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Effects of refrigeration or freezing on survival of Listeria monocytogenes Scott A in under-cooked ground beef
Food Control 14 (2003) 25–30 www.elsevier.com/locate/foodcont
Effects of refrigeration or freezing on survival of Listeria monocytogenes Scott A in under-cooked ground beef John S. Novak *, Vijay K. Juneja Food Safety Research Unit, Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA Received 23 March 2002; received in revised form 7 May 2002; accepted 9 May 2002
Abstract Listeria monocytogenes Scott A, inoculated into ground beef, was heat-shocked at 46 °C for 60 min to enhance stress adaptations and simulate sublethal minimal cooking conditions. The effects of refrigeration at 4 °C or freezing at 20 °C were examined on pathogen survival prior to or following mild cooking at 60 °C. D10 -values for heat-shocked samples were elevated as compared to nonheat-shocked controls. Refrigerated and frozen storage did not influence the observed effects. Cellular injury of survivors increased with timed exposure to 60 °C. The effects of refrigerated and frozen storage on heat-adapted L. monocytogenes in ground beef did not decrease the potential of the food-borne pathogen to survive additional low temperature cooking of food and raised concerns over the food safety of contaminated, temperature-abused foods. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Listeria; Sublethal cooking
1. Introduction Listeria monocytogenes is described as a gram-positive facultatively anaerobic, halotolerant rod with considerable variation in morphology dependent upon physiological and environmental conditions (Janda & Abbott, 1999). The pathogen has been reported to be the most frequently isolated Listeria species associated with human disease (Janda & Abbott, 1999). It has been isolated from a variety of foods and by-products and therefore, continues to be a serious public health concern (Beuchat, Brackett, Hao, & Conner, 1986; Watkins & Sleath, 1981). The United States alone, in 1999, had 92.2% of the 2518 illnesses from L. monocytogenes result in hospitalizations while accounting for 27.6% of the total foodborne deaths (Mead et al., 1999). At risk are the very young, the elderly, expectant mothers, and the immunocompromised. Growth at refrigerated temperatures and survivability in adverse environments, including those conditions that occur in minimally processed foods have made L. mono*
Corresponding author. Tel.: +1-215-836-3762; fax: +1-215-2336406. E-mail address: [email protected] (J.S. Novak).
cytogenes a challenge to control. The pathogen also has a higher reported heat resistance than many other nonspore-formers (Farber & Peterkin, 1991; Mackey & Bratchell, 1989). The versatility of L. monocytogenes to grow in a broad temperature range (2–45 °C) limits the effectiveness of restrictive measures following recent evidence of temperature acclimation and synthesis of protective proteins in the pathogen post-sublethal heat or cold shock (Bayles, Annous, & Wilkinson, 1996; Jorgensen, Panaretou, Stephens, & Knochel, 1996; Linton, Webster, Pierson, Bishop, & Hackney, 1992). This study was undertaken to examine the effects of refrigeration or freezing before and after exposure of L. monocytogenes to sublethal heating temperatures in ground beef simulating the conditions that might occur in prepared cook-chill foods reheated by consumers following chilled storage. Even though outbreaks of L. monocytogenes-related food poisoning have not been reported for undercooked ground beef, the potential does exist for contamination by multiple food handlers and extended chilled storage. These variations on pathogen survival are considered important from a food safety perspective with the increased marketing of mildly-cooked, quick-chilled foods for consumer convenience and time-economic home preparation.
0956-7135/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 6 - 7 1 3 5 ( 0 2 ) 0 0 0 4 8 - 8
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J.S. Novak, V.K. Juneja / Food Control 14 (2003) 25–30
2. Materials and methods
2.4. Refrigerated and then heat-treated
2.1. Bacterial strain
Prior to heat treatment at 60 °C, two replications of the study involving ground beef samples inoculated with L. monocytogenes Scott A in sterile filter bags were refrigerated at 4 °C for 24 h followed by heat treatment as for samples heat-treated alone. These samples were plated as previously stated. Other replicate samples following refrigeration were heat-shocked at 46 °C for 60 min and then immersed in the 60 °C water bath for heat treatment.
L. monocytogenes Scott A was obtained from the Eastern Regional Research Center (ERRC) culture collection. Cells were maintained in 20% glycerol at 80 °C and grown on tryptic soy agar (TSA) plates at 37 °C prior to study initiation. Freshly grown colonies were used to inoculate 10 ml brain heart infusion broth (BHIB) tubes and incubated at 37 °C for 20 h. Culture identity was confirmed by gram stain and incubated reactions read from gram-positive identification cards prepared for the Vitek Automicrobic System (BioMerieux Vitek, Inc., Hazelwood, MO). All microbiological media was prepared from dry chemical formulations (Difco Laboratories, Detroit, MI). 2.2. Ground beef, sample preparation and inoculation Stationary phase cultures of L. monocytogenes Scott A grown for 20 h at 37 °C in BHIB tubes were pelleted (5000 g for 15 min) and resuspended in 10 ml of 0.1% (w/v) peptone-water. Samples (100 g) of ground beef (7% fat) purchased from a local supermarket were irradiated (42 kGy/ 30 °C). These were then inoculated with 1 ml of culture and homogenized for 25 min at a high setting in 5 min intervals with a Stomacher lab-blender (Model 400, Spiral Systems, Inc., Cincinnati, OH). The inoculated ground beef (3 g portions) was then aseptically weighed into sterile filter bags, and heat-sealed. The sealed portions were then either heat-treated directly at 60 °C, refrigerated (4 °C ) and then heat-treated (60 °C), and then refrigerated (4 °C), frozen at (20 °C) and then heat-treated (60 °C), and then frozen at (20 °C).
