The objectives of this project are: 1) validate the effect of thermal processing interventions on the survival of Listeria monocytogenes, Salmonella, and shiga-toxin producing E. coli (STEC) in roast beef, turkey deli-breast, and boneless hams; 2) use the thermal destruction data to develop scientifically- validated, easy-to-use time-temperature tables as tools for assuring regulatory compliance and pathogen destruction for ready-to-eat roast beef, turkey deli-breast, and boneless hams and 3) develop the basis for a series of time-temperature tables organized in product categories that will cover the vast array of ready-to-eat meat products and thermal processes in the U.S. meat industry. <P>
Currently, U.S. processors of ready-to-eat meat and poultry products have limited science-based support documentation to validate pathogen destruction during thermal processing. USDA, FSIS Appendix A is widely used as validation support for thermal processes, but its time-temperature tables and humidity requirements were originally developed and validated for Salmonella in roast, cooked, and corned beef. However, Appendix A is routinely applied to a wide array of products including hams, hot dogs, luncheon meats and jerky, to name a few. The widespread use of a procedure originally developed only for roast beef has raised question about its appropriateness for non-roast beef products or for pathogens other than Salmonella. <P>
This study will focus on developing new Appendix A style time-temperature tables for non-beef RTE products such as turkey deli-breast and boneless ham. We will also compare the baseline results from the original work that was conducted to develop Appendix A to the results of this study for Salmonella in roast beef and confirm the validity of its effectiveness for pathogenic E. coli and L. monocytogenes strains.<P> The study will be divided into two phases. Phase 1 will determine D- and z-values for L. monocytogenes, Salmonella, and STEC using ground meat mixtures. Phase 2 will validate the D-values identified in Phase 1 using commercial products representing different product categories.
Phase 1: Determination of D- and z-values in model (ground) meat: April 2012: Three low-fat products selected for testing were categorized by moisture level and inclusion of sodium nitrite and will include 1) roast beef (low moisture, uncured), 2) deli-style turkey breast (high moisture, uncured) and 3) boneless ham (high moisture, cured). The formulas for each product were selected to represent the worst-case for thermal destruction in its category, and this thermal destruction data will be used as the basis for new Appendix A style time-temperature tables for those product categories.
Ground roast beef (containing 1.0% salt, 0.35% sodium phosphates, 0.75% sugar, 20% water), ground ham (containing 2.5% salt, 1.65% sugar, 0.35% sodium phosphates, 547 ppm Na erythorbate, 200 ppm Na nitrite, 20% water), or ground turkey breast (containing 1.5% salt, 1.5% dextrose, 20% water) were inoculated with 8 log CFU/g L. monocytogenes or Salmonella (5-strain mix) or STEC (7-strain mix). One-g portions (0.5-1.0 mm in moisture-impermeable vacuum pouches) were heated at one of four temperatures (54.4, 60, 65.6, or 71.1°C; 130, 140, 150 and 160°F, respectively) in a water bath. Triplicate samples were removed and immediately chilled to ≤4°C when meat reached target temperature and at seven additional times. Surviving L. monocytogenes, Salmonella, or STEC were enumerated using Modified Oxford, XLD, or Sorbitol MacConkey agar base, respectively, with thin layer overlay of nonselective media to enhance recovery of injured cells. Each study was replicated twice.
