Introduction
Sous vide is a cooking method where vacuum-packed meat is cooked in circulating water at relatively low temperatures for an extended period of time. Depending on the type and size of meat, the cooking temperature may range between 50 and 85°C, and the cooking duration may vary from 2 to 48 h (Latoch et al., 2023). This method has become popular in both the food industry and domestic settings due to multiple benefits, such as precise control over food preparation and extended shelf life (Kathuria et al., 2022). The sous vide cook-chill method allows marinating and cooking the meat in advance, followed by refrigeration for later consumption (Baldwin, 2012) to help manage logistics. Although sous vide is generally considered a safe cooking method, using low cooking temperatures with unvalidated durations to attain certain quality characteristics raises concerns regarding food safety (Ismail et al., 2022).
Listeria monocytogenes (L. monocytogenes) is a mesophilic organism, but it has the ability to grow at temperatures as low as −0.4°C (Bucur et al., 2018). This organism is a well-documented concern in ready-to-eat meat products due to its ability to persist and proliferate under cold storage conditions (Churchill et al., 2019; Hadjicharalambous et al., 2022). Although sous vide-cooked meat may not be immediately ready to eat due to the need for surface searing to achieve the desirable flavor, it is nonetheless a fully cooked product. L. monocytogenes is particularly concerning in sous vide applications due to its high heat tolerance (McMinn et al., 2018; Juneja et al., 2020; Manzoor et al., 2026). Additionally, heat stress can increase the expression of the opuC gene (van der Veen et al., 2007), which facilitates carnitine uptake, providing protective effects against cold stress (Tasara and Stephan, 2006). This suggests that surviving bacteria may be primed for subsequent survival or even growth during refrigeration.
In addition to L. monocytogenes, Salmonella enterica (S. enterica) also poses potential risks in sous vide cooking due to its thermal tolerance. S. enterica, a mesophilic organism, generally declines on fresh beef in vacuum packaging during refrigeration; however, some studies have reported a lack of inactivation (Silva et al., 2022). In a recent study by Kačániová et al. (2024b), S. enterica inoculated rabbit meat was cooked sous vide with or without thyme essential oil and stored for 7 d at 4°C. Mean S. enterica counts in samples without thyme essential oil were 1.90 log10 cfu/g on day 1 and 2.57 log10 cfu/g on day 7. This indicates that there is a potential for growth that may not be adequately addressed by subsequent surface browning with the brief searing. For instance, Patil et al. (2024) observed only 1.1–1.7 log cfu/g reduction by searing when sous vide-cooked steak was grilled to achieve an internal temperature of 57°C. The reduction may be even lower in less intense searing conditions. Furthermore, George et al. (1998) observed that heat-injured S. enterica and L. monocytogenes can recover more effectively under anaerobic conditions, particularly when exposed to less intense heat treatment (57 versus 60°C). This raises additional concerns about the recovery of sub-lethally injured pathogens during refrigeration of sous vide-cooked meat.
Refrigeration temperature control is one of the most critical factors in limiting the growth of foodborne pathogens. The World Health Organization recommends refrigerating perishable foods, including meat products, at temperatures below 5°C (WHO, 2023), while in the United States, the recommended limit is ≤ 4.4°C (40°F) (FDA, 2023). However, surveys have shown that in many countries, over 50% of home refrigerators operate above 5°C, with some exceeding 10°C (Kennedy et al., 2005; Evans and Redmond, 2016; Stefanou et al., 2023; van der Vossen-Wijmenga et al., 2024). Furthermore, Brown et al. (2018) found that 17% of the surveyed delis had at least one refrigerator exceeding 5°C, indicating that temperature abuse can occur in the food service settings as well. Given the increasing popularity of sous vide cooking and the preference for lower cooking temperatures or shorter durations to achieve specific sensory attributes, the combination of the cook-chill method and mild temperature abuse may present food safety risks.
This study aimed to determine whether sublethal heat injury resulting from inadequate sous vide cooking conditions (time-temperature combinations), combined with temperature abuse during post-cook storage, affects the survival and recovery of L. monocytogenes, S. enterica, and background microflora on beef steaks. The central hypothesis was that less intense sous vide cooking conditions could lead to incomplete pathogen inactivation and sublethal heat injury in the survivors, along with heat stress response, which could allow their recovery during refrigerated or mildly abusive storage conditions. This study will provide deeper insights into food safety risks associated with sous vide cook-chill practices.
Materials and Methods
Experimental design
This study was conducted in 2 independent experiments to assess the effect of sublethal injury caused by inadequate sous vide conditions and mild temperature abuse during storage of sous vide-cooked beef steaks on survival and recovery of L. monocytogenes, S. enterica, and aerobic plate counts (APC). While bacterial preparation, meat handling, and enumeration procedures were consistent across both experiments, the treatment variables and storage conditions differed.
The first experiment assessed the survival of L. monocytogenes, S. enterica, and APC in sous vide-cooked steaks stored under 2 refrigeration temperatures. Steaks were cooked in 3 different sous vide conditions, i.e., 52.5°C for 65 min, 57.5°C for 24 min, and 60.0°C for 18 min in preheated sous vide water baths (Anova Applied Electronics Inc., San Francisco, CA, USA) at a meat-to-water ratio of 1:9 (w/v). These temperatures represent the bottom, middle, and top of the common sous vide cooking range. We studied these temperatures for the refrigeration study, as bacteria exposed to different heating rates at these temperatures may express different heat stress responses and thereby different survival. The times were selected to achieve a 4-log reduction in Salmonella (Manzoor et al., 2026) and represent an under-cooking situation. The steaks were then immediately dipped in an ice bath for 30 mins to ensure the core temperature was below 4°C to stop the heat inactivation of pathogens. Chilled sample steaks were then stored at either 3 ± 1°C or 8 ± 1°C. Samples were collected on days 0, 2, 4, 6, and 8 for microbial enumeration. The bacterial counts immediately after sous vide cooking are represented as day 0.