2.5. Frozen and then heat-treated Prior to heat treatment, two replications of the study involving ground beef samples inoculated with L. monocytogenes Scott A in sterile filter bags were frozen at 20 °C for 24 h followed by heat treatment as before. These samples were plated as previously stated. Replicate samples following freezing were then heat-shocked at 46 °C for 60 min and finally immersed in the 60 °C water bath for heat treatment. 2.6. Heat-treated and then refrigerated Prior to plating, heat-treated and heat-shocked then heat-treated at 60 °C ground beef samples were refrigerated at 4 °C for 24 h followed by plating as previously described. 2.7. Heat-treated and then frozen Prior to plating, heat-treated and heat-shocked then heat-treated at 60 °C ground beef samples inoculated with L. monocytogenes Scott A were frozen at 20 °C for 24 h followed by plating as previously described.
2.3. Heat-treated samples
2.8. Determination of heating injury
Replicate samples of ground beef, inoculated with L. monocytogenes Scott A in sterile Stomacher filter bags (Nasco, Fort Atkinson, WI), were flattened to ensure even heat transfer, vacuum heat-sealed to 20 mbar in a vacuum packager (Multi-Vac Inc., Model A300, Kansas City, MO), and immersed in a 60 °C water bath. Samples were taken at 2.5 min time intervals for up to 35 min and cooled in an ice slurry. The heated bags were opened and 3 ml of phosphate-buffered saline with 0.05% Tween 20 (PBS-Tween; Sambrook, Fritsch, & Maniatis, 1989) were added and stomached for 2 min to form a 1:1 slurry. Dilutions were made in 0.1% (w/v) peptone-water and plated on TSA. The plates were incubated at 37 °C with counts taken 20 h later. A similar set of samples was first heat-shocked at 46 °C for 60 min and then immersed in the 60 °C water bath for heat treatment and sampled as for the other samples.
The percentage of heat injured survivors was determined using data from two replications for each experiment for all samples taken on TSA medium supplemented with 4.0% (w/v) NaCl. This selective plating medium restricted recovery of heat-injured survivors. The following formula was used to calculate percent injury for each time point: 1 ða=bÞ 100 ¼ %injury where a ¼ CFU/ml on TSA with 4% NaCl and b ¼ CFU/ml on TSA. 2.9. Determination of D10 -values All dilution platings were as previously described followed by incubation at 37 °C for 24 h. D10 -values (time for 10-fold reduction in viable cells), expressed in
J.S. Novak, V.K. Juneja / Food Control 14 (2003) 25–30
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minutes, were determined by plotting the log10 number of survivors against time point samplings for 60 °C using Lotus 1-2-3 software. The line of best fit for survivor plots was determined by regression analysis (Ostle & Mensing, 1975); a regression equation for the type y ¼ a þ bx was derived, where b is the slope of the best straight line and, when inverted the sign changed from to þ to give the D10 -value in min. For each treatment, an estimate of the D10 -value and its corresponding standard error was calculated from the slopes of the regressions in the analysis of covariance and their standard errors using SAS (1989). 2.10. Qualitative analyses of protein expression Samples from the different treatment conditions (excluding the 60 °C exposure kill) were pelleted (11,000 g for 3 min), resuspended in buffer consisting of 25 mM Tris (pH 7.5), 1 mM EDTA (pH 7.5), 5 mM b-mercaptoethanol, 6 mM MgCl2 , and 30 mM NH4 Cl (Rheinberger, Geigenmuller, Wedde, & Nierhaus, 1988). The cell pellets were then stored at 70 °C until use. The protease inhibitor, phenylmethanosulfane fluoride (PMSF), was added to 1 mM as was dithiothreitol to provide a stable reducing environment prior to cell lysis. Cells were broken using a Vibracell model VC130 ultrasonic processor (Sonics and Materials, Inc., Newtown, CT) amplitude setting of 60, pulser at 1 s, for 2 min on ice. DNAse was then added to a concentration of 0.2 mg/ml. Protein concentrations were determined using a modification of the Lowry procedure (Lowry, Rosebrough, Farr, & Randall, 1951; Markwell, Haas, Bieber, & Tolbert, 1978). Cell lysates were resuspended in gel loading buffer (Sambrook et al., 1989) and heated at 100 °C in a boiling water bath for 5 min. Standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) procedures involved (4% (w/v acrylamide stacking gel, 15% (w/v) acrylamide resolving gel; 30:0.8 acrylamide:bis acrylamide) and a modified Tris-glycine buffer system (Lugtenberg, Meijers, Peters, van der Hoek, & van Alphen, 1975). Protein standards were obtained from Bio-Rad Laboratories (Hercules, CA). All sample lanes contained 20 lg of total protein. Following electrophoretic resolution, the gels were stained with Coomassie blue and photographed.
