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Predicting the growth ofEscherichia colion displayed pork
Food Microbiology, 1998, 15, 235–242
ORIGINAL ARTICLE
Predicting the growth of Escherichia coli on displayed pork C. O. Gill*, G. G. Greer and B. D. Dilts
Samples of sterile pork fat or muscle tissue were inoculated with logarithmic phase cultures of Escherichia coli and were incubated in a display case at positions where they experienced average temperatures of 3·5, 6·0, 6·9, 8·0, 9·7 or 11·4°C. The temperature history of the tissue at each position was recorded. During incubation for up to 7 days, samples of each tissue were removed daily from each position for enumeration of E. coli and growth of E. coli at each position at each of those times was estimated by integrating the temperature history of the tissue with respect to a model describing the dependency on temperature of the aerobic growth of E. coli. When temperatures fluctuated above 7°C for periods <1 h, no growth of E. coli occurred on fat or muscle tissue although growth would be predicted by the model. When temperatures fluctuated above 7°C for periods >1 h, the predicted growth and that observed on fat tissue were similar. However, growth on muscle tissue occurred only when temperatures fluctuated above 9°C for lengthy periods, and then only after a lag of about 4 days when temperatures remained mainly below 12°C. When temperatures fluctuated above 12° for lengthy periods the predicted growth and that observed on both fat and muscle tissues were similar. Accurate prediction of the growth of E. coli on meat which experiences temperatures that fluctuate below 7°C will require the development of models to predict the lag before growth of E. coli on fat or muscle tissues during periods at growthpermitting temperatures.
Introduction Raw meat is inevitably contaminated with mesophilic enteric pathogens (Grau 1986). The risks to consumers from such organisms will increase if the bacteria grow as a result of product temperatures rising above the chiller temperature range (Bogh-Sorensen and Bertelsen 1988). During display most chilled meat will experience abusive temperatures, at least briefly, during the defrosting cycles of the display case refrigeration equipment, and the monitoring of the temperatures of commercial products has *Corresponding author. 0740-0020/98/020235+08 $25·00/0/fd970157
shown that substantial fractions of displayed meat experience abusive temperatures for prolonged periods (Malton 1980, Bogh-Sorensen and Olsson 1990, Olsson 1990). Certainly, growth of the indicator organism Escherichia coli in commercially displayed raw meat has been demonstrated (Gill and McGinnis 1993). E. coli is generally accepted as an indicator of meat contamination with enteric pathogens, and growth of that organism in meat would be indicative of possible increases in the numbers of most mesophilic, enteric pathogens. A recent evaluation of temperature histories from meat in commercial display cases indicated that the cases could not be operated to preclude the growth of E. coli
Received: 11 July 1997 Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C&E Trail, Lacombe, AB, Canada T4L 1W1
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on all products, which therefore suggested that the hygienic adequacy of display processes for raw meat could be assured only by specifying a maximum residence time related to the maximum, average product temperature consistently achieved in each display case (Greer et al. 1994). In that study, the aspect of microbiological safety was assessed by integrating each temperature history with respect to a model which describes the dependency on temperature of the growth of E. coli (Gill et al. 1991). In calculating the E. coli proliferation permitted by a temperature history it was assumed that no lag would delay growth when temperatures fluctuate above the minimum for growth, and that the composition of the meat would not affect the rate of aerobic growth. Such simplifying assumptions have allowed calculation of values for E. coli growth during cooling of meat which agreed closely with values determined by plating of samples (Reichel et al. 1991). However, recent studies of the growth of cold-tolerant pathogens on pork have shown that models derived from the growth of those organisms in broths may poorly describe their behaviours on pork fat and lean tissues at temperatures approaching their minima for growth (Gill et al. 1997). Similar unreliability in the model for aerobic growth of E. coli used for the integration of temperature histories could result in misleading estimates of E. coli growth on displayed meat. Therefore, a study was undertaken to ascertain if the growth of E. coli on displayed pork can be predicted by the temperature function integration technique previously employed for predicting the growth of that organism on cooling meat (Reichel et al. 1991).
Materials and methods The organism The strain of E. coli used for inoculating pork was isolated from product in a beef packing plant. Its identity was confirmed by the API 20E Gram-negative identification system ´ (bioMerieux, St Laurent, PQ, Canada). The IMViC pattern indicated it was Biotype I. The organism was maintained in cooked
meat medium (Difco Laboratories, Detroit, MI, USA) and was subcultured by transfer to tryptic soy broth (Difco) which was incubated for 18 h at 25°C. The stationary-phase culture so obtained was used to inoculate tubes containing 10 ml of tryptic soy broth, to obtain mid-log-phase cultures after 7 h incubation at 25°C. Such log-phase cultures were used for the inoculation of pork tissues samples.