Based on the absence of L. monocytogenes growth in the first experiment, and its known limited ability to compete in the presence of other microorganisms (Hwang and Sheen, 2011), a second experiment was conducted with only L. monocytogenes inoculated steaks. In this experiment, 2 sets of inoculated vacuum-packed steaks were prepared: one set was cooked sous vide at 57.5°C for 45 min, while the other remained uncooked and served as a control. Sous vide-cooked steaks were chilled in an ice bath and then stored alongside uncooked vacuum-sealed steaks for 14 d at 4 ± 1°C. Samples were collected every other day from day 0 to day 14. This approach was aimed at studying the effects of heat injury and stress, caused by inadequate sous vide cooking, on the recovery of L. monocytogenes during refrigerated storage.
Bacterial strains and culture conditions
Four S. enterica subsp. enterica serotypes, including Anatum (strain KRS 051404), Newport (strain ATCC 6962), Saintpaul (strain LJH1211), Typhimurium (strain ATCC13311); and 3 serotypes of L. monocytogenes, 1/2a, 1/2b, and 4c (Ribotypes: DUP 1039C, DUP 1042B, and 758453, respectively), were all kindly provided by Dr. Keith Schneider, and S. enterica subsp. Enterica serotype Dublin (strain 1724) was provided by Dr. Michelle Danyluk. Details regarding the origin of the strains used in this study are presented in Table 1.
Bacterial strains and serotypes used in this study
| Pathogen | Serotype | Strain | Description |
|---|---|---|---|
| S. enterica | Anatum | KRS 051404 | Unknown |
| Newport | ATCC 6962/NCTC 129 | Isolated from a food poisoning fatality in England | |
| Saintpaul | LJH1211 | Serovar Saintpaul recovered in a jalapeño outbreak; from Linda Harris | |
| Typhimurium | ATCC 13311/NCTC 74 | Isolated from feces in a food poisoning case | |
| Dublin | 1724 | Unknown | |
| L. monocytogenes | 1/2a | DUP-1039C | Seafood and processing environment |
| 1/2b | DUP-1042B | Deli meat and processing environment. | |
| 4c | DUP-1061A/758453 | Processing environment |
S. enterica and L. monocytogenes isolates were stored at −80°C in tryptic soy broth (TSB) (Difco Laboratories, Sparks, MD, USA) containing 15% glycerol (Thermo Fisher Scientific, Waltham, MA, USA). Before each replicate, the isolates were streaked on XLT-4 plates with XLT-4 agar supplement (Difco Laboratories) for isolating S. enterica and modified Oxford agar (MOX) plates with Remel 450551 moxalactam supplement (Remel Products, Thermo Fisher Scientific) for L. monocytogenes isolation and incubated at 35°C for 18–24 h.
Individual colonies from each plate were then suspended in 6 mL TSB and incubated at 35°C for approximately 18 h in a rotating incubator (Forma Orbital Shaker, Model 420, Thermo Electron Corp., Waltham, MA, USA) at 150 rpm. After 18 h incubation, 150 μL of the culture from 6 mL TSB was subcultured in 150 mL sterile TSB to obtain the required bacteria for meat inoculation. The 150 mL broth was incubated at 35°C for 20–24 h in the rotating incubator to obtain stationary-phase bacteria. Bacterial cultures were then centrifuged (Du-Pont Co., Newton, CT, USA) at 3,000 × g for 13 min at 4°C.
For the first experiment, S. enterica pellets were resuspended in 20 mL sterile 0.1% peptone water (Oxoid Ltd., Basingstoke, Hants, UK), all serotypes mixed, centrifuged, and then suspended in 9 mL 0.1% peptone water. L. monocytogenes pellets were resuspended in 20 mL 0.1% peptone water, mixed, centrifuged, and resuspended in 900 mL 0.1% peptone water. Equal volumes (7.2 mL) from each S. enterica and L. monocytogenes suspension were then mixed and centrifuged. The final pellet was resuspended in 7.2 mL sterile 0.1% peptone water. The suspension was vortexed and homogenized in a shaking incubator (150 rpm, 10 min). The 100 μL of this final cocktail mixture was inoculated per side of the steak (200 μL per steak) to achieve ca. 8 log10 cfu/g S. enterica and ca. 6 log10 cfu/g L. monocytogenes on raw steaks.
For the second experiment, pellets from a 150 mL subculture of each L. monocytogenes serotype were washed, mixed, and resuspended in 9 mL sterile 0.1% peptone water. For the sous vide set of samples, 100 μL of this cocktail suspension was inoculated per side of the steak to achieve a ca. 8 log10 cfu/g concentration. For the uncooked set of samples, 100 μL of this suspension was diluted in 100 mL of sterile 0.1% peptone water, and then 100 μL of this dilution was inoculated on each side of the steak to achieve a ca. 5 log10 cfu/g concentration. The concentrations were confirmed by plating raw samples on selective agar.