3. Results 3.1. Enhanced survival following sublethal heating temperatures Surviving L. monocytogenes Scott A heat-shocked in ground beef exhibited an elevated heat resistance at 60 °C as compared to nonheat-shocked controls (Fig. 1). Although heat injury was lower for heat-shocked sam-
Fig. 1. Thermal inactivation and survival of L. monocytogenes in ground beef and exposed to 60 °C over time. Symbols: ( ) control nonheat-shocked; ( ) heat-shocked at 46 °C for 60 min prior to heat treatments. The degree of heat injury measured on TSA þ 4% (w/v) NaCl plates as a percentage of the total plate counts on TSA medium is listed next to specific time points. Error bars designate standard deviations for means of two experimental trials.
ples, there was a parallel increase in cellular damage following growth on selective medium (TSA with 4% NaCl) when compared to controls grown on TSA medium (Fig. 1). Survival beyond 15 min at 60 °C was too low to give accurate estimations of heat injury. In order to examine the likelihood of L. monocytogenes survival in contaminated ground beef following mild heating, storage effects such as refrigeration at 4 °C or freezing at 20 °C for 24 h were examined. In one set of experiments, refrigeration or freezing effects on the Listeria-inoculated meat samples prior to heating at 60 °C were examined (Fig. 2A). Heat shock adaptations resulted in elevated survival profiles for the heat-shocked samples at 60 °C as compared with nonheat-shocked refrigerated or frozen samples (Fig. 2A). A similar trend was realized in the lowered degree of heat injury seen for heat-shocked versus nonheat-shocked samples (Fig. 2B). In another set of experiments, the refrigeration and freezing effects on ground beef inoculated with L. monocytogenes were recorded after cooking the meat at 60 °C over time (Fig. 3A). The heat-shocked samples refrigerated or frozen after minimal cooking at 60 °C produced indistinguishable survival curves with respect to one another, but significantly different profiles in comparison to refrigerated or frozen samples that were not heat-shocked, respectively (Fig. 3A). The heat shock effect appeared to be more pronounced when the refrigerated or frozen storage was incorporated after the heat treatments as compared to before. This finding would be significant with regards to the safety of precooked meals subsequently chilled for reheating. The degree of heat injury appeared comparable to that found for the experiments involving refrigerated or frozen
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Fig. 2. (A) Thermal inactivation and survival of L. monocytogenes in ground beef: (D) refrigerated at 4 °C for 24 h or () frozen at 20 °C for 24 h prior to heat exposures over time. Open symbols designate no prior heat shock, whereas closed symbols depict samples heat-shocked at 46 °C for 60 min. Error bars designate standard deviations for means of two experimental trials. (B) The degree of heat injury measured on TSA þ 4% (w/v) NaCl plates as a percentage of the total plate counts on TSA medium for L. monocytogenes Scott A inoculated ground beef exposed to 60 °C over time.
storage prior to heat shock (Figs. 2B and 3B). The frozen nonheat-shocked samples exhibited less injury following 10 min of heat treatment than any of the other treatments (Fig. 3B). This did not indicate a greater degree of repair to heat injury in the frozen samples as the survivors continued to decrease, concurrently (Fig. 3A). 3.2. Comparison of thermal resistance parameters at 60 °C D10 -values were significantly increased (P < 0:05) for the following heat-shocked samples: refrigerated prior to heat treatment (4.3) compared to 2.3 for nonheatshocked, frozen prior to heat treatment (3.6) compared to 1.7 for nonheat-shocked, and refrigerated following heat treatment (3.2) compared to 2.0 for nonheatshocked (Table 1). There were no significant differences at the P < 0:05 level among heat-shocked samples based
Fig. 3. (A) Thermal inactivation and survival of L. monocytogenes in ground beef: (D) refrigerated at 4 °C for 24 h or () frozen at 20 °C for 24 h after heat exposure over time. Open symbols designate no prior heat shock, whereas closed symbols depict samples heat-shocked at 46 °C for 60 min. Error bars designate standard deviations for means of two experimental trials. (B) The degree of heat injury measured on TSA þ 4% (w/v) NaCl plates as a percentage of the total plate counts on TSA medium for L. monocytogenes Scott A inoculated ground beef exposed to 60 °C over time.
Table 1 Summary of thermal characterizations Sample conditions
D10 -value at 60 °Ca
Control Heat-shocked control Refrigerated prior to heat treatments Refrigerated and heat-shocked prior Frozen prior to heat treatments Frozen and heat-shocked prior Refrigerated following heat treatments Heat-shocked and refrigerated following Frozen following heat treatments Heat-shocked and frozen following
2:1 0:2E 4:3 0:4A 2:3 0:2E 4:3 0:5AB 1:7 0:1F 3:6 0:2ABC 2:0 0:2E 3:2 0:3CD 2:6 0:5DE 3:4 0:3BCD
a
D10 -values in minutes at 60 °C represent the results from two separate experimental trials and were calculated as described in Section 2. These estimates were then used to form a series of pairwise comparisons. The results agreed with the output from the analysis of covariance used to test the homogeneity of the slopes. Any two D10 values with no letter in common are significantly different by the pairwise t-test technique at the (P < 0:05) level of confidence.
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Fig. 4. SDS-PAGE analysis of protein turnover in response to experimental conditions detailed in Section 2. Lanes: (1) and (10) protein molecular weight standards; (2) nonheat-shocked control; (3) heatshocked control; (4) refrigerated and nonheat-shocked; (5) refrigerated prior to heat shock; (6) frozen and nonheat-shocked; (7) frozen prior to heat shock; (8) heat-shocked and then refrigerated; and (9) heatshocked and then frozen.
on the method of storage (refrigeration at 4 °C for 24 h or freezing at 20 °C for 24 h) prior to or post-heat treatment (Table 1). However, it was evident that heat shock effects were lessened with respect to the control (4.3), if samples were refrigerated after heat treatment (3.2) or frozen after heat treatment (3.4). Therefore, storage conditions were found to influence the protective heat shock effects in L. monocytogenes Scott A to some extent. 3.3. Protein expression in response to treatment conditions All of the heat-shocked samples exhibited similar increases in proteins approximating 55, 44, 32, 27, and 24 kDa despite refrigeration or frozen storage (Fig. 4, lanes 3, 5, 7, 8, and 9). The nonheat-shocked samples also contained proteins approximating 33, 23, 22, 20, and 19 kDa that were repressed, denatured, or turnedover more rapidly under the heat-shock conditions (Fig. 4, lanes 2, 4, and 6). All samples contained large, but similar quantities of a protein product at 40 kDa that was not influenced by the heat shock or storage conditions.