Data has been collected for log reduction of pathogens for all treatment combinations (3 product types x3 pathogens; 4 temperatures) over time to attain time-temperature-log reduction relationships. Data has been analyzed using standard linear regression statistical methodology to generate linear regressions for each product type, pathogen and temperature combination incorporating at least 4 time-points for each combination to allow for generation of D- and z- values. To account for initial come-up time, to first time-point (0 seconds), time adjustments were made to all subsequent time-points. For example, in Figure 1, “0” time was adjusted 8 seconds for replication 1 and 11 seconds for replication 2 to signify the actual time the internal sample temperature reached the pre-determined experiment temperature. In total, 36 graphs were developed (data not shown) plotting pathogen log reduction over time. Figure 1 provides an example of a standard linear regression calculated for a ham/Salmonella treatment combination at 60°C (140°F) where populations of Salmonella were determined at 0, 120, 240, 360, and 480 seconds. From this linear regression, a calculated D- value of 90.1 seconds was less than the D- value of 102.6 seconds calculated from the USDA, FSIS Appendix A for the same temperature. This would suggest Appendix A is a conservative predictor of Salmonella reduction, even when raw materials sources were not the same. Similarly, the D-values calculated for Salmonella in turkey were less than those identified in the USDA-FSIS Time-Temperature Tables for Cooking Ready-To-Eat Poultry Products. Table 1 displays calculated D- values for all roasts beef, deli-style turkey breast, boneless ham, L. monocytogenes, Salmonella, and STEC combinations.
In all product types (roast beef, ham and turkey), inactivation rates for STEC were similar to Salmonella at 60, 65.6, or 71.1°C, (140, 150 and 160°F, respectively) and were comparable to or less than times reported in Appendix A. In contrast, L. monocytogenes showed greater thermotolerance than Salmonella and STEC under all conditions. For example, >5-log reduction of Salmonella and STEC in turkey was achieved instantaneously at 71.1°C, whereas L. monocytogenes was inactivated within 10 seconds. At 60°C (140°F), >5-log reduction of L. monocytogenes required 30 and 50 minutes in turkey and ham, respectively, as compared to <12 minutes for Salmonella and STEC. At the lowest temperature tested (54.4°C/130°F), >5-log reduction of Salmonella, STEC and L. monocytogenes in all product types was achieved in <2, 2.8 and 4.6 hours, respectively.
Results from Phase I support Appendix A as an acceptable tool for Salmonella and STEC lethality; as expected L. monocytogenes was more thermotolerant than Salmonella or STEC. Since Phase I utilized a model type approach using one gram meat samples for all lethality investigations, only immediate lethality was measured while integrated lethality was not accounted for nor considered to determine an actual and/or expected total lethality. Therefore, care and an understanding of appropriate use must be taken when interpreting Phase I data and results to recognize this important thermal processing component.
August 2012: Phase I data was reviewed and analyzed using an exponential non-linear regression model determined most appropriate for fitting the microbial data and providing a conservative and realistic output. Z- values were calculated for each treatment combination by plotting D- values against temperature.
Project Status- Phase 2: Validation of D- and z-values in commercial-type products. To validate the results found in Phase 1 and explore the impact of integrated lethality, all three products (roast beef, deli-style turkey breast, and boneless ham) will be manufactured following commercial production practices.
After manufacture at the meat science and muscle biology lab, chunked and formed roast beef and turkey deli-breast product will be inoculated with the target pathogen and stuffed into 4.5” diameter impermeable (turkey and beef) or permeable (ham) casings. Chubs of beef and turkey product will be thermally processed in a combi-oven (Alto Shaam) using smokehouse schedules similar to those commonly used in the meat industry (i.e. ramped steam cycles). Chubs of ham product will be thermally processed in a combi-oven (Alto Shaam) or a smokehouse oven (Alkar) using a smokehouse schedule similar to that commonly used in the meat industry containing appropriate wet bulb, dry bulb and relative humidity configured temperature ramping steps. Internal temperature probes (iButtons; DS1922T-F5) will be placed at three locations inside the chub to monitor the surface, midpoint (to center), and internal temperature. Temperature data will be collected each minute for use in integrated lethality calculations; humidity of the oven will also be recorded (as appropriate). Inactivation of pathogens will be determined by enumerating, with the same techniques previously described, at the pre- determined time intervals.
