Validation of D- and z-values for Salmonella, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli in processed meat products

Strain selection

As a validation study, the strains used in these experiments were identical to those used by McMinn et al. (2018). Five strains of Salmonella enterica (Heidelberg S13 [clinical isolate], Enteritidis 6424 [phage type 4, baked cheesecake isolate], Enteritidis E40 [chicken ovary isolate], Typhimurium M-09-0001-A1 [peanut butter isolate], and Typhimurium S9 [clinical isolate]); 5 strains of L. monocytogenes (LM 101 [hard salami isolate, serotype 4b], FSL-C1-109 [clinical isolate, processed turkey outbreak, serotype 4b], LM 310 [goat milk cheese isolate, serotype 4b], V7 [raw milk isolate, serotype 1/2b], and LM 108 [hard salami isolate, serotype 1/2a]); and 7 clinical isolates of Shiga toxin-producing E.coli (Strain designations: O157:H7 strain FRIK47 [ATCC 43895] [Riley et al., 1983; Wells et al., 1983], O111:H8-strain 00-3142, O103:H2–strain 01-3002, O121:H9-strain 01-3434, O45:H2-strain 01-3510, O145:NM-strain 99-3311, and O26:H11-strain H30), were used in this study. All strains were obtained from the University of Wisconsin-Madison Food Research Institute stock culture collection or the Wisconsin State Hygiene Laboratory (Madison, WI, USA).

Culture preparation

Culture preparation followed the methodology that was previously described by McMinn et al. (2018). Strains were grown individually in 9 ml of Trypticase^™ Soy Broth (TSB, Difco, BD Biosciences, Sparks, MD, USA) with shaking for 18 h at 37°C. For each strain, 0.2 ml aliquots of each overnight culture were spread onto 5 Trypticase Soy Agar (TSA, BD Biosciences) plates and incubated at 37°C for 18–22 h. Cells were harvested by scraping the surface of TSA plates with a sterile inoculating loop and suspending them in 4.5 ml of 0.1% peptone water (PW, pH 7.2) to achieve approximately 10 log colony-forming units (CFU)/ml. Approximately equal populations of each strain were combined and diluted with PW to provide a mixture with a concentration of approximately 10-log CFU/ml. Populations of each strain and the mixture were verified by plating on TSA, Xylose-Lysine-Desoxycholate agar (XLD, BD Biosciences), modified Oxford agar (MOX, Listeria Selective Agar base, BD Biosciences), or MacConkey-Sorbitol agar (SMAC, BD Biosciences) for Salmonella, L. monocytogenes, and E. coli, respectively. Plates were incubated at 37°C for 18–22 h.

Product manufacture

This study used the same product formulations as those described by McMinn et al. (2018). Three categories of low-fat, RTE products (deli-style uncured turkey breast, uncured roast beef, and boneless cured ham) were selected to represent a range of meat species, moisture contents, and levels of sodium nitrite incorporation. Formulations for all 3 products, listed in Table 1, were standardized for other critical factors, including pH, phosphate, fat, and salt, which have been shown to impact the heat tolerance of pathogens (Kang et al., 2018).

Products were manufactured using Good Manufacturing Practices in the Meat Science and Muscle Biology Laboratory at the University of Wisconsin-Madison. All muscle tissues described below were obtained from a local commercial supplier and stored at ≤ 4.0°C for ≤ 72 h prior to use. Uncured roast beef was manufactured using fresh, closely trimmed beef top round muscles (Semimembranosus); uncured, deli-style turkey breast was manufactured using boneless, skinless turkey breasts; and cured, deli-style ham was made using fresh, boneless, closely trimmed ham inside muscles (Semimembranosus). For all products, any visible external fat was removed, and the muscles were coarsely ground through a 19.05 mm plate attached to a grinder (Model 4732, Hobart Corp., Troy, OH).

