.0 0.00 10.5 28.0 0.00 42.5 82.0 19.0 57.0 0.00 19.0 42.5 82.0 0.00 57.0 0.00 0.00 42.5 57.0 82.0 0.00 0.00 0.00 19.0 0.00 0.00 0.00 19.0 23.0 34.0 45.0

0.00 37.0 18.0 40.0 23.0 0.00 0.00 65.0 21.0 0.00 37.5 76.5 21.0 36.0 64.0 39.0 0.00 0.00 58.0 21.5 0.00 56.4 0.00 11.0 20.0 58.0 64.0 56.4 0.00 0.00 0.00 0.00 0.00 21.0 0.00 0.00 0.00 0.00 0.00 21.0 21.0 0.00 0.00 0.00 0.00 21.0 0.00 0.00 18.0 38.0 0.00 0.00 60.0 34.0 18.0

81.0 63.0 82.0 35.0 39.0 45.0 20.0 35.0 79.0 100 62.5 23.5 79.0 54.5 25.5 13.0 38.0 18.5 42.0 58.0 76.0 15.6 56.0 40.0 8.00 42.0 25.5 15.6 100 57.5 18.0 81.0 43.0 79.0 81.0 57.5 18.0 100 43.0 79.0 79.0 57.5 43.0 18.0 100 79.0 100 81.0 82.0 62.0 100 81.0 17.0 32.0 37.0

0.00856 0.0199 0.00426 0.0220 0.0135 0.00626 0.00817 0.00196 0.0763 0.0406 0.0256 0.0170 0.000809 0.0139 0.0275 0.0240 0.00811 0.0133 0.0111 0.00409 0.00277 0.0291 0.00439 0.0228 0.0208 0.000879 0.00578 0.0222 0.0232 0.122 0.0964 0.506 0.0144 0.0295 0.0551 0.0405 0.0325 0.0143 0.0223 0.00904 0.00712 0.0168 0.00394 0.0133 0.0183 0.0206 0.0171 0.0272 0.0486 0.0497 0.0958 0.0907 0.0548 0.0311 0.0322

0.00141 0.00449 0.000576 0.00269 0.00172 0.00108 0.00111 0.00035 0.00907 0.0216 0.00752 0.00418 0.000232 0.00149 0.00831 0.00736 0.000917 0.00265 0.00151 0.000923 0.000288 0.00956 0.000553 0.0108 0.00321 0.000311 0.000744 0.00577 0.00353 0.0217 0.0164 3.28E-12 0.00294 0.00525 0.00988 0.00937 0.00322 0.00298 0.00339 0.00107 0.000985 0.00529 0.000366 0.000954 0.00229 0.00129 0.00124 0.0306 0.00539 0.00479 0.0158 0.0143 0.0105 0.00937 0.00385

4.0 4.0 4.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11

7.20 7.20 7.20 7.20 6.18 6.30 6.30 6.30 6.30 6.30 6.30 6.30 6.30 6.61 6.61 6.61 6.61 6.61 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 6.13 6.13 6.34 6.34 6.34 6.34 6.34 6.34 6.34 6.34 6.34 6.34 6.34 6.34 6.64 6.64 6.64 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20

57.0 60.0 71.0 83.0 0.00 0.00 0.00 0.00 9.50 20.5 48.0 49.0 72.0 0.00 24.0 44.0 62.0 81.5 0.00 0.00 0.00 20.0 21.0 23.0 43.0 46.0 58.0 80.0 0.00 19.0 0.00 0.00 0.00 0.00 17.0 17.0 26.0 38.0 51.5 57.0 57.0 64.0 19.0 42.5 82.0 0.00 0.00 0.00 0.00 21.0 25.0 28.0 46.0 56.0 80.0