Growth of E. coli on pork Sterile pork muscle and fat tissue discs (10 cm2) were aseptically excised from boneless pork loins of normal muscle quality as previously described (Greer and Dilts 1995). The pH values of five muscle and five fat tissue discs were determined before inoculation for each incubation temperature. Surface pH was measured using an Oakton portable pH meter (Anachemia Scientific, Calgary, AB, Canada) equipped with a flat-surface electrode. Inocula were prepared by the dilution of 10 ml of a log-phase culture of E. coli in 1 litre of 0·1% w/v peptone water. Tissue discs were suspended from alligator clips and immersed in the inocula for 15 s, to give an initial bacterial count approximating 105 bacteria cm−2 of tissue. After inoculation, samples were permitted to drain for 15 min at 20°C to remove excess liquid. Following this treatment, pork tissue discs were placed in square (9×9 cm) Petri plates with three fat and three lean tissue discs in each plate at each location in the case. The plates were then overwrapped with film with an oxygen transmission rate of about 8000 cc m−2 24 h−1 atm−1 at 25°C and 75% r.h. Tissue discs were displayed in a horizontal, fan-assisted, commercial display cabinet (Hill Refrigeration of Canada, Barrie, ON, USA) illuminated with 150 w incandescent floodlights to give a light intensity at the meat surface of 750 lx. The case was illuminated between 0700 and 1900 hrs each day, with defrosting being initiated automatically at each of those times. The characteristics of this display case have been described in detail (Greer and Jeremiah 1981). The positions within the retail display case
Escherichia coli on pork 237
at which mean temperatures of about 4, 6, 7, 8, 10 or 12°C were maintained had previously been determined. During the incubation of plates, the tissue temperatures at each position were recorded at 15 min intervals by a temperature data logger (Delphi logger; TruTest, Auckland, NZ) equipped with an external thermistor probe. On the first and on each of 7 subsequent days, a plate each of fat and muscle tissue discs was removed from each position. Three fat and three muscle tissue samples were evaluated on each sampling day for each temperature. Each disc of muscle or fat was homogenized in 90 ml of 0·1% w/v peptone water for 2 min, using a Colworth Stomacher 400 (Baxter Diagnostics Corp., Edmonton, AB, Canada). After serial 10-fold dilution of the homogenate in 0·1% w/v peptone water, 0·1 ml aliquots of appropriate dilutions were surface plated on plates of tryptic soy agar (Difco Laboratories, Detroit, MI, USA). E. coli were enumerated after incubation of the agar plates for 48 h at 25°C. On the last day of display at each temperature bacteria were also enumerated on lactose monensin gluconurate agar (LMGA, QA Life Sciences Inc., San Diego, CA, USA) and buffered 4-methylumbelliferyl-β-D-glucuronide agar (BMA, QA Life Sciences Inc.) using the hydrophobic grid membrane filtration method of Entis and Boleszczuk (1990) for the enumeration of coliforms and E. coli as applied to meat samples by Gill and McGinnis (1993).
Estimation of E. coli growth by E. coli temperature function integration The temperature history recovered from each logger was integrated with respect to a model that describes the dependency on temperature of the aerobic growth of E. coli (Gill et al. 1991). The triphasic model has the form: y=(0·0513x–0·17)2 when x is between 7 and 30; y=(0·027x+0·55)2 when x is between 30 and 40; y=2·66 when x is between 40 and 47; and y=0 when x is <7 or >47; where y is the growth rate expressed as
generations h−1 and x is the temperature in °C. In performing integrations it was assumed that growth commenced without lag when the temperature entered the range for growth of E. coli. The proliferation for each record interval was calculated on the assumption that the recorded temperature was maintained for the duration of the interval, and the total proliferation was obtained by summation of the incremental proliferations. For comparisons of observed and calculated growth, the calculated proliferations were converted to log values which were added to the numbers observed initially on that tissue, to estimate the numbers expected to be recovered after incubation of the tissues. The correlation between calculated and observed values for growth was calculated with Microsoft Excel (Version 4, statistical functions; Microsoft Corp., Redmond, WA, USA).