For all products, any non-meat ingredients were first dissolved in water to make a brine. Brines and pre-weighed coarsely ground meat were then placed in a vacuum tumbler (Lance Model LT-5, Koch Industries, Kansas City, KS) and continuously tumbled under vacuum at 24 rpm and at ≤ 4.0°C. Roast beef and turkey were tumbled for 90 min, while ham was tumbled for 120 min to maximize both pick-up of the brine and protein extraction. After tumbling, product mixtures were vacuum packaged (3-mil high-barrier pouches; Ultra Source LLC, Kansas City, MO) and transported to the Food Research Institute at the University of Wisconsin-Madison for subsequent inoculation and thermal inactivation testing.

Sample inoculation and preparation

For inoculation, 14.0 kg of the appropriate raw meat mixture was added to a paddle mixer (Buffalo Model 2VSS mixer, John E. Smith’s Sons and Co., Buffalo, NY) and mixed for 30 s. A 1.0% (v/w) inoculum targeting 8.0 log CFU/g meat was added and mixed for 2 min after which the mixer was stopped, and the sides were scraped using rubber spatulas sanitized with 70% ethanol. After mixing for an additional 2 min, the inoculated meat mixture was then stuffed into 10.61 cm diameter moisture-impermeable plastic (roast beef and turkey breast) or fibrous (boneless ham) casings (World Pac International USA, Sturtevant, WI) to a length of 35.56 cm using a manual piston stuffer (F. Dick, Deizisau, Germany), clipped, and stored at ≤ 4.0°C prior to thermal inactivation testing. Thermal processing of roast beef and deli-style turkey breast chubs began within 1 h of stuffing. Ham chubs were sealed in moisture-impermeable bags and placed in coolers with ice for transport to the Alkar-RapidPak Research and Technology Center (Lodi, WI) for subsequent thermal inactivation testing. Thermal processing of ham chubs began within 3 h of stuffing.

Temperature monitoring

Internal temperature probes (iButtons; DS1922T-F5, Maxim Integrated, San Jose, CA, USA) were placed at 3 latitudes inside an uninoculated chub to continuously monitor temperature at the surface, midpoint, and geometric center (core) of the product during cooking (Figure 1). The midpoint latitude was located halfway between the surface and the core of the chub, approximately 2.54 cm below the surface. Probes were inserted into the end of the chub to a longitudinal depth ≥6.35 cm before clipping. A plastic template was used to ensure that probes were placed at the same latitude for each trial. Thermal profiles were generated for each treatment/process combination by measuring product surface, midpoint, and core temperatures as well as environmental (steam or wet-bulb/dry-bulb) conditions at 1-min intervals. Real-time temperatures were monitored with a digital thermocouple (Fisher Scientific Traceable Thermometer and type K probe, Thermo Fisher Scientific, Waltham, MA, USA) inserted into the end of a separate, uninoculated chub.

Figure 1.

Cross-sectional diagram of 10.61-cm diameter meat product chubs indicating locations for pathogen sampling and temperature probe placement.

Cooking, testing, and microbial enumeration

The thermal processing parameters used for all 3 products are listed in Table 2. Roast beef and turkey breast chubs were cooked in a combination steam-convection oven (Alto-Shaam Combitherm, model 6.10 ESI SK, Alto-Shaam Inc., Menomonee Falls, WI, USA) using step-up steam cook processes. Ham chubs were cooked in a smokehouse (Alkar Model 1000, Alkar Engineering Corp., Lodi, WI, USA) at the Alkar-RapidPak Research and Technology Center using a controlled humidity cook process. Table 3 outlines the sampling points for the 9 different product/pathogen treatment combinations. Duplicate trials were conducted for each product/pathogen combination. For each sampling time point, one inoculated chub was removed from the oven when the treatment-prescribed target internal temperature or hold time was reached.

Once removed, the casing was aseptically peeled and discarded from each chub, and 5.1-cm pieces were removed from each end of the chub and discarded to ensure the uniform geometry of each sample. The remaining 25.36-cm central portion of the chub was cut into 3 pieces of equal length, and 25-g samples (8.45 cm long × 1.9 cm diameter) were removed from the geometric center (core) and the midpoint of each section using a sterile metal bore inserted parallel to the length of the chub. A 25-g sample was also extracted from the outside surface of each chub segment by aseptically shaving 2–3 mm-thick pieces with a sterile scalpel. From each chub, 3 surface, 3 midpoints, and 3 core samples were collected for microbial enumeration of surviving pathogens (n = 9). An additional chub was removed at the end of each cooking process, placed in an ice-water bath, and chilled to an internal temperature of ≤4.0°C (the approximate time to reach less than 4.0°C was 30 min). Samples were then collected from the cooled chub as described above to capture any additional lethality during cooling.