0.00 25.0 10.0 0.00 0.00 0.00 37.5 76.5 36.0 21.5 39.0 11.0 20.0 21.0 0.00 0.00 0.00 0.00 18.0 38.0 65.0 0.00 21.0 60.0 0.00 39.0 20.0 0.00 0.00 0.00 0.00 21.0 37.0 51.6 17.2 45.0 47.0 37.7 30.5 0.00 13.0 15.0 0.00 0.00 0.00 0.00 18.0 38.0 65.0 21.0 40.0 0.00 0.00 20.0 0.00

43.0 15.0 19.0 17.0 100 100 62.5 23.5 54.5 58.0 13.0 40.0 8.00 79.0 76.0 56.0 38.0 18.5 82.0 62.0 35.0 80.0 58.0 17.0 57.0 15.0 22.0 20.0 100 81.0 100 79.0 63.0 48.4 65.8 38.0 27.0 24.3 18.0 43.0 30.0 21.0 81.0 57.5 18.0 100 82.0 62.0 35.0 58.0 35.0 72.0 54.0 24.0 20.0

0.0405 0.0343 0.0525 0.0449 0.0351 0.0276 0.0539 0.0289 0.0160 0.0140 0.0385 0.0218 0.0373 0.0161 0.0530 0.0442 0.0139 0.0475 0.371 0.111 0.0751 0.380 0.239 0.0486 0.173 0.0746 0.0541 0.0688 0.0412 0.0163 0.113 0.0971 0.0875 0.0445 0.0669 0.0588 0.0342 0.0310 0.0342 0.0518 0.0416 0.0397 0.0522 0.0331 0.0615 0.385 0.258 0.366 0.309 0.474 0.106 0.288 0.267 0.121 0.149

0.00345 0.00124 0.0113 0.00322 0.00167 0.00249 0.00508 0.00561 0.00239 0.00141 0.0101 0.00173 0.00465 0.000959 0.00429 0.0259 0.00238 0.00432 0.167 0.0119 0.0121 0.17 0.0123 0.00309 0.029 0.00599 0.00359 0.00484 0.00423 0.00121 0.0123 0.0492 0.0113 0.0153 0.0054 0.0134 0.00538 0.00402 0.0048 0.0106 0.00313 0.00445 0.00461 0.00295 0.00665 0.0886 0.0262 0.0375 0.0119 0.0295 0.00463 0.0447 0.0352 0.0111 0.0157

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Maximum specic rate.

x (i.e., g is unbiased and minimally distanced from the data used to create it) (6). Therefore, the collected observations of the natural logarithms of the specic rate can be considered randomly in the environmental space and we can speak simply about the mean square error of observed f values with respect to the g predictions, as follows: MSE(g,f) E(f g)2. For this model, f is a random variable but g is not. If f is restricted to observations made in food, then MSE(g,ffood) E(ffood g)2 is an indicator of the accuracy of the model applied to food. For example, the accuracy factor of the model g as introduced by Ross (21) is approximately Ag

exp[MSE(g,ffood)] (inasmuch as we accept that the root MSE and the arithmetical average are close to each other). The percent discrepancy of Baranyi et al. (1) is %D {exp[MSE(g,ffood)] 1} 100. Extending the interpolation region. The model and its interpolation region are based on broth data. Inside the region, the predictions generally overestimate the observations made in food. We wished to consider as many food observations from outside of the interpolation region as possible to extend the interpolation (applicability) region of the model. Rearrange the above expression as follows:

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PIN ET AL. TABLE 2. Growth and death rates of A. hydrophila in seafoodd

APPL. ENVIRON. MICROBIOL.

