Department of Animal and Food Sciences, Texas Tech University, Box 42141, Lubbock, Texas 79409, USA

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1 1389 Journal of Food Protection, Vol. 67, No. 7, 2004, Pages Copyright, International Association for Food Protection Validation of Time and Temperature Values as Critical Limits for Salmonella and Background Flora Growth during the Production of Fresh Ground and Boneless Pork Products J. E. MANN, L. SMITH, AND M. M. BRASHEARS* Department of Animal and Food Sciences, Texas Tech University, Box 42141, Lubbock, Texas 79409, USA MS : Received 13 June 2003/Accepted 14 January 2004 ABSTRACT To provide pork processors with valuable data to validate the critical limits set for temperature during pork fabrication and grinding, a study was conducted to determine the growth of Salmonella serotypes and background flora at various temperatures. Growth of Salmonella Typhimurium and Salmonella Enteritidis and of background flora was monitored in ground pork and boneless pork chops held at various temperatures to determine growth patterns. Case-ready modified atmosphere packaged ground pork and fresh whole pork loins were obtained locally. Boneless chops and ground pork were inoculated with a cocktail mixture of streptomycin-resistant Salmonella to facilitate recovery in the presence of background flora. Samples were held at 4.4, 7.2C, and 10 C and at room temperature (22.2 to 23.3 C) to mimic typical processing and holding temperatures observed in pork processing environments. Salmonella counts were determined at regular intervals over 12 and 72 h for both room and refrigeration temperatures. No significant growth of Salmonella (P 0.05) was observed in boneless pork chops held at refrigeration temperatures. However, Salmonella in boneless pork chops held at room temperature had grown significantly by 8 h. Salmonella grew at faster rates in ground pork. Significant growth was observed at 6, 24, and 72 h when samples were held at room temperature, 10 C, and 7.2 C, respectively. No significant growth was observed at 4.4 C. Background flora in ground pork samples increased significantly after 10 h at room temperature and after 12 h for samples held at 10 and 7.2 C. Background flora in samples held at refrigeration temperatures did not increase until 72 h. Background flora in the boneless chops increased significantly after 6 h at room temperature and after 24 h when held at 10 and 4.4 C. These results illustrate that meat processors can utilize a variety of time and temperature combinations as critical limits to minimize Salmonella growth during production and storage of raw pork products. Salmonellosis is an important cause of foodborne illness. Approximately 95% of human Salmonella infections can be attributed to foodborne exposure (8). Foodborne infection with nontyphoid Salmonella usually results in a self-limiting gastroenteritis within 12 to 72 h of ingestion. In rare instances, gastroenteritis can lead to systemic infections such as septicemia or to chronic disease syndromes such as reactive arthritis or Reiter s syndrome (9, 13). Surveillance conducted for the FoodNet program at the Centers for Disease Control and Prevention indicated an increase in Salmonella infections in 2001; Salmonella surpassed Campylobacter as the most common cause of foodborne illness (4). Additionally, Salmonella is the leading cause of death from foodborne bacterial illnesses. Although less prevalent in pork than in poultry products, this pathogen is still of great concern to pork processors. Recent U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) regulatory requirements, including the implementation of the pathogen reduction and hazard analysis and critical control point (HACCP) systems rule (11), have led to an increased emphasis on scientific evaluations of production practices to determine safety limits. Included in the pathogen reduction rule are provisions * Author for correspondence. Tel: ; Fax: ; mindy.brashears@ttu.edu. for a Salmonella performance standard based on a national baseline determined by FSIS (12). Although performance standards have been determined for market hog carcasses, there are currently no performance standards for fresh ground or fabricated pork products. Numerous studies have revealed