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Growth of Listeria monocytogenes on Sliced and Inoculated Pastrami

Oleksandr A. Byelashov, Ifigenia Geornaras, Patricia A. Kendall1, and John N. Sofos SUMMARY A recent outbreak of listeriosis in North America triggered a massive recall of ready-to-eat meat products, including three different varieties of pastrami from a single plant implicated in the disease. This study evaluated the fate of L. monocytogenes on pastrami. Commercial pastrami (formulated without or with 1.44% sodium lactate and 0.1% sodium diacetate; SL/SD) was provided by a manufacturer, sliced, inoculated with a ten-strain mixture of L. monocytogenes, vacuum-packaged and stored at 39, 54, and 77 °F. The study was repeated twice using three samples per replication. Samples were analyzed periodically for pathogen numbers (PALCAM agar). Microbial counts vs. time were fitted using DMFit software. The initial levels of L. monocytogenes were 1.45±0.19 log CFU/cm2. Overall, lag phases were extended and growth rates of the pathogen were reduced by SL/SD and lower storage temperature. The longest lag phase (20 to 24 days) and the slowest growth rate (0.07 to 0.08 log CFU/cm2/day) was observed on pastrami formulated with SL/SD and stored at 39 °F. The pathogen grew 9 to 13 times faster on product without SL/SD that was stored at 77 °F, compared to product with SL/SD that was stored at 39 °F. These data show the effect of SL/SD against L. monocytogenes on pastrami, and highlight the importance of proper storage temperature. The findings of this study may be useful in new or

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updated L. monocytogenes risk assessments. Key Words: Listeria monocytogenes, Pastrami, Storage, Antimicrobials, Growth kinetics. INTRODUCTION Listeria monocytogenes is a psychrotrophic gram-positive salt- and pH-tolerant bacterium which is commonly found in the environment (Ryser and Marth, 2007). It is also a facultative intracellular pathogen which causes listeriosis, a potentially deadly disease, most commonly affecting immunocompromised people (Ryser and Marth, 2007). L. monocytogenes is of particular concern in ready-to-eat (RTE) meat and poultry products, as they may support growth of the pathogen even when stored at refrigeration temperatures (FDA, 2003). Between 1990 and 2000, the United States Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS) conducted microbiological testing in 1,800 federally inspected production facilities (Levine et al., 2001). It was reported that the cumulative 10-year prevalence of L. monocytogenes in deli meats was 5.16 % (Levine et al., 2001). It was also reported that the prevalence of L. monocytogenes in RTE products that were subjected to post-cooking handling, including peeling, slicing, dicing and packaging was higher compared to other products (Levine et al., 2001). Contaminated RTE meat and poultry products have been implicated in outbreaks and sporadic cases of listeriosis in North America. The most recent outbreak of listeriosis emerged in August of 2008 in Canada (PHAC, 2008). According to the Public Health Agency of Canada, as of October 17, 2008, there were 53 confirmed cases of the disease, including 20 deaths traced to RTE meat products manufactured by a single Canadian processing plant (PHAC, 2008). The number of confirmed cases may continue to rise

because the incubation period for the disease may be up to 70 days, and because of the lag in reporting of the symptoms by the consumers (PHAC, 2008). In response to the outbreak, the company voluntarily recalled 220 varieties of potentially contaminated products implicated in the outbreak, including pastrami (PHAC, 2008). In the quantitative L. monocytogenes risk assessment, the United Stated Food and Drug Administration and USDA-FSIS identified deli meats as a high risk product that may cause listeriosis (FDA, 2003). The agencies also emphasized the importance of time/temperature control during storage of RTE foods (FDA, 2003). This risk assessment was done using a risk-ranking model, which needs to be updated using L. monocytogenes growth kinetics data collected from specific commercially available products. This study was done to evaluate the behavior of L. monocytogenes on sliced, inoculated and then vacuum-packaged pastrami stored at 39, 54, and 77 °F. MATERIALS AND METHODS Inoculation of pastrami Pastrami (formulated without or with 1.44% sodium lactate and 0.1% sodium diacetate; SL/SD) was provided by a commercial manufacturer. Both types of product were cured with 2% of salt, sugar, sodium phosphate, flavorings, sodium nitrite, and sodium erythrobate. After curing, beef was coated with spices, caramel color, salt, dextrose, onion and garlic powder, and natural smoke flavor. The commercial products were sliced (0.08 inch thick; 2×2 inch size) in the Meat Laboratory of the Animal Sciences Department, and thereafter transferred to the Pathogen Reduction Laboratory for inoculation, packaging, storage and analysis. The L. monocytogenes inoculum consisted of ten strains of food, environmental or clinical origin: N1225, N1-227, R2-500, R2-501, R2763, R2-764, R2-765, 558, NA-1 and

Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, CO 80523

N-7150 (Fugett et al., 2006). Strains were activated, subcultured, habituated individually, and diluted in pastrami homogenate prior to inoculation of the product (Lianou et al., 2007). Sliced pastrami was inoculated on each side to obtain 1-2 log CFU/cm2, vacuum-packaged (2 slices/bag), and stored at 39, 54, and 77 °F for up to 90 days, or until the pathogen reached the stationary phase of growth. Microbiological analysis Before the analysis, samples were transferred into bags with 1.7 ounces of maximum recovery diluent, and shaken for 30 sec to dislodge the cells (Lianou et al., 2007). Appropriate dilutions were surface-plated onto PALCAM agar (Difco, Becton Dickinson, Sparks, MD) for enumeration of the pathogen. Inoculated agar plates were incubated at 86 °F for 48 h. The counts were expressed as log CFU/cm2. Data analysis The experiment was conducted twice using three samples per replication. DMFit software (Institute of Food Research, Norwich Research Park, Colney, UK) was used to fit the logarithm of microbial counts vs. storage time (Baranyi and Roberts, 1994). The output parameters included Y0 (lower asymptote of the sigmoid curve), lag phase duration (time needed for the pathogen to adjust to the new environment before growth is initiated), pathogen growth rate, and Yend (upper asymptote) (Baranyi and Roberts, 1994). RESULTS AND DISCUSSION The initial level of L. monocytogenes on inoculated product was 1.45±0.19 log CFU/cm2. The coefficient of determination, R2 for all treatments ranged between 0.71 and 0.99, indicating that the equation fit the data (Table 1). On product formulated without SL/SD and stored at 39°F, the pathogen grew at a rate of 0.15 to 0.26

log CFU/cm2/day after a lag phase of 3 to 4 days. As expected, the lag phase duration shortened and rates of pathogen growth increased with an increase in storage temperature. For example, at 77 °F, the pathogen grew 3 to 6 times faster than at 39 °F. This confirms that storage of deli meats at abusive temperatures in consumer refrigerators may substantially increase the risk of foodborne listeriosis. Le Marc et al. (2008) reported that the growth rate of L. monocytogenes on uncured roast beef formulated without antimicrobials and stored at 77 °F in vacuum-packages was 1.31 log CFU/cm2/day, which is higher than the highest growth rate observed on pastrami in our study. The lower growth rate of the pathogen on cured pastrami, compared to the uncured roast beef, may be explained by the presence of sodium nitrite as a curing agent, which inhibits metabolic processes in bacterial cells (Thompson et al., 2008). At all storage temperatures, the pathogen reached stationary phase at 6.9 to 7.7 log CFU/cm2. On product with SL/SD stored at 39 and 54 °F, pathogen growth rates were lower and lag phases were longer than those at corresponding storage conditions for product without SL/SD. However, lag phase was not observed on product with or without SL/SD stored at 77 °F, and the growth rates were similar. The longest lag phase duration (20 to 24 days) and the lowest growth rate (0.07 to 0.08 log CFU/cm2) was observed on product with SL/SD stored at 39 °F. Also, the stationary phase of the pathogen was not reached in these samples. Our results agree with previous findings which showed that salts of organic acids may suppress the growth of L. monocytogenes on sliced deli meats (Barmpalia et al., 2005). Results of this study confirm that the use of SL/SD in manufacturing of pastrami followed by the proper storage temperature at consumer homes may reduce the risk of listeriosis.