4. Discussion There have been previous reports of heat shock effects on thermotolerance in L. monocytogenes (Linton, Pierson, & Bishop, 1990; Linton et al., 1992), the incidence of protective heat shock proteins (Jorgensen et al., 1996), changes in L. monocytogenes thermotolerance affected by growth conditions and heating menstruum (Jorgensen, Hansen, & Knochel, 1999), and even pro-
29
tective effects due to cold stress proteins (Bayles et al., 1996). This represents the first report to our knowledge of cold storage and the subsequent survival attributes of L. monocytogenes in a meat product when used in combination with mild sublethal heat treatment used before or after storage for 24 h. All of these are possible consumer handling practices awaiting cook-chill foods. Heat shock effects (46 °C for 60 min) on L. monocytogenes in ground beef were statistically significant in increasing the subsequent survival characteristics of the pathogen with respect to D10 -values and reduced percent cellular injury at 60 °C. Refrigerated and frozen storage did not lessen these effects. This finding should have significant implications in the safety of cook-chilled products which are increasing, being produced and marketed in response to consumer demands. A very likely scenario involves pre-market contamination of heat-treated meat with L. monocytogenes. The heattreated meat can activate adaptations in L. monocytogenes to enable the foodborne pathogen to survive a second low temperature heating following chilled storage and prior to ingestion by the consumer. Documentation supports the belief that nearly all organisms respond to sublethal stress with the production of proteins to counteract and protect against subsequent stress (Cheville, Arnold, Buchrieser, Cheng, & Kaspar, 1996; Lindquist & Craig, 1988; Schlesinger, Ashburner, & Tissieres, 1982; Volker et al., 1994). Many of these heat shock proteins are molecular chaperones, such as GroESL gene products in Escherichia coli, that aid in proper folding or removal of damaged proteins (Georgopoulos & Welch, 1993). Although several proteins have been shown to be expressed in L. monocytogenes following 46 °C for 60 min at 55, 44, 32, 27, and 24 kDa an equal number of proteins at 33, 23, 22, 20, and 19 kDa appear repressed. Metabolic reactions in all living organisms exist in balance between anabolic and catabolic processes. A complete evaluation of heat adaptations in L. monocytogenes Scott A requires the consideration of repressed products that have not been previously identified and may negatively impact survival as well as those positively regulated. Of concern was the one log10 CFU/ml increase in the initial counts of L. monocytogenes in ground beef refrigerated or frozen after heat treatment as compared to heat treatment before. A possible explanation might have involved the release of intracellular molecules from cells weakened or killed by the heat treatment and followed by refrigeration or freeze-fracture of cell walls and membranes resulting in the protection of surviving cells of L. monocytogenes which were less stressed. Much of the safety associated with food preparations involves proper handling and temperature controls pre- and post-cooking. Therefore the results of this study increased concerns over the safety of cook-chill foods if
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such foods were contaminated with a psychrotrophic pathogen such as L. monocytogenes Scott A.
Acknowledgements We wish to thank Ms. Kenyetta J. Chaney for technical assistance in performing this study and Dr. John Phillips for statistical analyses of the data obtained.
References Bayles, D. O., Annous, B. A., & Wilkinson, B. J. (1996). Cold stress proteins induced in Listeria monocytogenes in response to temperature downshock and growth at low temperatures. Applied Environmental Microbiology, 62, 1116–1119. Beuchat, L. R., Brackett, R. E., Hao, D. Y.-Y., & Conner, D. E. (1986). Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice. Canadian Journal of Microbiology, 32, 791–795. Cheville, A. M., Arnold, K. W., Buchrieser, C., Cheng, C.-M., & Kaspar, C. W. (1996). RpoS regulation of acid, heat, and salt tolerance in Escherichia coli O157:H7. Applied Environmental Microbiology, 62, 1822–1824. Farber, J. M., & Peterkin, P. I. (1991). Listeria monocytogenes, a foodborne pathogen. Microbiological Reviews, 55, 476–511. Georgopoulos, C., & Welch, W. J. (1993). Role of the major heat shock proteins as molecular chaperones. Annual Review of Cell Biology, 9, 601–634. Janda, J. M., & Abbott, S. L. (1999). Unusual food-borne pathogens. Clinical Laboratory Medicine, 19, 553–582. Jorgensen, F., Hansen, T. B., & Knochel, S. (1999). Heat shockinduced thermotolerance in Listeria monocytogenes 13-249 is dependent on growth phase pH and lactic acid. Food Microbiology, 16, 185–194. Jorgensen, F., Panaretou, B., Stephens, P. J., & Knochel, S. (1996). Effect of pre- and post-heat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes. Journal of Applied Bacteriology, 80, 216–224. Lindquist, S., & Craig, E. A. (1988). The heat-shock proteins. Annual Review of Genetics, 22, 631–677.