A cocktail of 5 S. enterica serotypes and 3 L. monocytogenes serotypes was used in the first experiment, whereas only the mixture of 3 L. monocytogenes serotypes was used in the second experiment. Pathogens were inoculated at higher concentrations than typically encountered in natural contamination scenarios to ensure measurable survivors following sous vide cooking. However, we recognize that these elevated levels may reduce the influence of background microflora and therefore represent a limitation of the study design.
Meat sample preparation and bacterial inoculation
Eye of rounds (Musculus semitendinosus, IMPS 171C) were purchased from a local retailer, transported to the laboratory, and trimmed into steaks (2.54-cm thick, 100 ± 3 g). Steaks were then vacuum-packed at an absolute pressure of 20 mbar using an AMAC T-165 vacuum chamber machine (AMAC Technologies, Anaheim, CA, USA) and stored at 3 ± 1°C to be used the following day.
On the day of the experiment, the steaks were transferred into individual vacuum bags. 100 μL of the bacterial cocktail mixture was inoculated on each side of the steak, followed by hand massaging for approximately 1 min to ensure even distribution. The steaks were held at 3 ± 1°C for 30 mins to allow bacterial attachment before vacuum sealing individually for sous vide cooking. The vacuum-packed inoculated steaks were randomly allotted to each sous vide time-temperature combination as well as refrigeration temperature.
For the first experiment, 30 steaks were inoculated, including 15 steaks for each refrigeration temperature with 5 steaks per sous vide time-temperature combination. The experiment was replicated 5 times (n = 150). For the second experiment, 16 steaks were inoculated, including 8 steaks for each sous vide versus uncooked treatment. This experiment was replicated 3 times (n = 48).
Microbial enumeration
S. enterica and L. monocytogenes were enumerated by aseptically transferring each steak and its associated drip into a stomacher bag containing 100 mL sterile 0.1% peptone water. Samples were hand massaged for approximately 1 min, serially diluted 10-fold, and 1 mL diluted homogenate was mixed with 4 mL melted tryptic soy agar (TSA) (Difco Laboratories) maintained at 50°C to improve resuscitation of sub-lethally injured bacterial cells (Kang and Fung, 2000). This 5 mL mixture was overlaid onto XLT-4 (Difco Laboratories) for S. enterica and Modified Oxford Agar MOX (Remel, Thermo Fisher Scientific) for L. monocytogenes. To enumerate APC, 100 μL of diluent was spread over TSA plates. The plates were then incubated at 35°C for 18 to 24 h, and colonies were manually counted based on criteria defined by the culture media manufacturers. The detection limit for both these pathogens was 1 cfu/g, and for Total viable count it was 10 cfu/g. The plates containing 1–300 colony-forming units were accepted for data analysis. When counts on more than one dilution plate were within an acceptable range, an average of the counts was taken.
Statistical analysis
Microbial count data were log10-transformed before analysis. The data from 2 experiments were analyzed using a linear mixed effects model in R (version 4.3.3) (R Core Team, 2024) with the nlme package (Graves et al., 2024). In experiment one, fixed effects included cooking conditions (52.5°C for 65 mins, 57.5°C for 24 mins, and 60.0°C for 18 mins), storage temperature (3°C and 8°C), storage time (0–8 d), and all their interactions. Cooking conditions (3 levels) and storage temperatures (2 levels) were included as categorical variables, while storage time was included as a continuous variable. The model included replication as a random effect. In experiment 2, fixed effects included treatment (sous vide versus uncooked), refrigeration time (0–14 d), and their interaction. Treatment (sous vide versus uncooked) was considered categorical, while refrigeration time was included as a continuous variable. Replicate was again included as a random effect.
Model assumptions, including linearity, normality of residuals, homoscedasticity, and independence, were verified through residual diagnostics and were adequately met for both experiments. The data for S. enterica survival in experiment 1 included 4 influential data points, which were identified based on Cook’s D value and were removed when this value was greater than 4/(n − p) where n = 150 corresponds to the number of observations, and p = 12 to the number of parameters. The excluded data points are presented in Supplementary Table 1. Inference on fixed effects was based on Wald t-tests of model coefficients obtained from the mixed-model fit. Model-based (population) predictions at specified combinations of cooking conditions, storage temperature, and time were obtained from the fitted model; standard errors for the predictions were derived from the model’s fixed effect variance-covariance matrix (delta method) using the predictSE.lme() function from the AICcmodavg package (Mazerolle, 2023). Statistical significance was determined at P < 0.05. When the P value for the interaction term involving storage time was 0.05 ≤ P < 0.10 (tendency), we estimated the simple slope of time (Δlog10 cfu/g per day) within each cooking-condition × storage temperature combination using the estimated marginal trend from the mixed model, and we performed Tukey’s adjusted pairwise comparisons of these slopes using the emtrends() function from emmeans package (Lenth, 2025).