Harvested samples were immediately placed into sterile Whirl-pak^® bags (Nasco, Fort Atkinson, WI, USA) and chilled to ≤4.0°C in an ice-water bath. Samples were then diluted 1:1 with 25 ml of Butterfield phosphate buffer and homogenized using a lab blender (Stomacher 400, A.J. Steward, London, England) for 2 min. Appropriate serial (1:10) dilutions of the processed sample were spread plated on duplicate plates of appropriate selective media (XLD, MOX, or SMAC agar for Salmonella, L. monocytogenes, or STEC, respectively). Selective media for each pathogen were overlayed with a thin layer of Tryptic Soy agar (TSA) to enhance the recovery of injured cells (Kang and Fung, 2000). Plates were incubated for either 24 h (Salmonella and STEC) or 48 h (Listeria) at 35°C, after which colonies were counted (LOD = 1.0 Log CFU/g).

Sub-lethal heating experiments

To better understand the efficacy of thermal processes following the longer time/lower temperature cooking combinations offered in Appendix A, an additional series of experiments investigated the effects of sub-lethal heat exposure on Salmonella in roast beef during extended cook processes with a target final core temperature of 54.4°C. These experiments were conducted in 2 phases: Isothermal D-value determination with thermally adapted Salmonella, followed by validation of these D-values in a pilot plant-scale system. Experiments from both phases were replicated 3 times. Isothermal experiments followed the methodology previously described by McMinn et al. (2018) with amendments described below. For each replication, 3 sets of sample pouches were prepared. Prior to submersion in a circulating water bath heated to 54.4°C, one set of samples (cold-adapted) was held at 4.0°C for 3 h, and a second set of samples (thermally adapted) was gradually heated to 54.3°C over 3 h in a circulating water bath following a step-up schedule to simulate sub-lethal heat exposure experienced at the center of a chub during cooking to 54.4°C. For this gradual warming procedure, the water bath was manually adjusted at 2-min intervals, with the adjustments modeled after the temperature profiles observed at the core of the roast beef chubs cooked to 54.4°C in the previous validation experiments. A third set of samples serving as the control (non-adapted) was inoculated and cooked immediately (no hold at 4.0°C or slow heating). Triplicate samples from each treatment were pulled immediately after submersion, and then at 10 min intervals thereafter, up to 90 min. Microbial enumeration data were used as described by McMinn et al. (2018) to generate a D-value for each treatment (n = 9 for each timepoint per treatment). For validation experiments investigating sub-lethal heating, manufacturing, and inoculation methods followed those described above, with temperature monitoring and pathogen enumeration only occurring at the core of each chub. Inoculated roast beef chubs were cooked to an internal temperature of 54.4°C following the same cook schedule as the prior experiments. The hold time at the end of the process was extended to 6 h. Microbial populations were enumerated from triplicate core samples at 1-h intervals once the product core reached the target temperature (n = 9 per timepoint)

Data analysis

For validation experiments, colony counts were transformed to log CFU/g and plotted versus heating time along with concurrently collected thermal processing data to create integrated thermal lethality profiles (Figures 2–4). For sub-lethal heating experiments, colony counts were transformed to log CFU/g and plotted versus heating time to create survival curves. Linear regressions were fitted to the linear portion of the survival curve for each experiment. For each treatment, D-values were estimated as the average of the absolute inverse of the slopes of the regression lines for 3 replicate experiments. One-way analysis of variance (ANOVA) was used to test the effect of treatment on D-values. Mean D-values for each treatment were compared using the Tukey-Kramer pairwise comparison method (α = 0.05). Linear regressions and ANOVA were conducted using JMP Pro software (version 11.0, SAS Institute Inc., Cary, NC, USA, 1989–2024). Thermal process data was entered into the American Meat Institute (AMI) process lethality spreadsheet in order to generate a predicted lethality for each pathogen-product combination (Foundation for Meat and Poultry Research and Education, 2015).