Type of seafood

Temp (C)

pH

CO2 (%)

O2 (%)

A. hydrophila observed SE

Total ora observed SE

Probability of growth

Predicted

Modela

Distance to centroidb

Final decisionc

Hake Kuruma prawns Sole Striped tunny Salmon


a b c

5 5 5 5 5 5 5 5 5 5

6.8 6.8 7.2 7.2 6.3 6.3 6 6 6 6

38 0 80 0 0 38 38 45 0 59

0 0 0 18 18 0 0 34 18 0

0.0622 0.0817 0.0525 0.0380 0.000725 0.00762 0.0221 0.0122 0.0526 0.0167

0.0142 0.0135 0.0119 0.00587 0.00152 0.00104 0.00168 0.00352 0.00633 0.0111

0.0502 0.107 0.0231 0.0508 0.00369 0.0345 0.0285 0.0457 0.0481 0.0322

0.0159 0.0130 0.00342 0.00811 0.000848 0.0218 0.00198 0.00499 0.00718 0.00811

0.783 0.849 0.907 0.953 0.418 0.360 0.156 0.105 0.191 0.126

0.0499 0.0629 0.0558 0.0784 0.0349 0.0315 0.0239 0.0181 0.0266 0.0210

I I I I I O O O O O

1.2 2.6 2.6 2.6 2.7

U U U U D D U U U U

Environmental condition inside (I) or outside (O) the interpolation region of the growth model. Square distance between food conditions and the centroid of the set of data generated in broth. Observations were used to estimate the overall error (U) or discarded (D) according to their distance from the interpolation region and the quality of the model predictions for those conditions. d The study of these conditions was used to extend the interpolation region of the model.

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MSEg,ffood Eg Effood Effood ffood2 MSEg,ffood Eg Effood2 EEffood ffood2 2Eg EffoodEffood ffood Notice that the term (g Effood) is a constant (not a random variable); therefore, Eg EffoodEffood ffood g Effood EEffood ffood so the cross product in the binomial expression is equal to 0 and the equation can be rearranged as follows: MSEg,ffood g Effood2 EEffood ffood2 Since (g Effood) E(g Effood), the rst component of the MSE is the bias of the model for food observations and the second is the variance of food measurements (Fig. 2):
2 MSEg,ffood Biasg ffood Varffood

set used to t the model, and therefore its location is determined mainly by those intervals of the environmental factors for which there are many observations. A normalized square distance is introduced between any x (x1. . .xk) combination of environmental factors and the r centroid, according to the formula

d 2 x , r

k i1

1 x r i 2 Zi2 i

Our basic assumption was that the bias and the variance in food, as dened above, are constant not only in the interpolation region but also in its extension, for which only food data exist. Because the logarithmic transformation of the specic rate was found to be suitable for modeling, the relative error of the specic rate observations in food was constant, as was the variance of the log(specic rate) values observed for food. We used this criterion to extend the interpolation region: the extension is possible as long as the assumption holds. Algorithmically, this means that we add those points to the MCP, for which observations made in food do not contradict this assumption. The . . .R notation is used for situations when a statistical indicator is calculated in the R region. We will use . . .(x1. . .xn) notation in a similar way for cases when the indicator is calculated for the set of (x1. . .xn) points. 2 Our basic assumption was that Biasg (ffoodRg) and Var(ffoodRg) are constant in an Rg region which includes the original MCP of the g model. Testing this assumption is more reliable when many broth data are used for model creation. To identify those regions, we introduced a normalized distance concept. Let the vectors r1. . .rn denote those combinations of environmental factors for which measurements were made in broth and used for model creation: rj (r1j. . .rkj), where j 1. . .n. For this model, k is the number of environmental factors (temperature, pH, etc. [in this paper, k 4]). Let r be the centroid of these points, dened as follows:

where Zi is the average Z value for the ith environmental factor in the interpolation region, based on the model predictions. Let (y1. . .ym) be the set of observations in food that are outside the MCP, sorted in increasing order according to their (normalized) distances from the centroid r (Fig. 3). A subset of food observations, y1. . .yj (1 j m), will be used to extend the interpolation region if Var[ffood(y1. . .yj)] Var[ffood(r1. . .rn)]. Because of our basic assumption that the bias is constant, this is equivalent to MSE[ffood(y1. . .yj)] MSE[ffood(r1. . .rn)]. Therefore, test points for which observations are made outside the original MCP are used to extend the original MCP in a sequence dened by their distances to the centroid r . For one test point, the MSE between the predictions and observations is calculated by using all of those test points outside the MCP that are closer to the centroid than to the considered test point (Fig. 3). The procedure stops when the calculated MSE is larger than that measured inside the original interpolation region. Once certain test points are accepted, the extended MCP is determined as described previously (3). Estimation of the probability of growth at the growth-no growth interface. A logistic regression model, as introduced previously (20), was used to describe the probability of growth, p, as a function of the temperature, pH, and percent CO2 and O2 in the atmosphere. Only the observations in laboratory medium were used (Table 1). The tted model is p 1 1 eab Tempc pHd CO2e O2

r r 1 r k, where r i

n j1

rij i 1k

where a, b, c, d, and e are the parameters estimated by the maximum likelihood method. The predictive ability of this model was assessed by estimating the percentage of concordance between predicted probabilities and observed responses (22). To estimate this, we dened growth with a value of 1, while the value for no growth was 0. A pair of observations with different responses is said to be concordant or discordant if the observation with the response value 1 has a higher or lower predicted probability, respectively.

RESULTS The data in Table 1 show the maximum specic growth and death rates of A. hydrophila in broth. For all broth experiments, when the pH was 6, the viable counts decreased with time.

In other words, rij is the jth observation of the ith environmental factor (1 i k; 1 j n), where k is the dimension of the environmental space and n is the number of those observations that were used for model creation. Note that r is not the center of the interpolation region but is the center of gravity of the data

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TABLE 3. Average Z values: changes in the environmental variables that cause a twofold increase in the growth or death rate
Average Z value for: Rate Temp (C) pH CO2 (%) O2 (%)

Growth Death

4.25

0.538 0.971

80.0 234

90.1 84.5

FIG. 3. Relationship between MSE for observations outside the interpolation region and the distance between those observations and the centroid of the data set used to t the model. Numbers indicate the cumulative numbers of observations located at a shorter or equal distance. (a) Analysis of MSE. (b) Analysis of variance.

The tted model for the probability of growth, p, was as follows: log

p 25.33 0.3569 Temp 3.717 pH 1p 0.0117 CO2 0.0110 O2

cients, which was originally 14 with a standard quadratic surface, was reduced to 4 for the death model and 6 for the growth model. According to the Z values, a 4C decrease in the temperature caused a twofold decrease in the growth rate. The death rate seemed to be unaffected by temperature. The pH affected both growth and death, but had a larger effect on growth. A 1-U decrease in pH caused a twofold (or 100%) increase in the death rate and a fourfold decrease in the growth rate. The percentage of CO2 in the atmosphere also had a greater effect on the growth rate than on the death rate. An increase of 11% in the CO2 concentration caused a 10% decrease in the growth rate but only a 3% increase in the death rate. The effect of O2 was similar on both growth and death: an increase of 11 to 12% caused a 10% increase in the death rate and a 10% decrease in the growth rate. The data in Table 2 show the rates observed in seafood; A. hydrophila grew in all of the samples tested except for sole. The soles were eviscerated, but their heads, gills, and skin were left intact. The numbers of natural contaminating bacteria were higher than in the other samples at the time of inoculation, at ca. 104 to 105 CFU/g. A. hydrophila is not a strong competitor when growing with other bacteria (15), and the inoculum could not colonize and compete with the well-established indigenous ora. Consequently, these data were not used to estimate the overall error of the model. Five of the seafood conditions lay outside of the interpolation region of the model (Table 2). Of the rates measured in fresh meat at temperatures up to 11C in air, vacuum, and modied atmospheres obtained from ComBase, 33 of 56 were produced outside of the strict interpolation region of the model (Table 5). Figure 3 shows how the extension of the interpolation region was carried out with the ComBase data for meat. The error
TABLE 4. Parameter estimates for the models for the natural logarithm of the maximum specic growth and death rates
Term ln(g) Coefcient SE Coefcient lnd SE