In the original experimental design, the pathogen with the highest D-value for each of the three products was to be chosen for validation. However, to benefit the overall product applicability and industry impact of the study, the experimental design was modified with the following treatment combinations. For each treatment combination, initial (time 0) and at least 3 sampling points will be chosen to document additional integrated lethality for the target internal temperature. Each treatment combination will be manufactured/tested in duplicate.
<OL> <LI> Deli-style turkey breast – Salmonella – 160°F final internal temperature </LI> <LI>
Roast beef – Salmonella – 130°F final internal temperature</LI> <LI>
Roast beef – Salmonella – 145°F final internal temperature</LI> <LI>
Roast beef – Salmonella – 160°F final internal temperature</LI> <LI>
Roast beef – STEC – 130°F final internal temperature</LI> <LI>
Roast beef – STEC – 145°F final internal temperature</LI> <LI>
Roast beef – STEC – 160°F final internal temperature</LI> <LI>
Boneless ham – L. monocytogenes – 145°F final internal temperature</LI> <LI>
Boneless ham – L. monocytogenes – 160°F final internal temperature</LI> </ol>
August 2012: A preliminary test was conducted to confirm adequacy of experimental methodology and laboratory procedures. From this, pathogen inoculation techniques were modified and confirmed to ensure uniform distribution of inoculums.
Currently, we have completed replication 1 for the deli-style turkey breast. Turkey breast was inoculated with approximately 8 log cfu/g Salmonella, stuffed into 4.5” moisture impermeable casings, fitted with I-button time/temperature data logging probes in three chubs (each chub had data loggers located in the geometric center, surface and midway between center and surface), whereas a third chub was fitted with a FlashLink data logger with an LCD display to monitor real time core temperature. Chubs were thermal processed using a typical steam ramp-cook thermal processing schedule (130°F for 30 min; 145°F for 75 min; 160°F for 30 min; and 175°F until internal temperature of 160°F). An additional time/temperature data logging probe was also placed inside the Alto Shaam oven to measure ambient temperatures. A single inoculated chub was removed from the oven at each sampling interval when target internal temperatures of 130, 145, and, 160°F were reached. After removal at each time/temperature point, a 2 inch piece from the each end of the chub was removed and discarded. The remaining portion of the 12 inch long chub was cut into three, 4-inch long pieces, and 25 g core samples (4” long x 0.75” diameter) were removed from the geometric center and midpoint of each section using a metal bore, parallel to the length of the chub. Surface samples were extracted by first removing the casing and shaving 2-3 mm thick pieces from the outside surface. For each chub, three surface, three midpoint, and three center samples were collected for microbial enumeration. Samples were immediately chilled on ice and enumerated to determine cfu/g Salmonella. In addition, after the 160°F time point was achieved, a 4th remaining chub was removed from the oven and chilled by placing in an ice-bath for 2 hours followed by ambient cooling under refrigeration (36°F) to determine additional integrated lethality during stabilization.
Integrated lethality considerations are important for an overall understanding of pathogen reduction. For Trial 1 Turkey, when the internal temperature reached 130°F, a 4.12, 1.31, and 0.58 log cfu/g reduction for Salmonella was observed at the surface, midpoint, and center of the chub, respectively. However, as the internal temperature of the center increased to 145°F, at least a 6 log cfu/g reduction (conservative limit of detection by direct plating) occurred, regardless of location. No Salmonella was detected by direct plating for any of the samples removed from the chubs cooked to 160°F (either immediately sampled/cooled or chilled overnight).
Project Next Steps: D- and Z- value validation and integrated lethality investigation for the remaining treatment combinations (2-9) will continue to take place for Salmonella, STEC, and L. monocytogenes in roast beef and boneless ham. Thermal processing profile data will be collected for each treatment combination. Boneless ham treatment combinations will provide additional insight on the impact evaporative cooling will have on overall lethality, but especially surface log reductions. Data will be analyzed to begin exploring how new time-temperature tables and other thermal processing tools may be developed as a result of this research.