Linton, R. H., Pierson, M. D., & Bishop, J. R. (1990). Increase in heat resistance of Listeria monocytogenes Scott A by sublethal heat shock. Journal of Food Protection, 53, 924–927. Linton, R. H., Webster, J. B., Pierson, M. D., Bishop, J. R., & Hackney, C. R. (1992). The effect of sublethal heat shock and growth atmosphere on the heat resistance of Listeria monocytogenes Scott A. Journal of Food Protection, 55, 84–87. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275. Lugtenberg, B., Meijers, J., Peters, R., van der Hoek, P., & van Alphen, L. (1975). Electrophorectic resolution of the ‘major outer membrane protein’ of Escherichia coli K12 into four bands. FEBS Letters, 58, 254–258. Mackey, B. M., & Bratchell, N. (1989). The heat resistance of Listeria monocytogenes. Letters in Applied Microbiology, 9, 89–94. Markwell, M. A. K., Haas, S. M., Bieber, L. L., & Tolbert, N. E. (1978). A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analytical Biochemistry, 87, 206–210. Mead, P. S., Slutsker, L., Dietz, V., McCraig, L. F., Bresee, J. S., Shapiro, C., Griffin, P. M., & Tauxe, R. V. (1999). Food-related illness and death in the United States. Emerging Infectious Diseases, 5, 607–625. Ostle, B., & Mensing, R. W. (1975). Statistics in research. Ames, Iowa: Iowa State University Press. Rheinberger, H.-J., Geigenmuller, U., Wedde, M., & Nierhaus, K. H. (1988). Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding. Methods in Enzymology, 164, 658–662. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd ed.). New York: Cold Spring Harbor Laboratory Press. SAS Institute, 1989. SAS/STAT user’s guide, vol. 2, (4th ed.), Ver. 6 (pp. 891–996). Cary, NC: SAS Institute. Schlesinger, M. J., Ashburner, M., & Tissieres, A. (1982). Heat shock from bacteria to man. New York: Cold Spring Harbor Laboratory Press. Volker, U., Engelmann, S., Maul, B., Reithdorf, S., Volker, A., Schmid, R., Mach, H., & Hecker, M. (1994). Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology, 140, 741–752. Watkins, J., & Sleath, K. P. (1981). Isolation and enumeration of Listeria monocytogenes from sewage, sewage sludge, and river water. Journal of Applied Bacteriology, 50, 1–9.
Effects of refrigeration or freezing on survival of Listeria monocytogenes Scott A in under-cooked ground beef John S. Novak *, Vijay K. Juneja Food Safety Research Unit, Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA Received 23 March 2002; received in revised form 7 May 2002; accepted 9 May 2002
Abstract Listeria monocytogenes Scott A, inoculated into ground beef, was heat-shocked at 46 °C for 60 min to enhance stress adaptations and simulate sublethal minimal cooking conditions. The effects of refrigeration at 4 °C or freezing at 20 °C were examined on pathogen survival prior to or following mild cooking at 60 °C. D10 -values for heat-shocked samples were elevated as compared to nonheat-shocked controls. Refrigerated and frozen storage did not influence the observed effects. Cellular injury of survivors increased with timed exposure to 60 °C. The effects of refrigerated and frozen storage on heat-adapted L. monocytogenes in ground beef did not decrease the potential of the food-borne pathogen to survive additional low temperature cooking of food and raised concerns over the food safety of contaminated, temperature-abused foods. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Listeria; Sublethal cooking
1. Introduction Listeria monocytogenes is described as a gram-positive facultatively anaerobic, halotolerant rod with considerable variation in morphology dependent upon physiological and environmental conditions (Janda & Abbott, 1999). The pathogen has been reported to be the most frequently isolated Listeria species associated with human disease (Janda & Abbott, 1999). It has been isolated from a variety of foods and by-products and therefore, continues to be a serious public health concern (Beuchat, Brackett, Hao, & Conner, 1986; Watkins & Sleath, 1981). The United States alone, in 1999, had 92.2% of the 2518 illnesses from L. monocytogenes result in hospitalizations while accounting for 27.6% of the total foodborne deaths (Mead et al., 1999). At risk are the very young, the elderly, expectant mothers, and the immunocompromised. Growth at refrigerated temperatures and survivability in adverse environments, including those conditions that occur in minimally processed foods have made L. mono*
Corresponding author. Tel.: +1-215-836-3762; fax: +1-215-2336406. E-mail address: [email protected] (J.S. Novak).
cytogenes a challenge to control. The pathogen also has a higher reported heat resistance than many other nonspore-formers (Farber & Peterkin, 1991; Mackey & Bratchell, 1989). The versatility of L. monocytogenes to grow in a broad temperature range (2–45 °C) limits the effectiveness of restrictive measures following recent evidence of temperature acclimation and synthesis of protective proteins in the pathogen post-sublethal heat or cold shock (Bayles, Annous, & Wilkinson, 1996; Jorgensen, Panaretou, Stephens, & Knochel, 1996; Linton, Webster, Pierson, Bishop, & Hackney, 1992). This study was undertaken to examine the effects of refrigeration or freezing before and after exposure of L. monocytogenes to sublethal heating temperatures in ground beef simulating the conditions that might occur in prepared cook-chill foods reheated by consumers following chilled storage. Even though outbreaks of L. monocytogenes-related food poisoning have not been reported for undercooked ground beef, the potential does exist for contamination by multiple food handlers and extended chilled storage. These variations on pathogen survival are considered important from a food safety perspective with the increased marketing of mildly-cooked, quick-chilled foods for consumer convenience and time-economic home preparation.