Figure 2.

Integrated average temperatures and Salmonella lethality from the center, midpoint, and surface of 10.61-cm diameter chubs of roast beef steam cooked to a final internal temperature of 54.4°C and held for 2 h (n = 2).

Figure 3.

Integrated average temperatures and Shiga toxin-producing Escherichia coli (STEC) lethality from the center, midpoint, and surface of 10.61-cm diameter chubs of roast beef steam cooked to a final internal temperature of 54.4°C and held for 2 h (n = 2).

Proximate analysis

Triplicate uninoculated, raw samples from each validation (n = 54) and sub-lethal heating trial (n = 9) were assayed for moisture (5 h, 100°C, vacuum oven method AOAC 950.46) (AOAC, 2000), NaCl (measured as % Cl⁻, AgNO₃ potentiometric titration, Mettler DL22 food and beverage analyzer, Columbus, OH, USA), and water activity (Decagon AquaLab 4TE water activity meter, Pullman, WA, USA). In addition, the pH (Accumet Basic pH meter and Orion 8104 combination electrode, Thermo Fisher Scientific, Waltham, MA, USA) was measured on the slurry obtained by removing a representative of 10 g of the uninoculated sample and homogenizing with 90 ml deionized water using a lab blender (Stomacher 400, A.J. Steward, London, England). Results are reported in Table 4.

Process validation and integrated lethality

For this study, thermal processes were defined as valid for control of Salmonella and STEC if they provided a ≥7.0-log reduction. This limit was chosen both because it was the minimum limit for accurate enumeration of the target pathogens, and because it meets or exceeds the 6.5 and 7.0 log lethality cook times for beef and poultry, respectively, listed in FSIS Appendix A (USDA-FSIS, 2021). Because Appendix A does not offer guidance for the control of L. monocytogenes explicitly, thermal processes were defined as valid for the control of L. monocytogenes if they provided a ≥5.0-log reduction. The pathogen lethality results at the product core for each product-pathogen combination are listed in Table 5. In all products tested in this study, cooking to a core temperature of 71.1°C resulted in ≥6.-log reduction of all 3 pathogens. Regarding Salmonella, this result agrees with Appendix A, which predicts that a 6.5-log reduction would be achieved instantaneously at 71.1°C in a roast beef product. This result is also supported by the D- and z-values determined for Salmonella and STEC in a previous study (McMinn et al., 2018). From that study, roast beef and deli-style turkey breast, Salmonella, and STEC had a D-value of ≤0.02 min at 71.1°C. When the target cook temperature (T_F) is equal to the reference temperature for a particular D-value (D_TF), the hold time necessary to obtain a target log reduction can be estimated with the following equation:

• (1)

  Hold time at T_F = D_TF * Target log reduction

Using this equation with D_TF = 0.02 min, a hold time of 7.8 s at 71.1°C will produce a 6.5-log reduction of Salmonella or STEC in the roast beef and deli-style turkey breast products. Although samples were pulled from the oven for enumeration when the core temperature reached 71.1°C, a longer hold time was experienced by bacteria at the product core due to the amount of time required to process the samples. Similarly, the D_71.1°C of 0.27 min for L. monocytogenes in ham did not predict instantaneous lethality and would instead require a hold time of 1.76 min to achieve a 6.5-log reduction (McMinn et al., 2018). Although the thermal process used for this product/temperature combination did not have any intentional hold time at 71.1°C, the size of the product did not allow for instantaneous cooling at the core when cooking was completed, and the chub was removed for microbial sampling. As a result of this delay in cooling, reductions of L. monocytogenes still exceeded 7.0 log. This illustrates an additional margin of safety that arises from the dynamic range of temperatures experienced by the bacteria during the entirety of a thermal process, also known as integrated lethality.