the presence of Salmonella on pork carcasses and on finished retail products. Duffy et al. (5) found a mean Salmonella prevalence of between 5.8 and 9.6%, depending on product and production plant type, for whole muscle retail and ground pork products. In addition, FSIS collection data indicate a national Salmonella baseline of approximately 30% in fresh pork sausage (12). The presence of spoilage organisms on these types of products is also well established (3, 6). Control of the growth of Salmonella and other pathogens should also decrease the growth of spoilage organisms, which should in turn increase product quality and extend product shelf life. Control of time and temperature is especially important for the production of a safe final product. For many raw meat products, temperature control throughout the process is the only viable critical control point available for microbial growth. Because of a lack of pertinent scientific data, there has been debate over where to properly set critical limits to adequately control microbial growth during processing. In previous studies, Salmonella Typhimurium was

2 1390 MANN ET AL. J. Food Prot., Vol. 67, No. 7 able to grow at temperatures as low as 2 C in minced chicken (2). Modern meat processing environments generally exceed this temperature. Typically, refrigerated processing areas are maintained at temperatures ranging from 4.4 to 10 C. In some instances, raw product is even produced at ambient temperature. Critical limits are often established based on these values, usually in association with a time limit. With this research, we examined the effects of some commonly used product holding temperatures and times at these temperatures on the microbial safety of ground and sliced boneless pork to provide pork processors with time and temperature combinations that can be used during processing to produce a safe product. MATERIALS AND METHODS Experimental design. Ground pork samples were surveyed for microbial counts at 11 time intervals, and boneless pork chop samples were collected at 9 intervals. Three replications were performed for each experiment. Microorganisms. One isolate each of Salmonella Typhimurium ATCC and Salmonella Enteritidis phage 13, each resistant to 1,000 g/ml streptomycin, were obtained (Department of Food Science & Technology, University of Nebraska, Lincoln) and grown at 37 C for 18 h in tryptic soy broth (TSB) supplemented with 1,000 g/ml streptomycin. Antibiotic-resistant organisms, as validated in a previous study (1) were used to facilitate recovery in the presence of background flora. Resistance was developed (as described by Amezquita et al. (1)) by growing nonresistant strains in TSB (37 C) containing increasing serial concentrations of the antibiotic, beginning with 50 g/ml and doubling the concentration after each transfer. Generally, incubation times were 24 to 48 h, but additional incubation times were used when slow growth occurred at various concentrations. Before experimentation, cultures were removed from the TSB by centrifugation and serially diluted in buffered peptone water (BPW) to yield a population of 10 6 CFU/ml in the broth. Sample preparation. Fresh ground pork was obtained at a local retail establishment. A new sample was obtained for each replication. A portion of the meat was reserved to serve as the uninoculated control to determine growth of background flora on nonselective medium. The diluted Salmonella cocktail was thoroughly mixed into the remaining meat by placing inoculated meat into a sterile stomacher bag and kneading for 2 min to yield a final concentration of approximately 10 4 Salmonella cells per g of pork. Each treatment was further divided into approximately 100-g portions, which were aseptically placed into sterile whirlpak bags and placed on ice. Eight bags each of the inoculated sample and the uninocualted control were placed in refrigerated incubators at 4.4, 7.2, and 10.0 C. An additional six bags of each treatment were held at room temperature (22.2 to 23.3 C). Room temperature was measured using a calibrated thermometer in a glass of water near the sample holding area. One sample held under each refrigerated temperature was examined at 1, 4, 8, 12, 24, 32, 48, and 72 h. Samples held at room temperature were examined at 4, 