0956-7135/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 6 - 7 1 3 5 ( 0 2 ) 0 0 0 4 8 - 8
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J.S. Novak, V.K. Juneja / Food Control 14 (2003) 25–30
2. Materials and methods
2.4. Refrigerated and then heat-treated
2.1. Bacterial strain
Prior to heat treatment at 60 °C, two replications of the study involving ground beef samples inoculated with L. monocytogenes Scott A in sterile filter bags were refrigerated at 4 °C for 24 h followed by heat treatment as for samples heat-treated alone. These samples were plated as previously stated. Other replicate samples following refrigeration were heat-shocked at 46 °C for 60 min and then immersed in the 60 °C water bath for heat treatment.
L. monocytogenes Scott A was obtained from the Eastern Regional Research Center (ERRC) culture collection. Cells were maintained in 20% glycerol at 80 °C and grown on tryptic soy agar (TSA) plates at 37 °C prior to study initiation. Freshly grown colonies were used to inoculate 10 ml brain heart infusion broth (BHIB) tubes and incubated at 37 °C for 20 h. Culture identity was confirmed by gram stain and incubated reactions read from gram-positive identification cards prepared for the Vitek Automicrobic System (BioMerieux Vitek, Inc., Hazelwood, MO). All microbiological media was prepared from dry chemical formulations (Difco Laboratories, Detroit, MI). 2.2. Ground beef, sample preparation and inoculation Stationary phase cultures of L. monocytogenes Scott A grown for 20 h at 37 °C in BHIB tubes were pelleted (5000 g for 15 min) and resuspended in 10 ml of 0.1% (w/v) peptone-water. Samples (100 g) of ground beef (7% fat) purchased from a local supermarket were irradiated (42 kGy/ 30 °C). These were then inoculated with 1 ml of culture and homogenized for 25 min at a high setting in 5 min intervals with a Stomacher lab-blender (Model 400, Spiral Systems, Inc., Cincinnati, OH). The inoculated ground beef (3 g portions) was then aseptically weighed into sterile filter bags, and heat-sealed. The sealed portions were then either heat-treated directly at 60 °C, refrigerated (4 °C ) and then heat-treated (60 °C), and then refrigerated (4 °C), frozen at (20 °C) and then heat-treated (60 °C), and then frozen at (20 °C).
2.5. Frozen and then heat-treated Prior to heat treatment, two replications of the study involving ground beef samples inoculated with L. monocytogenes Scott A in sterile filter bags were frozen at 20 °C for 24 h followed by heat treatment as before. These samples were plated as previously stated. Replicate samples following freezing were then heat-shocked at 46 °C for 60 min and finally immersed in the 60 °C water bath for heat treatment. 2.6. Heat-treated and then refrigerated Prior to plating, heat-treated and heat-shocked then heat-treated at 60 °C ground beef samples were refrigerated at 4 °C for 24 h followed by plating as previously described. 2.7. Heat-treated and then frozen Prior to plating, heat-treated and heat-shocked then heat-treated at 60 °C ground beef samples inoculated with L. monocytogenes Scott A were frozen at 20 °C for 24 h followed by plating as previously described.
2.3. Heat-treated samples
2.8. Determination of heating injury
Replicate samples of ground beef, inoculated with L. monocytogenes Scott A in sterile Stomacher filter bags (Nasco, Fort Atkinson, WI), were flattened to ensure even heat transfer, vacuum heat-sealed to 20 mbar in a vacuum packager (Multi-Vac Inc., Model A300, Kansas City, MO), and immersed in a 60 °C water bath. Samples were taken at 2.5 min time intervals for up to 35 min and cooled in an ice slurry. The heated bags were opened and 3 ml of phosphate-buffered saline with 0.05% Tween 20 (PBS-Tween; Sambrook, Fritsch, & Maniatis, 1989) were added and stomached for 2 min to form a 1:1 slurry. Dilutions were made in 0.1% (w/v) peptone-water and plated on TSA. The plates were incubated at 37 °C with counts taken 20 h later. A similar set of samples was first heat-shocked at 46 °C for 60 min and then immersed in the 60 °C water bath for heat treatment and sampled as for the other samples.
The percentage of heat injured survivors was determined using data from two replications for each experiment for all samples taken on TSA medium supplemented with 4.0% (w/v) NaCl. This selective plating medium restricted recovery of heat-injured survivors. The following formula was used to calculate percent injury for each time point: 1 ða=bÞ 100 ¼ %injury where a ¼ CFU/ml on TSA with 4% NaCl and b ¼ CFU/ml on TSA. 2.9. Determination of D10 -values All dilution platings were as previously described followed by incubation at 37 °C for 24 h. D10 -values (time for 10-fold reduction in viable cells), expressed in
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minutes, were determined by plotting the log10 number of survivors against time point samplings for 60 °C using Lotus 1-2-3 software. The line of best fit for survivor plots was determined by regression analysis (Ostle & Mensing, 1975); a regression equation for the type y ¼ a þ bx was derived, where b is the slope of the best straight line and, when inverted the sign changed from to þ to give the D10 -value in min. For each treatment, an estimate of the D10 -value and its corresponding standard error was calculated from the slopes of the regressions in the analysis of covariance and their standard errors using SAS (1989). 2.10. Qualitative analyses of protein expression Samples from the different treatment conditions (excluding the 60 °C exposure kill) were pelleted (11,000 g for 3 min), resuspended in buffer consisting of 25 mM Tris (pH 7.5), 1 mM EDTA (pH 7.5), 5 mM b-mercaptoethanol, 6 mM MgCl2 , and 30 mM NH4 Cl (Rheinberger, Geigenmuller, Wedde, & Nierhaus, 1988). The cell pellets were then stored at 70 °C until use. The protease inhibitor, phenylmethanosulfane fluoride (PMSF), was added to 1 mM as was dithiothreitol to provide a stable reducing environment prior to cell lysis. Cells were broken using a Vibracell model VC130 ultrasonic processor (Sonics and Materials, Inc., Newtown, CT) amplitude setting of 60, pulser at 1 s, for 2 min on ice. DNAse was then added to a concentration of 0.2 mg/ml. Protein concentrations were determined using a modification of the Lowry procedure (Lowry, Rosebrough, Farr, & Randall, 1951; Markwell, Haas, Bieber, & Tolbert, 1978). Cell lysates were resuspended in gel loading buffer (Sambrook et al., 1989) and heated at 100 °C in a boiling water bath for 5 min. Standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) procedures involved (4% (w/v acrylamide stacking gel, 15% (w/v) acrylamide resolving gel; 30:0.8 acrylamide:bis acrylamide) and a modified Tris-glycine buffer system (Lugtenberg, Meijers, Peters, van der Hoek, & van Alphen, 1975). Protein standards were obtained from Bio-Rad Laboratories (Hercules, CA). All sample lanes contained 20 lg of total protein. Following electrophoretic resolution, the gels were stained with Coomassie blue and photographed.