Results The pH values for samples of tissue before inoculation were 6·3±0·3 for fat tissue and 5·6 ±0·2 for muscle tissue. The numbers of bacteria recovered using selective and differential media (LMGA, BMA) were not different than those recovered on tryptic soy agar. Results are reported with reference to the temperature 7°C, as this temperature was the lower limit of observed E. coli growth. In all temperature profiles, defrosting of the retail case was evident as transient peaks of temperature maxima at 12 h intervals. In the absence of defrosting, 12 h periods at which tissue temperatures were lowest occurred in the absence of display illumination while temperatures were somewhat higher during equivalent periods of illumination. These factors account for the shape of the profiles of the temperature history data reported in Figs 1b to 6b. At the coldest position, the mean tissue temperature was 3·5°C (Fig. 1). The mean temperatures during periods of case illumination were about 2°C higher than those at times when the case was not illuminated. Temperatures rose above 7°C only during
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Figure 1. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 3·5°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
Figure 2. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 6·0°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
defrosting cycles, for periods of <1 h and to temperatures <12°C. The growth predicted for the total incubation period of 167 h was < 0·5 log units, while no increases of E. coli numbers on fat or muscle tissues were apparent. At a position where the mean tissue temperature was 6·0°C, the temperatures of about 7°C during case illumination were about 4°C higher than the temperatures when the case was not illuminated (Fig. 2). Temperatures above 7°C persisted for no more than 1 h at any time, even during defrosting cycles when maximum temperatures about 20°C and 15°C were obtained during illumination and darkness, respectively. The growth predicted for the total incubation period was about 2 log units. However, no increases of E. coli numbers on fat or muscle tissues were apparent. At a position where the mean tissue temperature was 6·9°C, the temperatures of about 7°C during case illumination were about 2°C higher than the temperatures in the absence of illumination (Fig. 3). Temperatures were above 7°C about 45% of the time, during periods of illumination and defrosting cycles. During defrosting, maximum tem-
peratures were generally >15°C. However, temperatures above 7°C persisted for no more than 3 h at any time. The predicted growth was generally somewhat greater than that observed on fat. However, no increases of E. coli numbers on muscle tissue were apparent. At a position where the mean tissue temperature was 8·0°C, the temperatures of about 9°C during illumination were about 3°C higher than the temperatures in the absence of illumination (Fig. 4). Temperatures were above 7°C for about 50% of the time, during periods of illumination and defrosting cycles. During defrosting, maximum temperatures during periods of illumination and darkness were about 20 and 15°C, respectively. The predicted growth was somewhat greater than that observed on fat tissue. On muscle tissue there was apparently a lag of about 90 h before growth commenced at a rate which seemed similar to that observed on fat tissue. At a position where the mean tissue temperature was 9·7°C, the temperature remained above 7°C during the first 120 h, but fluctuated to temperatures below 7°C in the absence of illumination in the subsequent 47 h (Fig. 5). The predicted growth was generally similar to that observed on fat tissue. On muscle tissue there was a lag of about
Escherichia coli on pork 239
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Figure 3. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 6·9°C. Growth data are the means of three determinations on displayed tissue at each sampling time. 90 h before growth commenced at a rate which seemed similar to that observed on fat tissue. At a position where the mean tissue temperature was 11·4°C, the temperature remained above 7°C throughout the incubation period (Fig. 6). For values obtained during the first 60 h of incubation, the predicted growth was similar to that observed on muscle tissue and somewhat less than that observed on fat tissue. Subsequently, the predicted growth was similar to that observed on fat tissue and greater than that observed on muscle tissue. The coefficient of correlation (r) between the growth observed when growth commenced on fat or muscle tissues without apparent lag and the equivalent calculated values for growth was 0·97 (Fig. 7).
Discussion Most meat is prepared for retail display in cutting facilities at retail outlets. As meat will generally experience temperatures of 10°C or above in such facilities, any E. coli on the product may well have entered the logar-
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Figure 4. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 8·0°C. Growth data are the means of three determinations on displayed tissue at each sampling time. ithmic phase before the product is transferred to a retail case. In the retail case, some product may be exposed for prolonged periods to temperatures above the chiller temperature range, and most will experience air temperatures well above that range during defrosting cycles, at intervals of 12 h or less. The recovery of bacteria in equal numbers on selective and non-selective agars from samples at the end of incubation periods indicated that only E. coli were enumerated on the samples. The data show that when log-phase cells of E. coli on meat are exposed to chiller conditions for several hours before being returned to growth-permitting temperatures, there will be a lag before growth resumes. For E. coli on fat, that lag appears to be more than 1 h, to account for the absence of observed growth on fat tissue displayed at an average temperature of 6·0°C when a temperature above 7°C was maintained for 1 h or less at any time, but considerably less than 3 h, to account for the growth observed on fat tissue at an average temperature of 6·9°C when a temperature above 7°C was maintained for less than 3 h, but more than 1 h, at any time.