To illustrate the relationship between thermal processing and pathogen lethality, integrated thermal process/pathogen lethality profiles (Figures 2–5) were generated for selected treatments and time/temperature combinations by plotting microbial enumeration data along with the concurrently collected temperature data of the thermal process. These figures demonstrate how the temperature varied between the surface, midpoint, and core of the product during cooking and how pathogen populations at these 3 locations responded to varying rates of temperature increases. As expected, the rate of pathogen lethality generally increased with faster rates of heating.

For all thermal processes, the product surfaces spent the greatest amount of time at temperatures exceeding 54.4°C and at the respective target temperature. Consequently, the most rapid reductions for each pathogen occurred at the product surface compared to the core, regardless of treatment or thermal process. The effects of these higher temperatures and longer times are most apparent in Figures 2 and 3 with Salmonella and STEC, respectively, in roast beef heated to a final temperature of 54.4°C and held for 2 h. The product surfaces for these 2 processes experienced a temperature of 54.4°C for an average of 187.5 min compared with 120.0 min at the core. This longer dwell time at the target temperature resulted in greater reductions of 4.98 and 6.17 log for Salmonella and STEC on the product surfaces, respectively, compared with the lethality observed at the core of the product during the same process. For processes with target temperatures of 62.8 or 71.1°C, similar differences in populations between the surface and core were observed when the core temperature reached 54.4°C. However, the differences rapidly diminished as the core temperature exceeded 60.0°C and approached the target temperature. Adequate reductions (≥5.0 log for L. monocytogenes and ≥7.0 log for Salmonella and STEC) were achieved when pathogens were heated to a lethal temperature and held for a sufficient amount of time. These results suggest that both dwell time and surface temperature are integral to ensuring surface lethality. They also show that it is important to consider integrated lethality when estimating total process lethality.

The significance of integrated lethality is further demonstrated when target core temperatures were reduced to 62.8°C with a 5-min hold time. Appendix A lists hold times of 5.0 min and 10.6 min to achieve a 7.0-log reduction of Salmonella in beef and turkey, respectively. For both Salmonella and STEC in roast beef, population reductions >7.0 log were seen after the 5-min hold time (Table 5). For L. monocytogenes in ham, a reduction of only 5.0 log was seen after the hold time (Table 5); however, a 7.0-log reduction was observed in samples taken from chubs that were chilled to <4.0°C via submersion in an ice-water bath (data not shown). Core temperatures in these ham chubs remained above 54.4°C for 60.0 min after being removed from the oven. The D_54.4°C of 48.14 min for L. monocytogenes that was determined by McMinn et al. (2018) predicts that an additional 1.2-log reduction would occur during cooling. The lethality during cooling provides an additional measure of safety when targeting Salmonella and STEC at this temperature in roast beef, where sufficient log reduction requirements of 6.5 log and 5.0 log were easily met before cooling. It also shows that under the experimental conditions of this study, a target internal temperature of 62.8°C with a 5-min hold time may be sufficient for controlling L. monocytogenes as long as additional lethality that occurs during cooling is considered.