3. Results 3.1. Enhanced survival following sublethal heating temperatures Surviving L. monocytogenes Scott A heat-shocked in ground beef exhibited an elevated heat resistance at 60 °C as compared to nonheat-shocked controls (Fig. 1). Although heat injury was lower for heat-shocked sam-
Fig. 1. Thermal inactivation and survival of L. monocytogenes in ground beef and exposed to 60 °C over time. Symbols: ( ) control nonheat-shocked; ( ) heat-shocked at 46 °C for 60 min prior to heat treatments. The degree of heat injury measured on TSA þ 4% (w/v) NaCl plates as a percentage of the total plate counts on TSA medium is listed next to specific time points. Error bars designate standard deviations for means of two experimental trials.
ples, there was a parallel increase in cellular damage following growth on selective medium (TSA with 4% NaCl) when compared to controls grown on TSA medium (Fig. 1). Survival beyond 15 min at 60 °C was too low to give accurate estimations of heat injury. In order to examine the likelihood of L. monocytogenes survival in contaminated ground beef following mild heating, storage effects such as refrigeration at 4 °C or freezing at 20 °C for 24 h were examined. In one set of experiments, refrigeration or freezing effects on the Listeria-inoculated meat samples prior to heating at 60 °C were examined (Fig. 2A). Heat shock adaptations resulted in elevated survival profiles for the heat-shocked samples at 60 °C as compared with nonheat-shocked refrigerated or frozen samples (Fig. 2A). A similar trend was realized in the lowered degree of heat injury seen for heat-shocked versus nonheat-shocked samples (Fig. 2B). In another set of experiments, the refrigeration and freezing effects on ground beef inoculated with L. monocytogenes were recorded after cooking the meat at 60 °C over time (Fig. 3A). The heat-shocked samples refrigerated or frozen after minimal cooking at 60 °C produced indistinguishable survival curves with respect to one another, but significantly different profiles in comparison to refrigerated or frozen samples that were not heat-shocked, respectively (Fig. 3A). The heat shock effect appeared to be more pronounced when the refrigerated or frozen storage was incorporated after the heat treatments as compared to before. This finding would be significant with regards to the safety of precooked meals subsequently chilled for reheating. The degree of heat injury appeared comparable to that found for the experiments involving refrigerated or frozen
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Fig. 2. (A) Thermal inactivation and survival of L. monocytogenes in ground beef: (D) refrigerated at 4 °C for 24 h or () frozen at 20 °C for 24 h prior to heat exposures over time. Open symbols designate no prior heat shock, whereas closed symbols depict samples heat-shocked at 46 °C for 60 min. Error bars designate standard deviations for means of two experimental trials. (B) The degree of heat injury measured on TSA þ 4% (w/v) NaCl plates as a percentage of the total plate counts on TSA medium for L. monocytogenes Scott A inoculated ground beef exposed to 60 °C over time.
storage prior to heat shock (Figs. 2B and 3B). The frozen nonheat-shocked samples exhibited less injury following 10 min of heat treatment than any of the other treatments (Fig. 3B). This did not indicate a greater degree of repair to heat injury in the frozen samples as the survivors continued to decrease, concurrently (Fig. 3A). 3.2. Comparison of thermal resistance parameters at 60 °C D10 -values were significantly increased (P < 0:05) for the following heat-shocked samples: refrigerated prior to heat treatment (4.3) compared to 2.3 for nonheatshocked, frozen prior to heat treatment (3.6) compared to 1.7 for nonheat-shocked, and refrigerated following heat treatment (3.2) compared to 2.0 for nonheatshocked (Table 1). There were no significant differences at the P < 0:05 level among heat-shocked samples based
Fig. 3. (A) Thermal inactivation and survival of L. monocytogenes in ground beef: (D) refrigerated at 4 °C for 24 h or () frozen at 20 °C for 24 h after heat exposure over time. Open symbols designate no prior heat shock, whereas closed symbols depict samples heat-shocked at 46 °C for 60 min. Error bars designate standard deviations for means of two experimental trials. (B) The degree of heat injury measured on TSA þ 4% (w/v) NaCl plates as a percentage of the total plate counts on TSA medium for L. monocytogenes Scott A inoculated ground beef exposed to 60 °C over time.