IMPLICATIONS These data show the fate of L. monocytogenes on inoculated pastrami stored at various temperatures, highlighting the importance of time/temperature control during storage of pastrami at all stages from manufacturing to consumption. These results may be useful for future L. monocytogenes risk assessments. ACKNOWLEDGMENTS: This work was supported by the National Integrated Food Safety Initiative of the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service (agreements 2004-51110-02160 and 2005-51110-03278), and by the Colorado State University Agricultural Experiment Station. LITERATURE CITED Baranyi, J., and T. A. Roberts. 1994. A dynamic approach to predicting bacterial-growth in food. International Journal of Food Microbiology 23:277-294. Barmpalia, I. M., K. P. Koutsoumanis, I. Geornaras, K. E. Belk, J. A. Scanga, P. A. Kendall, G. C. Smith, and J. N. Sofos. 2005. Effect of antimicrobials as ingredients of pork bologna for Listeria monocytogenes control during storage at 4 or 10 degrees C. Food Microbiology 22:205211. FDA. 2003. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Food Safety and Applied Nutrition, and U.S. Department of Agriculture, Food Safety and Inspection Service. Quantitative assessment of the relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. Available at: http://www.foodsafety.gov/~dms/ lmr2-toc.html.

Fugett, E., E. Fortes, C. Nnoka, and M. Wiedmann. 2006. International Life Sciences Institute North America Listeria monocytogenes strain collection: Development of standard Listeria monocytogenes strain sets for research and validation studies. Journal of Food Protection 69:2929-2938. Le Marc, Y., I. Geornaras, B. A. Carlson, Y. Yoon, J. Baranyi, and J. N. Sofos. 2008. Predicting the effects of storage temperature on growth of Listeria monocytogenes on roast beef formulated with or without antimicrobials. International Association for Food Protection 95-th Annual Meeting. Poster presentation #P5-29.

Levine, P., B. Rose, S. Green, G. Ransom, and W. Hill. 2001. Pathogen testing of ready-to-eat meat and poultry products collected at federally inspected establishments in the United States, 1990 to 1999. Journal of Food Protection 64:1188-1193. Lianou, A., I. Geornaras, P. A. Kendall, K. E. Belk, J. A. Scanga, G. C. Smith, and J. N. Sofos. 2007. Fate of Listeria monocytogenes in commercial ham, formulated with or without antimicrobials, under conditions simulating contamination in the processing or retail environment and during home storage. Journal of Food Protection 70:378-385. PHAC (Public Health Agency of Canada). 2008. Listeria

monocytogenes outbreak. October 17, 2008 - 15:00 (EST). Available at: http://www.phacaspc.gc.ca/alertalerte/listeria/listeria_2008eng.php. Accessed November 3, 2008. Ryser, E. T., and E. H. Marth. 2007. Listeria, listeriosis and food safety, vol. CRC Press, Boca Raton, FL. Thompson, R. L., C. E. Carpenter, S. Martini, and J. R. Broadbent. 2008. Control of Listeria monocytogenes in ready-to-eat meats containing sodium levulinate, sodium lactate, or a combination of sodium lactate and sodium diacetate. Journal of Food Science 73:M239-M24.

Table 1. Growth kinetics of Listeria monocytogenes on sliced and inoculated pastrami stored in vacuum-packages at 39, 54, or 77°F. Formulation Without SL/SDd Storage temperature (°F) 39 54 77 With SL/SD 39 54 77

a b

Lag phase duration (days) 3-4 1-2 0 20-24 3-4 0

Growth rate (log CFU/cm2/day)a 0.15-0.26 0.33-0.43 0.72-0.91 0.07-0.08 0.13-0.14 0.29-0.91

Y0 (log CFU/cm2)b 1.40-1.45 1.28-1.38 1.34-1.38 1.36-1.48 1.34-1.42 1.48-2.01

Yend (log CFU/cm2)c 6.92-7.22 6.79-7.61 7.41-7.66

e

R2 0.95-0.98 0.96-0.99 0.97-0.98 0.71-0.85 0.88 0.92-0.97

-

6.33-6.76 7.09-7.66

Maximum potential rate of L. monocytogenes growth (Baranyi and Roberts, 1994). Lower asymptote of sigmoid curve estimated by the Baranyi model (Baranyi and Roberts, 1994). c Upper asymptote of sigmoid curve estimated by the Baranyi model (Baranyi and Roberts, 1994). d SL/SD-1.44% sodium lactate and 0.1% sodium diacetate. e The stationary phase was not reached.

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