6, 8, 10, and 12 h. In addition, a sample of each treatment was examined at time 0 to determine baseline microbial counts. Three replications were performed. Whole fresh boneless pork loins were obtained at the Texas Tech University meat laboratory. Three separate sets of loins were obtained for each replication. Loins were sliced into approximately 2-cm-thick portions. Boneless chops were dipped in the diluted Salmonella cocktail to yield a concentration of approximately 10 4 Salmonella cells per cm 2. Inoculated samples were placed on sterile drying racks in a biological safety cabinet for 20 min to allow bacterial attachment and then were aseptically placed in whirl-pak bags. A portion of the chops was reserved as an uninoculated control to determine the growth of the background flora. Six of each of the inoculated and control samples were placed in refrigerated incubators at 4.4, 7.2, and 10.0 C. Five additional samples of each were held at room temperature (22.2 to 23.3 C). Samples held under each refrigerated temperature were examined at 4, 8, 12, 24, 48, and 72 h. Samples held at room temperature were examined at 4, 6, 8, 10, and 12 h. In addition, a sample of each treatment was examined at time 0 to determine baseline microbial counts. Three replications were performed. Microbiological analysis. For each treatment and holding temperature at each of the specified times, a randomly chosen sample was placed on ice prior to analysis. For each ground pork sample, 11 g of sample and 99 ml of sterile BPW were placed into a sterile stomacher filter bag and stomached for 1 min to mix the contents. For each boneless chop sample, 99 ml of BPW was added to the whirl-pak bag, which was then stomached for 1 min. If necessary, samples were further diluted in sterile BPW. Each dilution was spiral plated using an Autoplate 4000 (Spiral Biotech, Inc., Norwood, Mass.). Samples were plated onto two different media: tryptic soy agar (TSA) plus 1,000 g/ml streptomycin to determine streptomycin-resistant Salmonella counts and TSA without streptomycin to determine total aerobic bacterial counts. Plates were incubated 24 to 48 h at 37.0 C, and colonies were counted using a Q-Count automatic plate counter (Spiral Biotech). Statistical analysis. Each experiment was carried out in triplicate. At each sampling time, samples were plated in duplicate for each dilution. Spiral plate counts were converted to units of log CFU per gram for ground pork and log CFU per square centimeter for boneless chops prior to statistical analysis. Data were analyzed using the mixed procedure of SAS (10). Mean separations were performed utilizing the least-squares means method. RESULTS Ground pork: Salmonella. At time zero (T 0), 3.95 log CFU/g streptomycin-resistant Salmonella were present in the inoculated samples (Table 1). There were no streptomycin-resistant bacteria recovered from the uninoculated controls at any sampling time. Inoculated samples held at room temperature showed no significant increase in Salmonella (P 0.05) for the first 4 h; however, a significant increase (P 0.05) was observed at 6 h. Significant increases in counts (P 0.05) were observed at 24 and 72 h for samples held at 10.0 and 7.2 C, respectively. For the duration of this study, no significant increase in Salmonella counts (P 0.05) was observed for samples held at 4.4 C. Ground pork: aerobic bacteria. A significant increase in aerobic plate count (APC) (P 0.05) was observed at 8 h for inoculated samples held at room temperature (Table 1), whereas uninoculated control samples showed a significant increase (P 0.05) at 10 h (Fig. 1). The APC of the inoculated samples also include counts for the Salmonella, whereas those of the uninoculated samples represent only the background flora. Samples held at 10 and 7.2 C showed a significant increase in APC at 24 h for both