Results
In the first experiment, we evaluated the impact of sous vide cooking conditions on the behavior of L. monocytogenes, S. enterica, and APC during post-cook refrigerated storage. L. monocytogenes results are presented in Figures 1 and Supplementary Tables 2 and 5. We did not see a three-way or two-way interaction (P > 0.05) for L. monocytogenes between sous vide cooking conditions, refrigeration temperatures, and storage duration, except there was a trend (P = 0.0674) of increasing L. monocytogenes counts during storage when steaks were cooked at 52.5°C for 65 mins and stored at 8°C compared to when stored at 3°C (Supplementary Table 2). Upon further analyzing the data, we observed a statistically significant increase (P = 0.029) of 0.05 ± 0.02 log10 cfu/g per day in L. monocytogenes counts over 8 d in steaks cooked at 52.5°C for 65 min and stored at 8°C (Supplementary Table 5). The model also revealed that L. monocytogenes counts at time zero were different among cooking conditions. The time zero counts were lower after cooking sous vide at 60.0°C–18 min (P < 0.001) than both lower temperature, longer time cook conditions. However, there was no difference (P = 0.079) between 52.5°C–65 min and 57.5°C–24 min (Supplementary Table 2). Mean L. monocytogenes counts along with standard errors during refrigeration at 3°C and 8°C are presented in Figure 1, respectively.
Behavior of L. monocytogenes, S. enterica, and aerobic plate counts (APC) on sous vide-cooked steaks during refrigerated storage at 3°C and 8°C. Steaks were inoculated with ca. 8 log10 cfu/g S. enterica and ca. 6 log10 cfu/g L. monocytogenes before being subjected to the indicated time and temperature combinations predicted to achieve a 4-log reduction of S. enterica. Solid line indicates the predicted values based on the selected model, shaded area indicates standard errors, and solid dots indicate observed bacterial counts (log10 cfu/g).
S. enterica results are presented in Figure 1 and Supplementary Tables 3 and 6. No three-way or two-way interaction was observed (P > 0.05) for S. enterica between sous vide cooking conditions, refrigeration temperatures, and storage duration (Supplementary Table 3). For S. enterica, the cooking time-temperature combination of 57.5°C–24 mins (P = 0.010) had significantly lower bacterial counts than 60.0°C–18 min at day zero. The day zero counts were not statistically different between 52.5°C–65 min and 57.5°C–24 min (P = 0.077) or between 52.5°C–65 min and 60.0°C–18 min (P = 0.402). The differences in the counts could be attributed to different inactivation during sous vide cooking. Overall, S. enterica counts significantly decreased (P < 0.019) from day 0 during refrigerated storage irrespective of cooking conditions and refrigeration temperatures (Supplementary Tables 3 and 6). The mean decline observed in this study, irrespective of refrigeration temperature in S. enterica counts after sous vide cooking at 52.5°C for 65 min, was 0.23 ± 0.07 log10 cfu/g per day of storage. While the decline after sous vide at 57.5°C for 24 min and 60.0°C for 18 min was 0.12 ± 0.07 log10 and 0.23 ± 0.07 log10 cfu/g per storage day, respectively (Supplemental Table 6). Mean S. enterica counts along with standard errors during refrigeration at 3°C and 8°C are presented in Figure 1, respectively.
APC results are presented in Figure 1 and Supplementary Tables 4 and 7. We did not see a three-way interaction for APC (Supplementary Table 3). Steaks cooked sous vide at 52.5°C for 65 min consistently exhibited higher APC (P < 0.001) compared to those cooked at 57.5°C for 24 min and 60.0°C for 18 min, regardless of the refrigeration temperature and time. APC on steaks cooked sous vide at 57.5°C for 24 min did not change over time, while the counts on steaks cooked at 60.0°C for 18 min and stored at 3°C decreased significantly (P = 0.021), i.e., 0.12 ± 0.05 log10 cfu/g per storage day (Figure 1, Supplementary Table 3 and 6). APC at day zero differed significantly only between 52.5°C for 65 min and 57.5°C for 24 min sous vide (P = 0.005). Mean APC counts along with standard errors during refrigeration at 3°C and 8°C are presented in Figure 1.
Results from the second experiment are presented in Figure 2, Supplementary Tables 8 and 9. In this experiment, we examined the behavior of L. monocytogenes during refrigerated storage following heat injury induced by sous vide cooking, compared to uninjured cells in vacuum-packed uncooked samples. The interaction between sous vide versus uncooked and storage duration was not significant (P = 0.11; Supplementary Table 8). The model results indicated that bacterial counts on sous vide-cooked steaks were not significantly different from those on uncooked steaks at day 0 (P = 0.443), indicating a similar starting point on day 0 of storage. However, upon examining the change over storage time, we observed a decreasing trend (P = 0.080) of 0.039 ± 0.022 log10 cfu per day in L. monocytogenes counts on sous vide-cooked steaks from 5.49 ± 0.20 to 4.95 ± 0.22 log10 cfu/g over 14 d of storage. In contrast, counts of uninjured L. monocytogenes on vacuum-packed uncooked samples remained unchanged (P = 0.600) during the same period (Figure 2, Supplementary Table 9).
Behavior of L. monocytogenes on sous vide-cooked (SV) and vacuum-packed uncooked (VP-UC) steaks during refrigerated storage at 4°C. Sous vide samples were inoculated at ca. 8 log10 cfu/g before cooking at 57.5°C for 45 min. Uncooked steaks were inoculated at ca. 5 log10 cfu/g to equal the remaining population in sous vide-cooked steaks. Solid line indicates the predicted values based on the selected model, shaded area indicates standard errors, and solid dots indicate observed bacterial counts (log10 cfu/g).