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Figure 5. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 9·7°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
For E. coli on pork muscle tissue of normal pH, the data indicate that the minimum temperature for growth of E. coli might be greater by 1 or 2°C than the minimum temperature for growth on fat, and that at growth-permitting temperatures up to about 10°C there will be a lag of 3 to 4 days before growth of E. coli is initiated by cells that were in the log phase when they were deposited on the meat. However, at temperatures of about 12°C or more growth on muscle tissue will occur with little or no lag at rates similar to rates of growth on fat. The differences in the behaviour of E. coli on fat and muscle tissues at temperatures approaching the minimum for growth, which have been ascribed to differences in the pH and lactate contents of the tissues, could be expected from previous reports of growth of the organism on meat (Grau 1983, Smith 1985). Despite that, such differences would not be predicted by models based on the growth of pathogenic strains of E. coli in broths (Gibson and Roberts 1986, Hughes and McDermott 1989, Buchanan and Klawitter 1992). It therefore appears that the model used in this study can be employed to predict with
Figure 6. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 11·4°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
reasonable accuracy the initial growth of E. coli on the fat tissue of displayed meats which periodically experiences temperatures of 7°C or above for times of 2 h or more. When temperatures of 12°C or above are similarly experienced, the predicted growth would reasonably represent the growth on muscle tissue as well. However, the model could not take account of possible perturbations of E. coli growth by competition from other species at the later stages of development of a natural flora on meat (Gill 1986). That, and other possible perturbing factors, would have to be considered when assessing the health risks implied by the predicted growth of E. coli. To predict properly growth of E. coli on fat when temperatures fluctuate below 7°C, a model for the lag before growth is initiated after movement from a non-permissive temperature to a temperature permissive of growth would have to be developed. This could then be substituted for the simple assumption of growth being initiated without lag. Such a model for the lag phase could be complex if the duration of the lag is dependent, as it may well be, on the time at non-
Escherichia coli on pork 241
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Figure 7. Relationship between observed and predicted (h) numbers of Escherichia coli on displayed fat or muscle tissues when growth was initiated without a lag. Each point is the mean of three fat or three lean tissue discs. permissive temperatures, and both the nonpermissive and permissive temperatures that are experienced (Zwietering et al. 1994). Whatever the relationships between the lag and those parameters, it is apparent that growth on fat cannot be predicted reliably from the mean temperature when temperatures are fluctuating above and below 7°C. Similar considerations would apply to modelling the growth of E. coli on muscle tissue when temperatures fluctuate above and below 12°C. Such conclusions with respect to predicting growth at fluctuating temperatures are similar to those arrived at by others from the study of the growth of pathogenic E. coli in broths (Rajkowski and Marmer 1995, Buchanan et al. 1997). Thus the procedure for calculating E. coli growth from temperature history data will have to be modified by incorporating models for the lag phase, to allow the realistic prediction of E. coli growth on displayed meat that does not experience persistent abusive temperatures.
Acknowledgements The authors gratefully acknowledge the technical assistance of Roberta Dyck, the clerical assistance of Loree Verquin and the financial support of Agriculture and Agri-Food Canada’s Food Safety Research Contract Fund.
Bogh-Sorensen, L. and Bertelsen, G. (1988) Temperatures in the chill chain for meat products. In Cold Chains in Economic Perspective, pp. 99–104. Paris, International Institute of Refrigeration. Bogh-Sorensen, L. and Olsson, P. (1990) The chill chain. In Chilled Foods: the State of the Art, (Ed. T. R. Gormley), pp. 245–267. London, Elsevier Applied Science. Buchanan, R. L. and Klawitter, L. A. (1992) The effect of incubation temperature, initial pH, and sodium chloride on the growth kinetics of Escherichia coli O157:H7. Food Microbiol. 9, 185–196. Buchanan, R. L., Whiting, R. C. and Damert, W. C. (1997) When is simple good enough: a comparison of the Gompertz, Baranyi and threephase linear models for fitting bacterial growth curves. Food Microbiol. 14, 313–326. Entis, P. and Boleszczuk, P. (1990) Direct enumeration of coliforms and Escherichia coli by hydrophobic grid membrane filter in 24 h using MUG. J. Food Protect. 53, 948–952. Gibson, A. M. and Roberts, T. A. (1986) The effect of pH, water activity, sodium nitrite and storage temperature on the growth of enteropathogenic Escherichia coli and Salmonella in a laboratory medium. Int. J. Food Microbiol. 3, 183–194. Gill, C. O. (1986) Control of microbial spoilage in fresh meats. In Advances in Meat Research, Vol. 2 (Eds A. M. Pearson and T. R. Dutson), pp. 49–88. Westport, CT, AVI Publishing Co. Gill, C. O. and McGinnis, C. (1993) Changes in the microflora on commercial beef trimmings during their collection, distribution and preparation for retail sale as ground beef. Int. J. Food Microbiol. 