3 J. Food Prot., Vol. 67, No. 7 HOLDING TIME AND TEMPERATURE AS CRITICAL LIMITS FOR PORK PROCESSING 1391 TABLE 1. Microbial growth in ground pork held at refrigerated and room temperatures Population (log CFU/g) a Time (h) Salmonella APC, inoculated AB 4.48 B 4.91 C 5.45 D 5.97 E 3.92 A 3.89 A 4.32 B 4.44 B 4.94 C 5.29 C 3.85 A 4.11 AB B B 4.62 BC 3.86 A 3.91 A 4.07 A 3.98 A 4.02 A 4.87 A 3.98 A 5.07 A 5.18 A 5.98 B 6.22 BC 6.8 C 5.12 A 5.17 A 4.88 A 5.51 A 6.47 B 6.9 BC 7.19 C 7.26 C 5.07 A 5.12 A 4.98 A 5.43 A 5.93 B 6.3 BC 6.74 CD 7.14 D SE A 4.83 A 4.76 A 5.04 A 5.5 B 5.5 B 6.1 C 6.42 C a Room temperature (RT) is 22.2 to 23.3 C. Different letters within columns indicate a significant difference in microbial counts (P 0.05). inoculated and control samples. At 4.4 C, a significant increase in APC was observed at 24 h for inoculated samples, but control samples did not show a significant increase in APC until 72 h. Boneless pork chops: Salmonella. At T 0, 4.10 to 4.45 log CFU streptomycin-resistant Salmonella per ml of diluent were present in the inoculated samples (Table 2). There were no streptomycin-resistant bacteria recovered from the uninoculated controls at any sampling time. Inoculated samples held at room temperature showed no significant increase in Salmonella (P 0.05) for the first 6 h. For the duration of this study, no significant increase in numbers of Salmonella was observed for samples held at refrigerated temperatures. Boneless pork chops: aerobic bacteria. A significant increase (P 0.05) in APC was observed at 6 h for both inoculated samples (Table 2) and control samples (Fig. 2) held at room temperature. Inoculated samples held at refrigerated temperatures showed a significant increase in APC at 24 h. Significant growth was observed at 24 h for FIGURE 1. Aerobic plate counts of uninoculated control ground pork held at refrigerated temperatures and room temperature (RT). No RT samples were collected after 12 h. Significant increases (P 0.05) occurred between 8 and 10 h at RT, between 12 and 24 h at 10 and 7.2 C, and between 24 and 32 h at 4.4 C. control samples held at 10 and 4.4 C, whereas those held at 7.2 C showed significant growth at 48 h. DISCUSSION This study was conducted because there is a lack of information on the growth of Salmonella in pork products. Processors of fresh pork products need this information to set critical limits for temperature-related critical control points in their HACCP plans. Data from this study can provide scientific validation for various time and temperature combinations. In ground pork samples, there were no increases in Salmonella loads during the 72 h of the study for samples held at 4.4 C. Therefore, holding the samples at this temperature is sufficient to prevent growth of the pathogen. These data agree with information published by Mackey and Roberts (7), who reported that no Salmonella growth occurred at temperatures at or below 7 to 8 C in intact beef. Generation times of Salmonella were 5.2 h at 12.5 C and 2.9hat15 C in beef samples, with no growth occurring at 7to8 C. After 24 h, the APC of both the inoculated and the uninoculated samples increased significantly, indicating that psychrotrophic organisms are typically present and will grow in ground pork samples over time. These results indicate that processing ground pork at 4.4 C is acceptable during a normal processing day and no Salmonella growth should occur. Salmonella populations did increase in ground pork samples held at 10 and 7.2 C after 24 and 32 h, respectively. However, a typical processing day is unlikely to exceed 24 or 32 h. Holding samples at or near 10 C for 24 h should not result in significant increases in the pathogen loads. The APCs on these samples increased between 12 and 24 h, depending on the temperature and inoculation temperatures. However, processing at either 7.2 or 10 C would be acceptable, and no significant growth of Salmonella or increase in APC for the product would be expected

4 1392 MANN ET AL. J. Food Prot., Vol. 67, No. 7 TABLE 2. Microbial growth on boneless pork chops held at refrigerated and room temperatures Population (log CFU/ml diluent) a Time (h) Salmonella APC, inoculated SE 4.2 A 4.48 AB 4.74 BC 4.88 BC 5.13 C A 4.17 A 4.19 A 4.46 A 4.5 A 4.47 A A 4.37 A 4.6 A 4.48 A 4.51 A 4.58 A 4.53 A A 4.14 A 3.26 A 4.34 A 4.17 A AB 5.92 B 6.42 C 6.87 CD 7.08 D A 5.43 A 5.7 A 6.71 B 7.56 C 7.61 C A 5.67 A 5.84 AB 5.64 A 6.35 BC 7.26 D 7.47 D A 5.61 AB 5.36 A 6.02 BC 6.48 C 7.07 D 0.33 a Room temperature (RT) is 22.2 to 23.3 C. Different letters within columns indicate a significant difference in microbial counts (P 0.05). as long as the processing day or the time spent in the processing room held at these temperatures did