Discussion
The present study provides insights into the dynamics of heat-injured L. monocytogenes, S. enterica, and APC in beef steaks during low-temperature sous vide cooking, refrigerated storage, and mild temperature abuse. In experiment one, we chose a storage duration of 8 d because, according to the FDA Code (FDA, 2022), sous vide-cooked food that is chilled to below 5°C and kept at this temperature or below must be consumed or discarded within 7 d, unless the food establishment has obtained a variance. Despite the ability of L. monocytogenes to grow at refrigeration temperatures, no growth was observed during 8 d of storage at either temperature, except in steaks cooked at 52.5°C for 65 min and stored at 8°C. Although L. monocytogenes is known to grow at refrigerated temperatures in cooked meat products, recovery from heat injury can require extended lag phases, needing at least 14 d at 4°C and 6 d at 8°C (McKellar et al., 1997). Furthermore, its generation time is long, ranging from 10.2 h at 8°C to 121 h at 0°C, on cooked beef under vacuum (Nyati, 2000). Similarly, no growth of heat-injured L. monocytogenes was observed in a study conducted by Beck Hansen and Knøchel (2001) when stored at 3°C for 30 d, while mildly injured cells showed recovery at 10°C after 10 d. The mild growth observed at 8°C in our study following cooking at 52.5°C for 65 min is practically insignificant; however, more research is needed for longer storage of ≥15 d. This minor growth in L. monocytogenes in samples cooked at 52.5°C for 65 min could be pointing towards potentially less severe injury caused by less intense cooking conditions and the relatively higher storage temperatures, which could have facilitated cellular repair.
Moura-Alves et al. (2020) investigated the survival of L. monocytogenes in sous vide-cooked beef inoculated with or without sage essential oil (SEO). They applied varying cooking durations at 55°C to achieve 1, 2, and 3-log reductions in bacterial counts, followed by storage at 2 and 8°C. Their results showed a decline in L. monocytogenes counts at 2°C over 28 d, but an increase at 8°C after a similar duration. Although the trend was similar, samples inoculated with SEO showed lower counts than control samples during storage. Similarly, Gál et al. (2023) observed an increase in L. monocytogenes counts during 12 d of storage at 6°C when beef with sage essential oil was cooked sous vide at 55°C for 5 to 20 min. In another study, Shamsuzzaman et al. (1995) reported no increase in L. monocytogenes counts during the first week of storage at 8°C when chicken breasts were cooked sous vide to an internal temperature of 71.1°C, but observed growth in subsequent weeks. These findings are consistent with our results, where L. monocytogenes did not grow in steaks cooked at higher temperatures of 57.5 or 60.0°C and stored for 8 d, suggesting that these more intense cooking conditions potentially either induced sufficient heat injury to prevent cellular repair or resulted in an extended post-injury lag phase that exceeded the 8-d storage period, thereby preventing detectable proliferation. Whereas a mild but practically insignificant growth in L. monocytogenes was observed in steaks cooked at 52.5°C and stored at 8°C.
Few studies have examined the survival of heat-injured S. enterica at refrigeration temperatures. Although the mixed effects model indicated a significant mean decline in counts over the 8-d storage period, irrespective of cooking conditions and refrigeration temperature, the large between-sample variability in this study suggests that these inferences should be interpreted with caution. Variation in S. enterica survival has been documented in other studies evaluating heat-injured cells. Kačániová et al. (2024b) reported S. enterica counts during refrigerated storage after sous vide cooking of rabbit meat. Although they did not statistically test the change in numbers during the 7 d, there was a numeric increase of up to 0.8 log10 cfu/g. Hsu-Ming et al. (2012) induced injury to S. enterica cells by heating at 55°C for 15 min and observed that S. enterica was able to recover and increase in number at 37°C after 3 h in TSB and after 1 h in selective agar. Other studies have reported reductions in S. enterica counts on fresh beef during vacuum storage at 4–7.5°C (Erkmen and Barazi, 2008; Muras et al., 2012; Djordjević et al., 2018), while some found no change in counts during storage (Dykes et al., 2001; İncili et al., 2021).
Our observed declines are consistent with the expectation that S. enterica, a mesophilic organism, does not grow below 8°C on chilled beef (Mackey et al., 1980; Huang, 2020). Failure to repair heat injury under cold or mildly abusive conditions may explain the gradual reductions. However, due to the magnitude of variability in our dataset, especially compared to the more stable patterns of L. monocytogenes, the precision of the estimates for S. enterica is limited, and these results should not be considered definitive. Additional investigation is needed to determine whether the variability reflects heterogeneous heat injury, differential survival among serotypes, microenvironmental differences on the beef surface, or stochastic survival dynamics. Until these factors are better understood, the practical implications of the S. enterica findings should be regarded as tentative.
In this study, APC represents the combined microbial load from both the background microflora and the inoculated pathogens. We included APC in accordance with the National Advisory Committee on Microbiological Criteria for Foods (NACMCF, 2010) recommendations, which emphasize monitoring background populations in challenge studies to ensure that pathogen behavior is not confounded by changes in microbial ecology. This aggregate measure is informative because it indicates whether native microflora remained suppressed or increased to levels that could influence pathogen survival. Accordingly, by comparing L. monocytogenes and S. enterica counts with the aggregate APC, we verified that the inoculated pathogens remained the predominant microbial component and were not outcompeted by rapidly proliferating spoilage organisms. According to NACMCF, specific pathogen behavior is only relevant if all conditions, including ecology, remain consistent (NACMCF, 2010). In this study, the mean APC remained stable except for steaks cooked at 60.0°C for 18 min and stored at 3°C, where it declined. These trends indicate that native microorganisms did not increase to levels that would limit shelf life or alter pathogen behavior via nutrient depletion. Moreover, previous co-culture studies have shown that background microflora can suppress L. monocytogenes under refrigeration and mild abuse temperatures (Hwang and Sheen, 2011), further supporting the value of monitoring aggregate aerobes when interpreting pathogen survival.