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The percentage of concordance between the predicted probabilities and observations in broth used to t the model was 91%. For validation of this model, 250 observations in broth, with temperatures of 11C, water activities of 0.99, pHs of 7, and different concentrations of CO2 and O2, were selected from ComBase. The percentage with concordance with these data was 85%. According to this model, the probability of growth of A. hydrophila at pH 6 reaches 0.5 only at a relatively high temperature of ca. 8 to 9C; the probability is 0.7 at 11C. The effect of the environmental variables in the region studied was quantied by using the average Z values (Table 3), which were estimated from the models for the growth and the death rates described in Table 4. The number of model coef-

Intercept Temp Temp2 pH pH2 Temp CO2 Temp pH pH O2 O2 CO2 Ra2a MSE
a

4.50 0.829 0.0151 0.00122 0.184 0.00114 0.746 0.212

0.345 0.169 0.00766 0.000286 0.017 0.000444

51.8 17.3 1.30

11.7 3.80 0.305

0.000804 0.488 0.773

0.000276

Adjusted coefcient of determination.

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PIN ET AL. TABLE 5. Growth rates observed for meat, obtained from ComBasea

APPL. ENVIRON. MICROBIOL.

ComBase code

Reference

Origin

Temp (C)

pH

CO2 (%)

O2 (%)

observed

Probability of growth

predicted

Modelb

Distance to centroidb

Final decisionb

M715_Ah M714_Ah M193_Ah M713_Ah M712_Ah M196_Ah M716_Ah M155_Ah M734_Ah M735_Ah M738_Ah M739_Ah M151_Ah M199_Ah M150_Ah M198_Ah M197_Ah M154_Ah M152_Ah M153_Ah M138_Ah M195_Ah M194_Ah M719_Ah M718_Ah M717_Ah M189_Ah M190_Ah M139_Ah M191_Ah M192_Ah M743_Ah M742_Ah SA118 SA67 SA124 SA68 M731_Ah M730_Ah SA130 M133_Ah M131_Ah M188_Ah M132_Ah SA74 SA5 SA80 SA10 SA112 SA111 M134_Ah M721_Ah M723_Ah M135_Ah M727_Ah M726_Ah
a b

5 5 20 5 5 20 5 21 18 18 18 18 21 20 12 20 20 21 21 21 11 20 20 5 5 5 20 20 11 20 20 18 18 19 19 19 19 18 18 19 9 9 20 9 19 19 19 19 19 19 9 5 5 9 18 18

Lamb Lamb Ground pork Lamb Lamb Ground pork Lamb Ground pork Meat extract Meat extract Meat extract Meat extract Ground pork Ground pork Pork Ground pork Ground pork Ground pork Ground pork Ground pork Beef fat Ground pork Ground pork Lamb Lamb Lamb Ground pork Ground pork Beef Ground pork Ground pork Meat extract Meat extract Turkey Pork Turkey Pork Meat extract Meat extract Turkey Beef Beef Ground pork Beef Turkey Pork Turkey Pork Turkey Turkey Beef Lamb Lamb Beef Meat extract Meat extract

5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 7 7 7 7 5 5 7 5 10 5 10 1 1 1 1 1 1 2 0 0 0 5 5

6.2 6.2 6.4 6.2 6.2 6.4 6.2 6.8 6.5 6.5 7.0 7.0 6.8 6.4 6.5 6.4 6.4 6.8 6.8 6.8 7.1 6.4 6.4 6.2 6.2 6.2 6.1 6.1 6.1 6.1 6.1 7.5 7.5 6.0 6.0 6.0 6.0 6.0 6.0 6 6 6 5.9 6 6.2 6.2 6.2 6.2 6.2 6.2 6 6.2 6.2 6 5.5 5.5