18, 321–332. Gill, C. O., Harrison, J. C. L. and Phillips, D. M. (1991) Use of a temperature function integration technique to assess the hygienic adequacy of a beef carcass cooling process. Food Microbiol. 8, 83–94. Gill, C. O., Greer, G. G. and Dilts, B. D. (1997) The aerobic growth of Aeromonas hydrophila and Listeria monocytogenes in broths and on pork. Int. J. Food Microbiol. 35, 67–74. Grau, F. M. (1983) Growth of Escherichia coli and Salmonella typhimurium on beef tissue at 25°C. J. Food Sci. 48, 1700–1704. Grau, F. M. (1986) Microbial ecology of meat and poultry. In Advances in Meat Research, Vol. 2 (Eds A. M. Pearson and T. R. Dutson), pp. 1–48. Westport, CT AVI Publishing Co. Greer, G. G. and Jeremiah, L. E. (1981) Proper control of retail case temperature improves beef shelf life. J. Food Protect. 44, 297–299. Greer, G. G., Gill, C. O. and Dilts, B. D. (1994) Evaluation of the bacteriological consequences of the temperature regimes experienced by
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fresh chilled meat during retail display. Food Res. Int. 27, 371–377. Greer, G. G. and Dilts, B. D. (1995) Lactic acid inhibition of the growth of spoilage bacteria and cold tolerant pathogens on pork. Int. J. Food Microbiol. 25, 141–151. Hughes, A. M. and McDermott, J. C. (1989) The effect of phosphate, sodium chloride, sodium nitrite, storage temperature and pH on the growth of enteropathogenic Escherichia coli in a laboratory medium. Int. J. Food Microbiol. 9, 215–223. Malton, P. (1980) Displaying wrapped meat in refrigerated cabinets: the problems. Meat (UK) 53, 37–43. Olsson, P. (1990) Chill cabinet surveys. In Processing and Quality of Foods, Vol. 3 (Eds P. Zeuthen, J. C. Cheftel, C. Eriksson, T. R. Gormley, P. Linko and K. Paulus), pp. 3·279–3·288. London, Elsevier Applied Science.
Rajkowski, K. T. and Marmer, B. S. (1995) Growth of Escherichia coli O157:H7 at fluctuating incubation temperatures. J. Food Protect. 58, 1307–1313. Reichel, M. P., Phillips, D. M., Jones, R. and Gill, C. O. (1991) Assessment of the hygienic adequacy of a commercial hot boning process for beef by a temperature function integration technique. Int. J. Food Microbiol. 14, 27–42. Smith, M. G. (1985) The generation time, lag time and minimum temperature of growth of coliform organisms on meat, and the implications for codes of practice in abattoirs. J. Hyg. Camb. 94, 284–300. Zwietering, M. H., deWit, J. C., Cuppers, H. G. A. M. and van’t Riet, K. (1994) Modeling of bacterial growth with shifts in temperature. Appl. Environment. Microbiol. 60, 204–213.
ORIGINAL ARTICLE
Predicting the growth of Escherichia coli on displayed pork C. O. Gill*, G. G. Greer and B. D. Dilts
Samples of sterile pork fat or muscle tissue were inoculated with logarithmic phase cultures of Escherichia coli and were incubated in a display case at positions where they experienced average temperatures of 3·5, 6·0, 6·9, 8·0, 9·7 or 11·4°C. The temperature history of the tissue at each position was recorded. During incubation for up to 7 days, samples of each tissue were removed daily from each position for enumeration of E. coli and growth of E. coli at each position at each of those times was estimated by integrating the temperature history of the tissue with respect to a model describing the dependency on temperature of the aerobic growth of E. coli. When temperatures fluctuated above 7°C for periods <1 h, no growth of E. coli occurred on fat or muscle tissue although growth would be predicted by the model. When temperatures fluctuated above 7°C for periods >1 h, the predicted growth and that observed on fat tissue were similar. However, growth on muscle tissue occurred only when temperatures fluctuated above 9°C for lengthy periods, and then only after a lag of about 4 days when temperatures remained mainly below 12°C. When temperatures fluctuated above 12° for lengthy periods the predicted growth and that observed on both fat and muscle tissues were similar. Accurate prediction of the growth of E. coli on meat which experiences temperatures that fluctuate below 7°C will require the development of models to predict the lag before growth of E. coli on fat or muscle tissues during periods at growthpermitting temperatures.
Introduction Raw meat is inevitably contaminated with mesophilic enteric pathogens (Grau 1986). The risks to consumers from such organisms will increase if the bacteria grow as a result of product temperatures rising above the chiller temperature range (Bogh-Sorensen and Bertelsen 1988). During display most chilled meat will experience abusive temperatures, at least briefly, during the defrosting cycles of the display case refrigeration equipment, and the monitoring of the temperatures of commercial products has *Corresponding author. 0740-0020/98/020235+08 $25·00/0/fd970157
shown that substantial fractions of displayed meat experience abusive temperatures for prolonged periods (Malton 1980, Bogh-Sorensen and Olsson 1990, Olsson 1990). Certainly, growth of the indicator organism Escherichia coli in commercially displayed raw meat has been demonstrated (Gill and McGinnis 1993). E. coli is generally accepted as an indicator of meat contamination with enteric pathogens, and growth of that organism in meat would be indicative of possible increases in the numbers of most mesophilic, enteric pathogens. A recent evaluation of temperature histories from meat in commercial display cases indicated that the cases could not be operated to preclude the growth of E. coli
Received: 11 July 1997 Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C&E Trail, Lacombe, AB, Canada T4L 1W1