Since L. monocytogenes has a limited ability to grow while competing against background microorganisms (Hwang and Sheen, 2011) and S. enterica was cohabitating the same steaks, the second experiment studied the effects of heat injury caused by sous vide cooking on L. monocytogenes recovery during refrigeration in the absence of S. enterica. We did not see any significant change (P = 0.600) in L. monocytogenes counts on vacuum-packed uncooked steaks over 14 d of storage at 4°C. Previous studies have reported both stable (Tsafrakidou et al., 2023) and increasing L. monocytogenes counts during storage of vacuum-packed meat (Kačániová et al., 2024a). Pennone et al. (2021) and Walker et al. (1990) have reported different growth rates for different strains of L. monocytogenes at refrigeration temperatures. Lack of growth of L. monocytogenes on uncooked steaks in our study could be due to differences in generation times for the strains used in this study, a long lag phase of L. monocytogenes (McKellar et al., 1997), or potential growth of lactic acid-producing bacteria hindering L. monocytogenes proliferation during storage (Tsafrakidou et al., 2023). Although L. monocytogenes counts remained stable in non-heat-treated steaks, a decreasing trend (P = 0.08) was observed in sous vide-cooked steaks, suggesting a potentially reduced survival of heat-injured cells. This finding is consistent with previous reports (Shamsuzzaman et al., 1995; Sibanda and Buys, 2017; Moura-Alves et al., 2020), indicating that heat injury compromises the ability of L. monocytogenes to recover during refrigerated storage. However, the effect observed here was only a tendency (P = 0.08); future work should extend storage to ≥30 d to establish whether a true time-dependent effect exists beyond the trend detected in this study. A longer observation window would also help distinguish whether the difference in counts between sous vide and uncooked samples reflects reduced survival of heat-injured cells or an extended lag phase that delays recovery beyond the 14-d period.
Conclusion
This study examined how different sous vide conditions (52.5°C for 65 min, 57.5°C for 24 min, and 60.0°C for 18 min) influenced the subsequent behavior of surviving heat-injured L. monocytogenes and S. enterica during refrigerated storage (3°C and 8°C), with APC reported as an aggregate aerobic indicator. Within the 8-d storage window of Experiment 1, L. monocytogenes and APC remained relatively stable, whereas S. enterica counts tended to decline on average during refrigeration. This suggests that in a risk assessment of the sous vide cook-chill method, no growth of L. monocytogenes and S. enterica should be expected, and the combination of low temperature and vacuum packaging prevents the growth of aerobic bacteria at least out to 8 d.
In Experiment 2, comparing heat-injured and uninjured L. monocytogenes at 4°C over 14 d, we observed no clear evidence of growth in either group. A modest decreasing trend was noted for heat-injured cells, while counts on uncooked samples remained unchanged. Taken together, these findings indicate that under these specific time-temperature profiles and storage durations evaluated, L. monocytogenes showed limited recovery and largely persisted at post sous vide levels, whereas S. enterica exhibited a mean decline with heterogeneous responses across replicates and conditions.
Conflict of Interests
The authors have no declared conflicts of interest.
Acknowledgments
The authors would like to thank the Florida Beef Council for providing funding to support this research.
Author Contributions
Adeel Manzoor: conceptualization, study design, data acquisition, analysis, interpretation, writing – original draft preparation, Isabel Ribeiro: data acquisition, Gabrielle Allen: data acquisition, Arie H. Havelaar: data analysis, interpretation, presentation and writing – review and editing, and Jason M. Scheffler: conceptualization, study design, funding acquisition, project administration, supervision, and writing – review and editing.
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Influential data points for S. enterica that were excluded from Experiment 1 model
| Replicate | Cook_cond | Ref_temp (°C) | Ref_time (day) | Log10 cfu/g |
|---|---|---|---|---|
| 1 | 60.0 °C—18 min | 8 | 8 | 0.00 |
| 2 | 60.0 °C—18 min | 8 | 6 | 7.89 |
| 3 | 52.5 °C—65 min | 3 | 8 | 5.95 |
| 4 | 57.5 °C—24 min | 3 | 8 | 5.70 |
Cook_cond is cooking condition (levels: 52.5 for 65 min, 57.5 for 24 min, and 60.0 °C for 18 min). Ref_temp is refrigeration temperature (levels: 3 and 8 °C). Ref_time is the storage duration, which is a continuous variable.