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 80 0 0 16 16 20 20 20 0 60 80 0 16 0 0 16 16

0 16 16 16 16 16 0 16 16 16 16 16 16 16 16 0 16 16 16 16 16 0 16 80 80 80 16 16 16 16 16 16 16 20 20 0 0 16 16 20 0 0 0 100 0 0 0 0 40 20 0 0 0 0 0 0

0.0138 0.0190 0.0240 0.0242 0.0260 0.0301 0.0323 0.0360 0.0449 0.0452 0.0483 0.0483 0.0499 0.0548 0.0642 0.0697 0.0697 0.0702 0.0757 0.0758 0.0800 0.0959 0.110 0.0101 0.0185 0.0200 0.0383 0.0383 0.0479 0.0479 0.0513 0.0522 0.0584 0.101 0.106 0.0293 0.785 0.0433 0.0446 0.0327 0.0384 0.112 0.0284 0.0260 0.0221 0.0499 0.0586 0.0117 0.0353 0.0353 0.0230 0.0118 0.0153 0.0128 0.0175 0.0194

0.38 0.34 0.52 0.34 0.34 0.52 0.38 0.82 0.61 0.61 0.91 0.91 0.82 0.52 0.52 0.56 0.52 0.82 0.82 0.82 0.94 0.56 0.52 0.16 0.16 0.16 0.26 0.26 0.26 0.26 0.26 0.98 0.98 0.32 0.32 0.37 0.37 0.19 0.19 0.16 0.22 0.63 0.14 0.32 0.10 0.10 0.10 0.13 0.044 0.044 0.090 0.078 0.092 0.046 0.036 0.036

0.0362 0.0323 0.0387 0.0323 0.0323 0.0387 0.0362 0.0555 0.0424 0.0424 0.0665 0.0665 0.0555 0.0387 0.0336 0.0435 0.0387 0.0555 0.0555 0.0555 0.0728 0.0435 0.0387 0.0182 0.0182 0.0182 0.0295 0.0295 0.0295 0.0295 0.0295 0.104 0.104 0.0317 0.0317 0.0363 0.0363 0.0270 0.0270 0.0180 0.0304 0.0391 0.0249 0.0103 0.0130 0.0130 0.0130 0.0150 0.00933 0.00830 0.0182 0.00991 0.0111 0.0111 0.0173 0.0173

I I I I I I I I I I I I I I I I I I I I I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

2.0 2.0 2.0 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.4 2.4 2.4 2.4 2.6 2.6 2.7 2.7 2.8 3.2 3.4 3.6 3.6 3.6 3.7 3.7 4 4 4.4 4.4 5.4 6.3 6.3

U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U D D D D D D D D D D D D D D D D D D D D D D D

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The study of these conditions was used to extend the interpolation region of the model. See the footnotes to Table 2 for details.

between predictions and observations increased when the distance from the observations to the centroid of the broth data increased. The MSE and the variance in food inside the interpolation region were 0.223 (Fig. 3a) and 0.222 (Fig. 3b), respectively. The closest observations to the centroid were a group of three observations at an equal distance from the

centroid. The MSE and the variance calculated for these three observations were 0.119 and 0.0932, respectively. The next step was a group of seven observations. The MSE for all 10 outside observations was 0.208 and the variance was 0.207. The next group of observations increased the MSE up to 0.394 and the variance up to 0.341, which were already higher than the error

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TABLE 6. MSE(g,ffood), Bias2 (g,ffood), and Var(ffood) of the growth model on the original and extended MCP for seafood and meat
Sample type MSE(g,ffood) for MCP Original Extended Bias2 (g,ffood) for MCP Original Extended Var (ffood) for MCP Original Extended