Surface dehydration

D- and z-values for pathogens are most often determined under hydrated conditions and are therefore most useful for predicting lethality in the interior of products. Conditions on the product surface during cooking are more dynamic, and as a result bacteria on the product surface may exhibit heat tolerance not predicted by experimentally derived D-values. Low water activity and desiccation of the product surface are both known to reduce Salmonella lethality during cooking (Goepfert et al., 1970; Hiramatsu et al., 2005; Mattick et al., 2000; Mattick et al., 2001; van Asselt and Zwietering, 2006). Desiccation of the product surface can occur when the surface temperature exceeds the wet-bulb temperature of the cooking environment. Such conditions were present in the thermal processes for the ham/L. monocytogenes treatment combinations (Table 2), which were conducted in a smokehouse where steam was injected to adjust the wet-bulb temperature during cooking. Gruzdev et al. (2011) found that the increased heat tolerance of desiccated Salmonella could be reversed by rehydrating the cells. The thermal processes for both the 62.8 and 71.1°C target temperatures for the ham/L. monocytogenes combination in the current study therefore incorporated a “surface lethality” step where the wet-bulb temperature was increased to a thermally lethal temperature to provide surface lethality for the product. This can be seen in Figure 4, which shows the integrated lethality profile for ham/L. monocytogenes cooked to 62.8°C with a 5-min hold. Cook processes for both temperatures demonstrated similar patterns of increasing lethality with increasing temperatures as was observed for Salmonella and STEC in roast beef and turkey breast. Despite achieving similar reductions, the times needed to achieve these results were considerably longer for L. monocytogenes in ham. This result can be partially explained both by differences in the cook schedules themselves and the considerably greater heat tolerance of L. monocytogenes, evidenced by the D- and z-values published in numerous studies (Juneja, 2003; McMinn et al., 2018; Murphy and Berrang, 2002; Murphy et al., 2003; Murphy et al., 2004c; Murphy et al., 2004d). When comparing pathogen reductions only at the surface for ham and roast beef, approximately 225 min were required to observe a 5-log reduction of L. monocytogenes for ham heated to 62.8°C compared with the 130 min and 125 min needed to achieve a similar reduction for Salmonella and STEC, respectively. The ham products were cooked in a fibrous casing which allowed moisture to escape the product while the use of moisture-impermeable casings for roast beef meant that no surface dehydration occurred during cooking. Although not a direct comparison, these results suggest that surface dehydration of the hams may have allowed for increased survival of L. monocytogenes. More research is necessary to determine the extent to which desiccation may confer increased heat tolerance to L. monocytogenes.

Figure 4.

Integrated average temperatures and Listeria monocytogenes lethality from the center, midpoint, and surface of 10.61-cm diameter chubs of ham cooked with controlled humidity to a final internal temperature of 62.8°C and held for 5 min (n = 2).

Sub-lethal heating

When sub-lethal temperatures persisted during long come-up times, overall pathogen reduction was lower or failed to achieve target reduction goals, regardless of sampling location. In roast beef cooked to 54.4°C and held for 2 h, reductions of Salmonella and STEC (3.21 and 3.44 log, respectively) failed to reach the target reductions necessary for that thermal process to be validated. (Table 5, Figures 2 and 3). These results suggest that the 121-min hold at 54.4°C listed in Appendix A may not produce a sufficient reduction of either pathogen. To both confirm these results and determine the amount of time necessary to ensure safety, validation experiments were repeated in this product for Salmonella with the hold time at 54.4°C extended to 6 h (Figure 5). With the longer hold times, a 4.69-log reduction was observed after 3 h, and a ≥ 6.5-log reduction was confirmed after 4 h (Figure 5). The effect of an extended hold at this temperature was also observed in the pathogen reductions seen on the product surface. For the thermal process with a 2-h hold time, Salmonella on the surface of the roast beef chubs experienced a 3.08 h hold at 54.4°C during cooking, resulting in a 4.98-log reduction. This result was comparable to the reduction found in the core samples after a 3-h hold. The surface of the STEC chubs spent an additional 70 min at 54.4°C but had a considerably greater reduction of 6.17 log. These comparisons support extending the hold time at 54.4°C beyond 3 h to ensure sufficient reductions for Salmonella and suggest that STEC may need ≤3 h at this temperature to achieve similar lethality.

Figure 5.

Integrated average temperatures and Salmonella lethality from the center of 10.61-cm diameter chubs of roast beef steam cooked to a final internal temperature of 54.4°C and held for up to 6 h (n = 3).