Fixed effects estimates for L. monocytogenes in experiment 1
| Value | SE | DF | t-value | P-value | |
|---|---|---|---|---|---|
| (Intercept) | 4.978 | 0.14628 | 134 | 34.03066 | 8.33E−68 |
| Cook_cond57.5 | −0.284 | 0.160332 | 134 | −1.77133 | 0.078779 |
| Cook_cond60 | −0.9096 | 0.160332 | 134 | −5.67324 | 8.28E−08 |
| Ref_temp8 | −0.1196 | 0.160332 | 134 | −0.74595 | 0.457002 |
| Ref_time | −0.0094 | 0.023142 | 134 | −0.40619 | 0.685251 |
| Cook_cond57.5:ref_temp8 | 0.0884 | 0.226743 | 134 | 0.389869 | 0.697253 |
| Cook_cond60:ref_temp8 | 0.0056 | 0.226743 | 134 | 0.024698 | 0.980333 |
| Cook_cond57.5:ref_time | −0.01 | 0.032728 | 134 | −0.30555 | 0.760419 |
| Cook_cond60:ref_time | −0.0201 | 0.032728 | 134 | −0.61416 | 0.54015 |
| Ref_temp8:ref_time | 0.0604 | 0.032728 | 134 | 1.84554 | 0.067166 |
| Cook_cond57.5:ref_temp8:ref_time | −0.0653 | 0.046284 | 134 | −1.41086 | 0.160602 |
| Cook_cond60:ref_temp8:ref_time | −0.0399 | 0.046284 | 134 | −0.86207 | 0.390187 |
Cook_cond is cooking condition (levels: 52.5 for 65 min, 57.5 for 24 min, and 60.0 °C for 18 min). Ref_temp is refrigeration temperature (levels: 3 and 8 °C). Ref_time is the storage duration, which is a continuous variable. SE: Standard error. DF: Denominator degrees of freedom. Significance determined at P-value <0.05.
Fixed effects estimates for S. enterica in experiment 1
| Value | SE | DF | t-value | P-value | |
|---|---|---|---|---|---|
| (Intercept) | 3.83429 | 0.575557 | 130 | 6.661876 | 6.94E−10 |
| Cook_cond57.5 | −1.12007 | 0.627235 | 130 | −1.78572 | 0.076475 |
| Cook_cond60 | 0.52531 | 0.624789 | 130 | 0.84078 | 0.402015 |
| Ref_temp8 | −0.04349 | 0.624789 | 130 | −0.06961 | 0.944613 |
| Ref_time | −0.22405 | 0.093939 | 130 | −2.38501 | 0.018523 |
| Cook_cond57.5:ref_temp8 | 0.074468 | 0.883609 | 130 | 0.084277 | 0.932966 |
| Cook_cond60:ref_temp8 | −0.30551 | 0.88357 | 130 | −0.34577 | 0.730074 |
| Cook_cond57.5:ref_time | 0.101634 | 0.13289 | 130 | 0.764798 | 0.445778 |
| Cook_cond60:ref_time | −0.08735 | 0.129977 | 130 | −0.67208 | 0.502727 |
| Ref_temp8:ref_time | −0.00385 | 0.129977 | 130 | −0.02966 | 0.976385 |
| Cook_cond57.5:ref_temp8:ref_time | 0.008966 | 0.183845 | 130 | 0.048769 | 0.961178 |
| Cook_cond60:ref_temp8:ref_time | 0.158894 | 0.184432 | 130 | 0.861531 | 0.390532 |
Cook_cond is cooking condition (levels: 52.5 for 65 min, 57.5 for 24 min, and 60.0 °C for 18 min. Ref_temp is refrigeration temperature (levels: 3 and 8 °C). Ref_time is the storage duration, which is a continuous variable. SE: Standard error. DF: Denominator degrees of freedom. Significance determined at P-value <0.05.
Fixed effects estimates for total viable counts in experiment 1
| Value | SE | DF | t-value | P-value | |
|---|---|---|---|---|---|
| (Intercept) | 4.8864 | 0.263184 | 134 | 18.56649 | 7.22E−39 |
| Cook_cond52.5 | 1.0668 | 0.369059 | 134 | 2.890592 | 0.004487 |
| Cook_cond60 | 0.522 | 0.369059 | 134 | 1.414407 | 0.159562 |
| Ref_temp8 | −0.0852 | 0.369059 | 134 | −0.23086 | 0.817778 |
| Ref_time | 0.0519 | 0.053269 | 134 | 0.974298 | 0.331664 |
| Cook_cond52.5:ref_temp8 | 0.0916 | 0.521929 | 134 | 0.175503 | 0.860949 |
| Cook_cond60:ref_temp8 | −0.0436 | 0.521929 | 134 | −0.08354 | 0.93355 |
| Cook_cond52.5:ref_time | −0.0867 | 0.075334 | 134 | −1.15088 | 0.251833 |
| Cook_cond60:ref_time | −0.1762 | 0.075334 | 134 | −2.33892 | 0.020818 |
| Ref_temp8:ref_time | −0.0411 | 0.075334 | 134 | −0.54557 | 0.586268 |
| Cook_cond52.5:ref_temp8:ref_time | 0.0746 | 0.106538 | 134 | 0.700218 | 0.485005 |
| Cook_cond60:ref_temp8:ref_time | 0.1339 | 0.106538 | 134 | 1.256826 | 0.211003 |
Cook_cond is cooking condition (levels: 52.5 for 65 min, 57.5 for 24 min, and 60.0 °C for 18 min). Ref_temp is refrigeration temperature (levels: 3 and 8 °C). Ref_time is the storage duration, which is a continuous variable. SE: Standard error. DF: Denominator degrees of freedom. Significance determined at P-value <0.05.