not exceed 12 h. Accelerated Salmonella growth was observed in ground pork compared with that on boneless chops at refrigeration temperatures. This difference was expected because the greater surface area of the ground pork is more conducive to microbial growth and distribution throughout the product. Under refrigerated processing conditions of 10 C, 1 log increase in numbers of Salmonella at 72 h for boneless chops occurred. However, the significant increases observed in the APC for chops held at 7.2 and 10 C indicate that even during pork processing at temperatures 10 C, the time the product spends in the processing room should be 12 h. Some smaller processors operate at room temperature when processing ground pork and other pork products. Therefore, it is important to determine critical limits at room temperature (22 to 23 C). For both ground pork and pork chops, there were significant increases in Salmonella populations after 6 h, and after 8 h there were increases in APCs in both inoculated and uninoculated samples. Therefore, at room temperature a processor producing a ground pork product should take measures to ensure that the product enters a refrigerated area within 6 h because microbial growth will start to occur at that time. Growth could start FIGURE 2. Aerobic plate counts of uninoculated control boneless pork chops held at refrigerated temperatures and room temperature (RT). No RT samples were collected after 12 h. Significant increases (P 0.05) occurred between 4 and 6 h at RT and between 12 and 24 h at 10, 7.2, and 4.4 C. at elevated temperatures and then stop under refrigeration conditions. Exposing pork to elevated temperatures an additional time after refrigeration could result in accelerated growth, but this scenario was not evaluated in the present study. After production, the product should remain at temperatures 4.4 C to ensure safety. Numerous time and temperature combinations can be used as critical limits for Salmonella growth and spoilage due to other bacteria during the processing and storage of fresh and ground pork. ACKNOWLEDGMENT The authors acknowledge the National Pork Producers Council for financial support of this study. REFERENCES 1. Amezquita, A., M. M. Brashears, and J. Stratton Recovery of injured pathogens on selective and non-selective media. J. Food Prot. 64: Baker, R. C., R. A. Qureshi, and J. H. Hotchkins Effect of an elevated level of carbon dioxide containing atmosphere on the growth of spoilage and pathogenic bacteria. Poult. Sci. 65: Borch, E., M. L. Kant-Muermans, and Y. Blixt Bacterial spoilage of meat and cured meat products. Int. J. Food Microbiol. 33: Centers for Disease Control and Prevention Preliminary FoodNet data on the incidence of foodborne illnesses selected sites, United States, Morb. Mortal. Wkly. Rep. 51(15): Duffy, E. A., K. E. Belk, J. N. Sofos, G. R. Bellinger, A. Pape, and G. C. Smith Extent of microbial contamination in United States pork retail products. J. Food Prot. 64: Gill, C. O., and J. Bryant The contamination of pork with spoilage bacteria during commercial dressing, chilling and cutting of pig carcasses. Int. J. Food Microbiol. 16: Mackey, B. M., and T. A. Roberts Growth of Salmonella on chilled meat. J. Hyg. Camb. 85: Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe Food-related illness and death in the United States. Emerg. Infect. Dis. 5: Samuel, M. P., S. H. Zwillich, G. T. Thomson, M. Alfa, K. B. Orr, D. C. Brittain, J. R. Miller, and P. E. Phillips Fast food arthritis: a clinico-pathologic study of post-salmonella reactive arthritis. J. Rheumatol. 22: SAS Institute, Inc SAS/STAT user s guide, version Statistical Analysis System Institute, Cary, N.C.

5 J. Food Prot., Vol. 67, No. 7 HOLDING TIME AND TEMPERATURE AS CRITICAL LIMITS FOR PORK PROCESSING U.S. Department of Agriculture Pathogen reduction: hazard analysis and critical control point (HACCP) systems: final rule. Fed Regist. 61: U.S. Department of Agriculture Contamination with microorganisms; pathogen reduction performance standards for Salmonella, final rule. Fed. Regist. 63: Ziprin, R. L., and M. H. Hume Human salmonellosis: general medical aspects, p In Y. H. Hui, M. D. Pierson, and J. R. Gorham (ed.), Foodborne disease handbook, vol. 1. Bacterial pathogens. Marcell Dekker, Basel, Switzerland.