Impact of Storage and Cooking Temperature on Listeria Innocua Growth in Plant-Based and Beef Proteins

Authors: Esther VanOverbeke, High School Student, Waverly High School, Waverly, NE; Olivier Munezero, Graduate Student; Gary Sullivan, Associate Professor; Samodha Fernando, Professor, Animal Science, Lincoln. 

Summary with Implications

Listeriosis, caused by Listeria monocytogenes and other Listeria spp., is a food-borne illness, that accounts for 260 deaths annually. The objective of this study was to identify how storage method and cooking temperature impacts Listeria innocua growth in ground beef and plant-based protein patties. Patties (162 ground beef; 162 plant-based) were used in 3 replications. For each replication, patties were either inoculated (1×108 CFU) or not inoculated and stored for 26 hr at refrigeration, freezer, or room temperature. Three patties per storage method were cooked to either 145°F or 165°F and were plated before/after cooking. Nine patties without inoculation were plated before/after cooking; and 9 patties were held at room temperature for 4 h after inoculation and plated before/after cooking. The results from this study showed cooking to an internal temperature of 165°F is more effective in eliminating L. innouca in ground beef. However, for plant-based patties, no difference in L. innocua abundance was observed based on cooking temperature or storage method. 

Introduction

Listeriosis is a foodborne infection caused by consuming food products contaminated with Listeria monocytogenes. Annually, an estimated1,600 individuals contract the bacterium resulting in 260 deaths, causing Listeriosis to be the third largest foodborne infection in the United States. Listeria species are facultative organisms that grow at a wide temperature range from 29°F to 113°F, making it a challenge to control. Therefore, Listeria species can grow at frozen/refrigerated and at room temperature conditions.  This project evaluated the growth and inhibition of Listeria innocua in ground beef and plant-based protein patties at different storage methods and to different cooking temperatures. This research is expected to help identify safe storage and cooking temperatures to reduce growth of Listeria innocua a surrogate organisms used to evaluate the growth and persistence of Listeria monocytogenes. As such, findings from this study can be applied to the common and dangerous food-borne pathogen, Listeria monocytogenes, as Listeria innocua and Listeria monocytogenes occupy the same environmental niche during colonization and growth.

Procedure

Twenty-seven packages of frozen Impossible burgers, and 41 packages of frozen ground beef burgers, resulting in 162 individual patties of each protein source was utilized in this study. Packages were stored at -4°F until use. Modified Oxford Agar plates which are selective for Listeria growth were used for quantification of pathogen load. The experiment was performed as 3 independent replicates with 3 technical replicates within each independent replicate giving 9 observations for each treatment. 
Briefly, the study procedure included storage of patties at 39°F overnight to thaw. Listeria innocua at a concentration of 1×108 Colony Forming Units (CFU)/ml was used for inoculating the beef and plant-based patties. The patties were placed in their respective treatment groups (Table 1)  in triplicate. Twenty-seven patties were inoculated with 1×108 CFUs of Listeria innocua and were incubated at respective storage temperatures which included, 39°F for 26 hr, -4°F for 26 hr or at room temperature (77°F) for either 4 or 26 hr. Control, uninoculated patties were placed in trays (3 patties per tray) and placed at their respective storage temperatures. Additional patties were inoculated immediately before cooking. After storage treatments, patties were either plated before cooking to determine growth or after cooking to measure lethality. Treatments identified for cooking were cooked to an internal temperature of 145°F or 165°F, depending on the treatment group. Cooking time was recorded for each patty.

All patties, cooked and uncooked, were macerated using a stomacher with 200 ml of PBS and a 1.5 ml aliquot of the sample was serially diluted in PBS (10-fold dilutions) and plated on selective media. Fifty microliters of each serial dilution was plated using the EddyJet serial plating machine and were incubated at 98°F overnight. Following incubation, plates were enumerated to identify Listeria abundance. This entire process was repeated for 3 replicate trials of ground beef patties and plant-based protein patties. 

Once all plates for all replicates were enumerated, Listeria abundance was reported CFU/g. All statistical analyses were performed using R (Version 4.4.2).  One-way ANOVA was used to assess significant differences in mean values among treatments and means were separated using Tukey’s HSD test. The CFU counts were reported as mean and the standard error of the mean (SEM) values. A P-value < 0.05 was considered significant. 

Results and Discussion

For patties stored in refrigeration (39°F), Listeria abundance was significantly decreased (P < 0.05) by cooking patties to an internal temperature of both 145°F and 165°F. The Listeria abundance was similar in treatments inoculated immediately prior to plating and those inoculated 26 hr prior to plating (P > 0.05). There was no difference (P > 0.05) in abundance levels for treatments cooked to 145°F and 165°F. However, a 1.5-2 log reduction was observed during cooking with a numerical decrease with increasing temperature.