Seafood Meat

0.161 0.223

0.165 0.218

0.00575 0.00167

0.00163 0.00141

0.155 0.222

0.164 0.217

and variance inside the interpolation region and indicated a noticeable increase in the bias. The seafood data were analyzed in the same way (not shown). Hence, the extension of the interpolation region was done with 10 observations in meat selected as indicated above and with data for all ve seafood groups tested. The data in Table 6 show that the error of the model inside the original interpolation region was practically equal to the error measured in the extended MCP. The MSE, estimated for the predictions and observations that were used to t the data, was 0.22. Since the model is unbiased for these data and assuming that the dependence of the growth rates on the environment is described well enough by the model, the MSE is due to the variance of the bacterial response in broth. The analysis of the error when applying the model to food is shown in Table 6. Because the bias of the model is very small, the main source of error can be identied as the variance in food. Moreover, note that the variances of the bacterial responses in food and in laboratory media were very similar. DISCUSSION In experiments at pHs of 6 selected from the ComBase database (http://www.combase.cc; http://wyndmoor.arserrc.gov /combase/), A. hydrophila died in 31 but grew in 80. pH values close to 6 may be inhibitory enough to prevent the growth of the organism at refrigeration temperatures. In addition, in CO2-enriched atmospheres, low temperatures favor the dissolution of CO2 as carbonic acid into the medium, and consequently the pH value drops (8). On the other hand, as indicated by the Z values in Table 3, the pH had a signicant effect not only on the growth but also on the death of A. hydrophila. According to the Z values, in the range from 1.5 to 11C, a decrease in temperature does not noticeably increase the rate of death. Low refrigeration temperatures can prevent bacterial growth, but they do not accelerate bacterial death. The effect of CO2 was much larger on the growth rates than on the death rates. The percent O2 had a noticeable effect on both growth and death. It has also been reported that the growth of A. hydrophila is slower in air than in atmospheres saturated with nitrogen (10). In situations of oxygen stress, as reported for Escherichia coli (7), the growth rate can be limited by the low intracellular level of superoxide dismutase, which provides effective protection against superoxide ion toxicity. Both the faster death rates and the slower growth rates observed with increasing O2 concentrations in the atmosphere can be attributed to the toxic consequences of oxygen metabolism (23). The data in Tables 2 and 5 show the probability of growth of A. hydrophila and the predicted maximum specic rate under different conditions. The dominant part of the original inter-

polation region was in the environmental space where the probability of growth was close to or higher than 0.5. After the extension, reliable predictions could also be obtained for conditions with lower probabilities of growth. The extension of the model was carried out in a region for which a relatively high number of model-generating data existed. However, this does not imply that the probability of growth in this region is the highest. In fact, we could not generate reliable predictions of the specic growth rate in the region of the highest probability of growth because of the lack of growth data for that region. As shown in Fig. 3a, the further the predictions were from the interpolation region, the higher the MSE was. Compared to the increase in the MSE, the increase in the variance was relatively small (Fig. 3b), so we deduced that when we extrapolated, the increase in the bias was the major component responsible for the increase in the MSE. If laboratory media simulate the conditions in food perfectly, then the MSE between food observations and model predictions is equal to the error obtained when tting the model to laboratory data. When the latter error is smaller, it indicates more variability in the growth parameters in food and/or the bias of the model when applied to food. This paper focused on quantication of the components of the error of a predictive model for A. hydrophila. The model presented here was practically unbiased for both meat and seafood and the variances of the bacterial response in food and in laboratory media were very similar. As a consequence, the data generated in laboratory media can be utilized efciently to study bacterial responses to food environments.
ACKNOWLEDGMENTS We acknowledge the ComBase consortium for making data available. The support of the European Commission, Quality of Life and Management of Living Resources, Key Action 1 (KA1) on Food, Nutrition and Health, project no. QLK1-CT-2002-300513 is thankfully acknowledged. G.D.G.F. acknowledges the support of the Comisio n Interministerial de Ciencia y Tecnologa (CICYT, Spain) through project ALI99-0405/98 and project AGL2000-0692.
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