236 C. O. Gill et al.
on all products, which therefore suggested that the hygienic adequacy of display processes for raw meat could be assured only by specifying a maximum residence time related to the maximum, average product temperature consistently achieved in each display case (Greer et al. 1994). In that study, the aspect of microbiological safety was assessed by integrating each temperature history with respect to a model which describes the dependency on temperature of the growth of E. coli (Gill et al. 1991). In calculating the E. coli proliferation permitted by a temperature history it was assumed that no lag would delay growth when temperatures fluctuate above the minimum for growth, and that the composition of the meat would not affect the rate of aerobic growth. Such simplifying assumptions have allowed calculation of values for E. coli growth during cooling of meat which agreed closely with values determined by plating of samples (Reichel et al. 1991). However, recent studies of the growth of cold-tolerant pathogens on pork have shown that models derived from the growth of those organisms in broths may poorly describe their behaviours on pork fat and lean tissues at temperatures approaching their minima for growth (Gill et al. 1997). Similar unreliability in the model for aerobic growth of E. coli used for the integration of temperature histories could result in misleading estimates of E. coli growth on displayed meat. Therefore, a study was undertaken to ascertain if the growth of E. coli on displayed pork can be predicted by the temperature function integration technique previously employed for predicting the growth of that organism on cooling meat (Reichel et al. 1991).
Materials and methods The organism The strain of E. coli used for inoculating pork was isolated from product in a beef packing plant. Its identity was confirmed by the API 20E Gram-negative identification system ´ (bioMerieux, St Laurent, PQ, Canada). The IMViC pattern indicated it was Biotype I. The organism was maintained in cooked
meat medium (Difco Laboratories, Detroit, MI, USA) and was subcultured by transfer to tryptic soy broth (Difco) which was incubated for 18 h at 25°C. The stationary-phase culture so obtained was used to inoculate tubes containing 10 ml of tryptic soy broth, to obtain mid-log-phase cultures after 7 h incubation at 25°C. Such log-phase cultures were used for the inoculation of pork tissues samples.
Growth of E. coli on pork Sterile pork muscle and fat tissue discs (10 cm2) were aseptically excised from boneless pork loins of normal muscle quality as previously described (Greer and Dilts 1995). The pH values of five muscle and five fat tissue discs were determined before inoculation for each incubation temperature. Surface pH was measured using an Oakton portable pH meter (Anachemia Scientific, Calgary, AB, Canada) equipped with a flat-surface electrode. Inocula were prepared by the dilution of 10 ml of a log-phase culture of E. coli in 1 litre of 0·1% w/v peptone water. Tissue discs were suspended from alligator clips and immersed in the inocula for 15 s, to give an initial bacterial count approximating 105 bacteria cm−2 of tissue. After inoculation, samples were permitted to drain for 15 min at 20°C to remove excess liquid. Following this treatment, pork tissue discs were placed in square (9×9 cm) Petri plates with three fat and three lean tissue discs in each plate at each location in the case. The plates were then overwrapped with film with an oxygen transmission rate of about 8000 cc m−2 24 h−1 atm−1 at 25°C and 75% r.h. Tissue discs were displayed in a horizontal, fan-assisted, commercial display cabinet (Hill Refrigeration of Canada, Barrie, ON, USA) illuminated with 150 w incandescent floodlights to give a light intensity at the meat surface of 750 lx. The case was illuminated between 0700 and 1900 hrs each day, with defrosting being initiated automatically at each of those times. The characteristics of this display case have been described in detail (Greer and Jeremiah 1981). The positions within the retail display case
Escherichia coli on pork 237
at which mean temperatures of about 4, 6, 7, 8, 10 or 12°C were maintained had previously been determined. During the incubation of plates, the tissue temperatures at each position were recorded at 15 min intervals by a temperature data logger (Delphi logger; TruTest, Auckland, NZ) equipped with an external thermistor probe. On the first and on each of 7 subsequent days, a plate each of fat and muscle tissue discs was removed from each position. Three fat and three muscle tissue samples were evaluated on each sampling day for each temperature. Each disc of muscle or fat was homogenized in 90 ml of 0·1% w/v peptone water for 2 min, using a Colworth Stomacher 400 (Baxter Diagnostics Corp., Edmonton, AB, Canada). After serial 10-fold dilution of the homogenate in 0·1% w/v peptone water, 0·1 ml aliquots of appropriate dilutions were surface plated on plates of tryptic soy agar (Difco Laboratories, Detroit, MI, USA). E. coli were enumerated after incubation of the agar plates for 48 h at 25°C. On the last day of display at each temperature bacteria were also enumerated on lactose monensin gluconurate agar (LMGA, QA Life Sciences Inc., San Diego, CA, USA) and buffered 4-methylumbelliferyl-β-D-glucuronide agar (BMA, QA Life Sciences Inc.) using the hydrophobic grid membrane filtration method of Entis and Boleszczuk (1990) for the enumeration of coliforms and E. coli as applied to meat samples by Gill and McGinnis (1993).