One possible explanation for the decreased lethality observed in the validation experiments is that exposure to sub-lethal temperatures (≤54.4°C) before reaching a core temperature of 54.4°C allowed for the target pathogen to express a heat shock response, resulting in increased survival (Jorgensen et al., 1996; Mackey and Derrick, 1986; Stasiewicz et al., 2008; Tenorio-Bernal et al., 2013; Wesche et al., 2005). While the effects of sub-lethal heating during slow cooking are well known, it is difficult to quantify this exposure and its impact on process lethality. Mackey and Derrick (1990) reported increased survivability of Salmonella Typhimurium at 55.0°C in tryptone soy broth after a heat shock of 30 min at 48.0°C. Wesche et al. (2005) demonstrated that Salmonella in ground turkey held at 43.0°C for 30 or 60 min showed increased survival when cooked to 60.0°C. The integrated lethality profiles for Salmonella in roast beef and STEC in roast beef (Figures 2 and 3, respectively) show that the core temperatures in these chubs remained below 54.4°C for 174 and 183 min for Salmonella and STEC, respectively. Within that timeframe, core temperatures exceeded 40.0°C for 93 and 96 min for Salmonella and STEC, respectively. These previous reports suggest that this level of sub-lethal heat exposure would be adequate for inducing a heat shock response in both pathogens and could, therefore, be responsible for reduced process lethality.

Multiple studies have shown that predictive models, which do not account for sub-lethal heat exposure, may overestimate total lethality for thermal processes with long come-up times, however, little research has been done to define any sub-lethal heating effect on D-values in meat (Corradini and Peleg, 2009; Stasiewicz et al., 2008; Valdramidis et al., 2007). This result can be seen in Table 6, where the observed log reductions of Salmonella and L. monocytogenes are compared to a predicted lethality generated using the AMI process lethality spreadsheet (Foundation for Meat and Poultry Research and Education. 2015.). For the roast beef product cooked to an internal temperature (IT) = 54.4°C and held for 2 h, only a 3.21-log reduction of Salmonella was observed, yet the predicted log reduction of this process ranged from 15.1–38.1 log units (Table 6). The wide range for predicted lethality here is a result of the reference temperatures (Tref), D-values, and z-values that were used as inputs for lethality estimation. A log reduction of Salmonella ≥7.00 was only observed in roast beef after a 4-h hold time at 54.4°C.

D-values determined for pathogens following exposure to sub-lethal temperatures could be used to develop validated time-temperature tables for use with slow-cook processes and large-diameter products. Along with the validation experiments examining extended hold time, D-values for Salmonella at 54.4°C were determined in 3 sets of roast beef samples subjected to different pre-cook storage treatments D-values for all 3 treatments were significantly different (P < 0.05) from each other. The non-adapted treatment had a D-value of 10.3 min, which was similar to the D-value of 9.34 min determined for Salmonella in McMinn et al. (2018). The cold-adapted samples had a D-value of 14.4 min, and the thermally adapted samples had a D-value of 23.3 min. A 1.58-log reduction was observed during the thermal adaptation process; however, >5.0-log reduction was still observed before populations dropped below the limit of detection (2.0 log CFU/g) The elevated D-value following thermal adaptation offers some preliminary evidence that D-values for sub-lethal heat exposure could be developed. This higher D-value for the sub-lethal adapted samples suggests that a hold time of 155 min would be necessary to achieve a 6.5-log reduction of Salmonella, which is longer than the current Appendix A recommendation of 112 min (USDA-FSIS, 2021), which assumes no prior exposure to sub-lethal heat and aligns with the low lethality seen in validation experiments in the current study. However, following a 2-h hold at 54.4°C, the observed 3.21 ± 0.21-log reduction of Salmonella is considerably lower than the 5.02-log reduction predicted using this elevated D-value. Thus, the effects of sub-lethal heat exposure cannot fully explain the low lethality seen with Salmonella in these initial validation experiments.

In addition to sub-lethal heat exposure, the target temperature used for achieving lethality should also be considered when assessing low process lethality. While the effects of sub-lethal heating were only observed in cook processes with a target temperature of 54.4°C, it is important to note that as temperatures decrease, the potential lethality offered by these temperatures can diminish rapidly. As a result, small deviations from a low cooking temperature may produce significant reductions in pathogen lethality relative to those produced by similar deviations at higher cooking temperatures. Research reviewed by O’Bryan et al. (2006) illustrates this trend. Their review of D- and z-values for various processed meat products reported D-values for STEC in ground beef of 42.3 min at 51.6°C, 12.5 min at 55.0°C, and 2.8 min at 57.2°C. These reported D-values confirm that small temperature changes can significantly reduce pathogen lethality at low temperatures where reduced lethality is already expected.