Slopes for L. monocytogenes count over 8 days of refrigerated storage in experiment 1
| Cook_cond | Ref_temp | Ref_time.trend | SE | DF | lower.CL | upper.CL | t.ratio | P-value |
|---|---|---|---|---|---|---|---|---|
| 60 | 3 | −0.0295 | 0.023142 | 134 | −0.07527 | 0.016271 | −1.27475 | 0.204605 |
| 57.5 | 3 | −0.0194 | 0.023142 | 134 | −0.06517 | 0.026371 | −0.83831 | 0.403351 |
| 52.5 | 3 | −0.0094 | 0.023142 | 134 | −0.05517 | 0.036371 | −0.40619 | 0.685251 |
| 60 | 8 | −0.009 | 0.023142 | 134 | −0.05477 | 0.036771 | −0.38891 | 0.697963 |
| 57.5 | 8 | −0.0243 | 0.023142 | 134 | −0.07007 | 0.021471 | −1.05004 | 0.295588 |
| 52.5 | 8 | 0.051 | 0.023142 | 134 | 0.005229 | 0.096771 | 2.203798 | 0.029246 |
Cook_cond is cooking condition (levels: 52.5 for 65 min, 57.5 for 24 min, and 60.0 °C for 18 min). Ref_temp is refrigeration temperature (levels: 3 and 8 °C). Ref_time_trend is change in bacterial counts over storage duration. SE: Standard error. DF: Denominator degrees of freedom. Lower.CL and upper.CL: 95% confidence interval. Significance determined at P-value <0.05.
Slopes for S. enterica count over 8 days of refrigerated storage in experiment 1
| Cook_cond | Ref_time.trend | SE | DF | lower.CL | upper.CL | t.ratio | P-value |
|---|---|---|---|---|---|---|---|
| 52.5 | −0.22597 | 0.064989 | 130 | −0.35454 | −0.0974 | −3.47711 | 0.00069 |
| 57.5 | −0.11986 | 0.064989 | 130 | −0.24843 | 0.008717 | −1.84425 | 0.067423 |
| 60 | −0.23388 | 0.065457 | 130 | −0.36338 | −0.10438 | −3.57305 | 0.000496 |
Cook_cond is cooking condition (levels: 52.5 for 65 min, 57.5 for 24 min, and 60.0 °C for 18 min). Ref_time_trend is change in bacterial counts over storage duration. SE: Standard error. DF: Denominator degrees of freedom. Lower.CL and upper.CL: 95% confidence interval. Results are averaged over the levels of refrigeration temperature. Significance determined at P-value <0.05.
Slopes for total viable counts over 8 days of refrigerated storage in experiment 1
| Cook_cond | Ref_temp | Ref_time.trend | SE | DF | lower.CL | upper.CL | t.ratio | P-value |
|---|---|---|---|---|---|---|---|---|
| 52.5 | 3 | −0.0348 | 0.053269 | 134 | −0.14016 | 0.070557 | −0.65329 | 0.514691 |
| 57.5 | 3 | 0.0519 | 0.053269 | 134 | −0.05346 | 0.157257 | 0.974298 | 0.331664 |
| 60 | 3 | −0.1243 | 0.053269 | 134 | −0.22966 | −0.01894 | −2.33343 | 0.021114 |
| 52.5 | 8 | −0.0013 | 0.053269 | 134 | −0.10666 | 0.104057 | −0.0244 | 0.980566 |
| 57.5 | 8 | 0.0108 | 0.053269 | 134 | −0.09456 | 0.116157 | 0.202744 | 0.839643 |
| 60 | 8 | −0.0315 | 0.053269 | 134 | −0.13686 | 0.073857 | −0.59134 | 0.555291 |
Cook_cond is cooking condition (levels: 52.5 for 65 min, 57.5 for 24 min, and 60.0 °C for 18 min). Ref_temp is refrigeration temperature (levels: 3 and 8 °C). Ref_time_trend is change in bacterial counts over storage duration. SE: Standard error. DF: Denominator degrees of freedom. Lower.CL and upper.CL: 95% confidence interval. Significance determined at P-value <0.05.
Fixed effects estimates for L. monocytogenes in experiment 2
| Value | SE | DF | t-value | P-value | |
|---|---|---|---|---|---|
| (Intercept) | 5.493706 | 0.201622 | 79 | 27.24752 | 6.37E−42 |
| Trtvp-UC | −0.18475 | 0.239738 | 79 | −0.77064 | 0.443218 |
| Ref_time | −0.0388 | 0.021882 | 79 | −1.77313 | 0.080062 |
| Trtvp-UC:ref_time | 0.050317 | 0.030718 | 79 | 1.638005 | 0.105398 |
Treatments (categories: SV (sous vide) and VP_UC (vacuum packed uncooked)). Ref_time is a storage duration, which is a continuous variable. SE: Standard error. DF: Denominator degrees of freedom. Significance determined at P-value <0.05.
Slopes for L. monocytogenes over 14 days of refrigerated storage in experiment 2
| Trt | Ref_time.trend | SE | DF | lower.CL | upper.CL | t.ratio | P-value |
|---|---|---|---|---|---|---|---|
| SV | −0.0388 | 0.021882 | 79 | −0.08235 | 0.004755 | −1.77313 | 0.080062 |
| VP-UC | 0.011518 | 0.021882 | 79 | −0.03204 | 0.055072 | 0.526362 | 0.600111 |
Trt is treatment (categories: SV (sous vide) and VP_UC (vacuum packed uncooked)). Ref_time_trend is change in bacterial counts over storage duration. SE: Standard error. DF: Denominator degrees of freedom. Lower.CL and upper.CL: 95% confidence interval. Significance determined at P-value <0.05.