Estimation of E. coli growth by E. coli temperature function integration The temperature history recovered from each logger was integrated with respect to a model that describes the dependency on temperature of the aerobic growth of E. coli (Gill et al. 1991). The triphasic model has the form: y=(0·0513x–0·17)2 when x is between 7 and 30; y=(0·027x+0·55)2 when x is between 30 and 40; y=2·66 when x is between 40 and 47; and y=0 when x is <7 or >47; where y is the growth rate expressed as
generations h−1 and x is the temperature in °C. In performing integrations it was assumed that growth commenced without lag when the temperature entered the range for growth of E. coli. The proliferation for each record interval was calculated on the assumption that the recorded temperature was maintained for the duration of the interval, and the total proliferation was obtained by summation of the incremental proliferations. For comparisons of observed and calculated growth, the calculated proliferations were converted to log values which were added to the numbers observed initially on that tissue, to estimate the numbers expected to be recovered after incubation of the tissues. The correlation between calculated and observed values for growth was calculated with Microsoft Excel (Version 4, statistical functions; Microsoft Corp., Redmond, WA, USA).
Results The pH values for samples of tissue before inoculation were 6·3±0·3 for fat tissue and 5·6 ±0·2 for muscle tissue. The numbers of bacteria recovered using selective and differential media (LMGA, BMA) were not different than those recovered on tryptic soy agar. Results are reported with reference to the temperature 7°C, as this temperature was the lower limit of observed E. coli growth. In all temperature profiles, defrosting of the retail case was evident as transient peaks of temperature maxima at 12 h intervals. In the absence of defrosting, 12 h periods at which tissue temperatures were lowest occurred in the absence of display illumination while temperatures were somewhat higher during equivalent periods of illumination. These factors account for the shape of the profiles of the temperature history data reported in Figs 1b to 6b. At the coldest position, the mean tissue temperature was 3·5°C (Fig. 1). The mean temperatures during periods of case illumination were about 2°C higher than those at times when the case was not illuminated. Temperatures rose above 7°C only during
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Figure 1. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 3·5°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
Figure 2. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 6·0°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
defrosting cycles, for periods of <1 h and to temperatures <12°C. The growth predicted for the total incubation period of 167 h was < 0·5 log units, while no increases of E. coli numbers on fat or muscle tissues were apparent. At a position where the mean tissue temperature was 6·0°C, the temperatures of about 7°C during case illumination were about 4°C higher than the temperatures when the case was not illuminated (Fig. 2). Temperatures above 7°C persisted for no more than 1 h at any time, even during defrosting cycles when maximum temperatures about 20°C and 15°C were obtained during illumination and darkness, respectively. The growth predicted for the total incubation period was about 2 log units. However, no increases of E. coli numbers on fat or muscle tissues were apparent. At a position where the mean tissue temperature was 6·9°C, the temperatures of about 7°C during case illumination were about 2°C higher than the temperatures in the absence of illumination (Fig. 3). Temperatures were above 7°C about 45% of the time, during periods of illumination and defrosting cycles. During defrosting, maximum tem-
peratures were generally >15°C. However, temperatures above 7°C persisted for no more than 3 h at any time. The predicted growth was generally somewhat greater than that observed on fat. However, no increases of E. coli numbers on muscle tissue were apparent. At a position where the mean tissue temperature was 8·0°C, the temperatures of about 9°C during illumination were about 3°C higher than the temperatures in the absence of illumination (Fig. 4). Temperatures were above 7°C for about 50% of the time, during periods of illumination and defrosting cycles. During defrosting, maximum temperatures during periods of illumination and darkness were about 20 and 15°C, respectively. The predicted growth was somewhat greater than that observed on fat tissue. On muscle tissue there was apparently a lag of about 90 h before growth commenced at a rate which seemed similar to that observed on fat tissue. At a position where the mean tissue temperature was 9·7°C, the temperature remained above 7°C during the first 120 h, but fluctuated to temperatures below 7°C in the absence of illumination in the subsequent 47 h (Fig. 5). The predicted growth was generally similar to that observed on fat tissue. On muscle tissue there was a lag of about
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Figure 3. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 6·9°C. Growth data are the means of three determinations on displayed tissue at each sampling time. 90 h before growth commenced at a rate which seemed similar to that observed on fat tissue. At a position where the mean tissue temperature was 11·4°C, the temperature remained above 7°C throughout the incubation period (Fig. 6). For values obtained during the first 60 h of incubation, the predicted growth was similar to that observed on muscle tissue and somewhat less than that observed on fat tissue. Subsequently, the predicted growth was similar to that observed on fat tissue and greater than that observed on muscle tissue. The coefficient of correlation (r) between the growth observed when growth commenced on fat or muscle tissues without apparent lag and the equivalent calculated values for growth was 0·97 (Fig. 7).