Table 1 Summary of thermal characterizations Sample conditions
D10 -value at 60 °Ca
Control Heat-shocked control Refrigerated prior to heat treatments Refrigerated and heat-shocked prior Frozen prior to heat treatments Frozen and heat-shocked prior Refrigerated following heat treatments Heat-shocked and refrigerated following Frozen following heat treatments Heat-shocked and frozen following
2:1 0:2E 4:3 0:4A 2:3 0:2E 4:3 0:5AB 1:7 0:1F 3:6 0:2ABC 2:0 0:2E 3:2 0:3CD 2:6 0:5DE 3:4 0:3BCD
a
D10 -values in minutes at 60 °C represent the results from two separate experimental trials and were calculated as described in Section 2. These estimates were then used to form a series of pairwise comparisons. The results agreed with the output from the analysis of covariance used to test the homogeneity of the slopes. Any two D10 values with no letter in common are significantly different by the pairwise t-test technique at the (P < 0:05) level of confidence.
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Fig. 4. SDS-PAGE analysis of protein turnover in response to experimental conditions detailed in Section 2. Lanes: (1) and (10) protein molecular weight standards; (2) nonheat-shocked control; (3) heatshocked control; (4) refrigerated and nonheat-shocked; (5) refrigerated prior to heat shock; (6) frozen and nonheat-shocked; (7) frozen prior to heat shock; (8) heat-shocked and then refrigerated; and (9) heatshocked and then frozen.
on the method of storage (refrigeration at 4 °C for 24 h or freezing at 20 °C for 24 h) prior to or post-heat treatment (Table 1). However, it was evident that heat shock effects were lessened with respect to the control (4.3), if samples were refrigerated after heat treatment (3.2) or frozen after heat treatment (3.4). Therefore, storage conditions were found to influence the protective heat shock effects in L. monocytogenes Scott A to some extent. 3.3. Protein expression in response to treatment conditions All of the heat-shocked samples exhibited similar increases in proteins approximating 55, 44, 32, 27, and 24 kDa despite refrigeration or frozen storage (Fig. 4, lanes 3, 5, 7, 8, and 9). The nonheat-shocked samples also contained proteins approximating 33, 23, 22, 20, and 19 kDa that were repressed, denatured, or turnedover more rapidly under the heat-shock conditions (Fig. 4, lanes 2, 4, and 6). All samples contained large, but similar quantities of a protein product at 40 kDa that was not influenced by the heat shock or storage conditions.
4. Discussion There have been previous reports of heat shock effects on thermotolerance in L. monocytogenes (Linton, Pierson, & Bishop, 1990; Linton et al., 1992), the incidence of protective heat shock proteins (Jorgensen et al., 1996), changes in L. monocytogenes thermotolerance affected by growth conditions and heating menstruum (Jorgensen, Hansen, & Knochel, 1999), and even pro-
29
tective effects due to cold stress proteins (Bayles et al., 1996). This represents the first report to our knowledge of cold storage and the subsequent survival attributes of L. monocytogenes in a meat product when used in combination with mild sublethal heat treatment used before or after storage for 24 h. All of these are possible consumer handling practices awaiting cook-chill foods. Heat shock effects (46 °C for 60 min) on L. monocytogenes in ground beef were statistically significant in increasing the subsequent survival characteristics of the pathogen with respect to D10 -values and reduced percent cellular injury at 60 °C. Refrigerated and frozen storage did not lessen these effects. This finding should have significant implications in the safety of cook-chilled products which are increasing, being produced and marketed in response to consumer demands. A very likely scenario involves pre-market contamination of heat-treated meat with L. monocytogenes. The heattreated meat can activate adaptations in L. monocytogenes to enable the foodborne pathogen to survive a second low temperature heating following chilled storage and prior to ingestion by the consumer. Documentation supports the belief that nearly all organisms respond to sublethal stress with the production of proteins to counteract and protect against subsequent stress (Cheville, Arnold, Buchrieser, Cheng, & Kaspar, 1996; Lindquist & Craig, 1988; Schlesinger, Ashburner, & Tissieres, 1982; Volker et al., 1994). Many of these heat shock proteins are molecular chaperones, such as GroESL gene products in Escherichia coli, that aid in proper folding or removal of damaged proteins (Georgopoulos & Welch, 1993). Although several proteins have been shown to be expressed in L. monocytogenes following 46 °C for 60 min at 55, 44, 32, 27, and 24 kDa an equal number of proteins at 33, 23, 22, 20, and 19 kDa appear repressed. Metabolic reactions in all living organisms exist in balance between anabolic and catabolic processes. A complete evaluation of heat adaptations in L. monocytogenes Scott A requires the consideration of repressed products that have not been previously identified and may negatively impact survival as well as those positively regulated. Of concern was the one log10 CFU/ml increase in the initial counts of L. monocytogenes in ground beef refrigerated or frozen after heat treatment as compared to heat treatment before. A possible explanation might have involved the release of intracellular molecules from cells weakened or killed by the heat treatment and followed by refrigeration or freeze-fracture of cell walls and membranes resulting in the protection of surviving cells of L. monocytogenes which were less stressed. Much of the safety associated with food preparations involves proper handling and temperature controls pre- and post-cooking. Therefore the results of this study increased concerns over the safety of cook-chill foods if
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such foods were contaminated with a psychrotrophic pathogen such as L. monocytogenes Scott A.
Acknowledgements We wish to thank Ms. Kenyetta J. Chaney for technical assistance in performing this study and Dr. John Phillips for statistical analyses of the data obtained.
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