For treatments inoculated with Listeria before freezing, before storing at 39°F, no difference (P > 0.05) in CFU counts were observed compared to treatments inoculated immediately prior to plating. There was also no difference (P > 0.05) in Listeria abundance between treatment that were plated before cooking and that were frozen and cooked to 145°F. There was significantly reduced Listeria abundance (P < 0.05) in treatments after cooking to 165°F compared to uncooked treatments.There was no difference in Listeria abundance (P > 0.05) between treatments cooked to 145°F and 165°F.

Ground beef patties stored at room temperature for 26 hr and cooked to 145°F had similar (P > 0.05) abundances of Listeria to the samples inoculated and stored at room temperature before cooking (Figure 1). There was a trend (P = 0.077) where the samples inoculated and stored at room temperature had higher abundance of Listeria before cooking compared to samples inoculated and stored at room temperature (77°F) for 26 hr and cooked to 145°F.  Additionally, a trend of decreased Listeria abundance (P = 0.066) was also seen for patties that were inoculated and stored at room temperature for 4 hr and cooked to 165°F compared to patties inoculated and stored at 39°F before cooking. There was a trend (P = 0.082) for patties inoculated, stored at room temperature for 4 hr and cooked to 165°F to have less Listeria abundance than the treatment that was inoculated and stored at room temperature for 4 hr.  Patties that were inoculated and stored at room temperature for 26 hr and cooked to 165°F had less abundance (P < 0.05) of Listeria than patties that were inoculated immediately prior to plating and patties inoculated and stored at room temperature for 4 or 26 hr prior to plating.

There were no significant differences (P > 0.05) in plant-based protein patties that were inoculated immediately prior to plating and those that were inoculated and stored under refrigeration for 26 hr before plating or cooking. No significant difference (P > 0.05) in Listeria abundance levels was observed for treatments that were inoculated immediately prior to plating and those that were inoculated and stored in -4°F for 26 hr before plating or cooking.

There was no difference (P > 0.05) between treatments with different incubation times before cooking (Figure 2). Decrease abundance (P < 0.05) of Listeria was observed in treatments of plant-based protein patties that were inoculated and stored for 4 hr and cooked to 145°F and 165°F as well as treatments stored for 26 hr and cooked to 165°F compared to the treatments that were inoculated and stored at room temperature for 26 hr (P < 0.05).

Regardless of storage temperature, ground beef patties, when cooked to 165°F, had significantly (P < 0.05) lower Listeria abundance than the inoculated patties that were stored at the same temperature and not cooked. Ground beef patties stored under refrigeration decreased from 5.23 log CFU/g to 2.74 log CFU/g when cooked to 165°F and to 3.61 log CFU/g when cooked to 145°F. When stored in the freezer for 26 hr, Listeria abundance decreased from 5.28 log CFU/g to 3.79 log CFU/g when cooked to 165°F. Additionally, when stored at room temperature for 26 hr Listeria abundance decreased from 5.9 log CFU/g to 2.37 log CFU/g when cooked to 165°F. There was no significant change in Listeria abundance with storage when stored at room temperature for 4 hr, however a 2-3 log reduction in abundance was observed in response to cooking to 165°F. 

Plant-based protein patties did not result in the same decrease in Listeria abundance with cooking as seen in ground beef patties. When stored under refrigeration, plant-based protein patties had 5.61 log CFU/g before cooking and decreased to 4.4 log CFU/g when cooked to 165°F. This decrease in Listeria abundance was not significant (P > 0.05). When stored in the freezer for 26 hr, plant-based proteins patties had 5.86 log CFU/g before cooking and 5.6 log CFU/g when cooked to 165°F the decrease. However, when stored at room temperature for 26 hr, plant-based proteins patties had 7.05 log CFU/g prior to cooking and decreased (P < 0.05) to 4.81 log CFU/g. 

Conclusions

Cooking did not result in the same impact of killing Listeria in ground beef patties and plant-based proteins. The USDA recommends that plant-based proteins and ground beef patties should be cooked to 160°F to kill bacteria and promote food safety. However, the data in this study do not support this for plant-based meat, suggesting that higher temperatures may be needed to kill Listeria in plant-based proteins. The reason for no significant decrease in Listeria in plant-based proteins after cooking is unknown. It can be noted that plant-based protein patties cooked at a faster rate than ground beef patties. Still, it is unknown if there is a relationship between time and temperature and interactions between the plant-based protein matrix and the pathogen that affects Listeria innocula killing. Additionally, the plant-based protein matrix may have protected Listeria from heat. The lower cooking time in plant-based proteins compared to ground beef may have resulted in decreased killing of Listeria. Current guidelines for cooking temperatures appear to be sufficient for ground beef patties but may need to be further explored for plant-based options. 

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