Discussion Most meat is prepared for retail display in cutting facilities at retail outlets. As meat will generally experience temperatures of 10°C or above in such facilities, any E. coli on the product may well have entered the logar-
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Figure 4. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 8·0°C. Growth data are the means of three determinations on displayed tissue at each sampling time. ithmic phase before the product is transferred to a retail case. In the retail case, some product may be exposed for prolonged periods to temperatures above the chiller temperature range, and most will experience air temperatures well above that range during defrosting cycles, at intervals of 12 h or less. The recovery of bacteria in equal numbers on selective and non-selective agars from samples at the end of incubation periods indicated that only E. coli were enumerated on the samples. The data show that when log-phase cells of E. coli on meat are exposed to chiller conditions for several hours before being returned to growth-permitting temperatures, there will be a lag before growth resumes. For E. coli on fat, that lag appears to be more than 1 h, to account for the absence of observed growth on fat tissue displayed at an average temperature of 6·0°C when a temperature above 7°C was maintained for 1 h or less at any time, but considerably less than 3 h, to account for the growth observed on fat tissue at an average temperature of 6·9°C when a temperature above 7°C was maintained for less than 3 h, but more than 1 h, at any time.
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Figure 5. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 9·7°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
For E. coli on pork muscle tissue of normal pH, the data indicate that the minimum temperature for growth of E. coli might be greater by 1 or 2°C than the minimum temperature for growth on fat, and that at growth-permitting temperatures up to about 10°C there will be a lag of 3 to 4 days before growth of E. coli is initiated by cells that were in the log phase when they were deposited on the meat. However, at temperatures of about 12°C or more growth on muscle tissue will occur with little or no lag at rates similar to rates of growth on fat. The differences in the behaviour of E. coli on fat and muscle tissues at temperatures approaching the minimum for growth, which have been ascribed to differences in the pH and lactate contents of the tissues, could be expected from previous reports of growth of the organism on meat (Grau 1983, Smith 1985). Despite that, such differences would not be predicted by models based on the growth of pathogenic strains of E. coli in broths (Gibson and Roberts 1986, Hughes and McDermott 1989, Buchanan and Klawitter 1992). It therefore appears that the model used in this study can be employed to predict with
Figure 6. (a) Growth of Escherichia coli observed on pork fat (s) or muscle (d) tissues, or predicted (h) from (b) the temperature history for a mean tissue temperature of 11·4°C. Growth data are the means of three determinations on displayed tissue at each sampling time.
reasonable accuracy the initial growth of E. coli on the fat tissue of displayed meats which periodically experiences temperatures of 7°C or above for times of 2 h or more. When temperatures of 12°C or above are similarly experienced, the predicted growth would reasonably represent the growth on muscle tissue as well. However, the model could not take account of possible perturbations of E. coli growth by competition from other species at the later stages of development of a natural flora on meat (Gill 1986). That, and other possible perturbing factors, would have to be considered when assessing the health risks implied by the predicted growth of E. coli. To predict properly growth of E. coli on fat when temperatures fluctuate below 7°C, a model for the lag before growth is initiated after movement from a non-permissive temperature to a temperature permissive of growth would have to be developed. This could then be substituted for the simple assumption of growth being initiated without lag. Such a model for the lag phase could be complex if the duration of the lag is dependent, as it may well be, on the time at non-
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Figure 7. Relationship between observed and predicted (h) numbers of Escherichia coli on displayed fat or muscle tissues when growth was initiated without a lag. Each point is the mean of three fat or three lean tissue discs. permissive temperatures, and both the nonpermissive and permissive temperatures that are experienced (Zwietering et al. 1994). Whatever the relationships between the lag and those parameters, it is apparent that growth on fat cannot be predicted reliably from the mean temperature when temperatures are fluctuating above and below 7°C. Similar considerations would apply to modelling the growth of E. coli on muscle tissue when temperatures fluctuate above and below 12°C. Such conclusions with respect to predicting growth at fluctuating temperatures are similar to those arrived at by others from the study of the growth of pathogenic E. coli in broths (Rajkowski and Marmer 1995, Buchanan et al. 1997). Thus the procedure for calculating E. coli growth from temperature history data will have to be modified by incorporating models for the lag phase, to allow the realistic prediction of E. coli growth on displayed meat that does not experience persistent abusive temperatures.
Acknowledgements The authors gratefully acknowledge the technical assistance of Roberta Dyck, the clerical assistance of Loree Verquin and the financial support of Agriculture and Agri-Food Canada’s Food Safety Research Contract Fund.
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