Effect of Lactate and Diacetate-based Antimicrobial Agents on Listeria Monocytogenes in Ready-To-Eat Meat Products
Survival and growth of Listeria monocytogenes on RTE meat products formulated with a combination of lactate and diacetate-based antimicrobial agents was investigated. Cured (large diameter) and cured and smoked (small diameter) commercial RTE meat containing varying levels of sodium lactate/sodium diacetate, potassium lactate/sodium diacetate, sodium-potassium lactate/sodium diacetate were surface inoculated with approximately 100 CFU/100g of a five-strain mixture of L. monocytogenes. Products were vacuum-packaged and stored at 4±2ºC for up to 12 and 14 weeks for large and small diameter products, respectively. L. monocytogenes was recovered by surface plating method. A 2-log outgrowth of L. monocytogenes over the study period was used as a threshold of acceptance. Growth of L. monocytogenes was inhibited in cured chicken products containing sodium diacetate/sodium lactate at 1.46/0.10%, cured pork product with 2.07/0.148%, pork product with high water content containing 2.4/0.17%, cured turkey breast containing 2.24/0.16%, and cured chicken-beef-pork combination with 1.12/0.08 percent sodium lactate/sodium diacetate. Similarly, cured-smoked products with sodium lactate/sodium diacetate levels of 1.90/0.136%, 1.68/0.12% (beef-pork-chicken products), and 1.29/0.09% (chickenpork-beef skinless sausage) showed suppressed growth of L. monocytogenes. One cured-smoked beef product containing 1.12% sodium and potassium lactate and 0.08% sodium diacetate also maintained the level of L. monocytogenes below 2 logs. In contrast, a cured chicken product containing 1.90% potassium lactate and 0.136% sodium diacetate had more than 2 logs outgrowth of L. monocytogenes.
Listeria monocytogenes continues to be a pathogen of concern in ready-to-eat (RTE) meat products. Efforts to keep food free from Listeria spp. contamination include adequate product handling, sanitation practices and use of antimicrobial agents approved for meat products. Survival and growth of L. monocytogenes on RTE meat products formulated with a combination of lactate and diacetate-based antimicrobial agents was investigated. Less than two logs outgrowth of L. monocytogenes was used as the criteria of formulation acceptance.
Six small diameter and nine large diameter RTE meat products were formulated to contain a combination of sodium lactate, potassium lactate and sodium diacetate as antimicrobial agents in the meat and one product (corn dog) was formulated to contain antimicrobial agents in the batter/breading (Tables 1 and 2). All products (links and sliced) were shipped on ice to IEH Laboratories (Seattle, WA) by overnight delivery following production at Bar-S Foods facilities. Products were aseptically transferred to 15×20 cm, 2.48 mil high-barrier bags (Deli-Gold High-Barrier, 2.48 mil).
Listeria monocytogenes Inoculum
Five strains (IEH No. 4610, 5405, 4599, 4601, and 4547) of L. monocytogenes isolated from commercial meat processing environments were used to create a cocktail inoculum. Each bacterial strain was revived from frozen (-80°C) stock cultures into Listeria Enrichment Broth (LEB, BD), streaked on TSA with 5% blood agar and incubated at 35oC for 24 h. Colonies from overnight cultures of each strain were suspended in PBS. Equal volumes of each pathogen strain suspension were mixed to obtain a pathogen cocktail with a concentration of approximately 108 cells/ml. Appropriate dilutions were made immediately before inoculation.
Inoculation and Packaging
Three replicate samples of each product type were inoculated with 1 ml of the L. monocytogenes suspension. Products were hand-massaged for 30 s to distribute the inoculum on the product surface. All packages (including uninoculated) were vacuum sealed using a Multivac C20 packaging machine.
Large diameter products were stored at 4±2ºC throughout the study and evaluated at seven different times for up to 13 weeks (day-0, -42, -56, -70, -77, -84, and 91 (week-0, -6, -8, -10, -11, -12, and -13)). All small diameter products were stored at 4±2ºC during the 22 weeks shelf life study with the exception of Beef Franks which was stored at 0±2ºC for the first 6 weeks. Products were evaluated at ten different times for up to 22 weeks (day-0, -56, -70, -84, -91, 98, -105, -140, -147, -154 (week-0, -8, -10, -12, -13, -14, -15, -20, -21, -22)).
At each time point, triplicate samples of inoculated and one uninoculated sample were evaluated for each product type. Bags containing inoculated product were opened aseptically and approximately 50 ml of sterile 0.1% peptone water (0.1% PW, Becton Dickinson) was added to the bag and hand-massaged for 30 s to facilitate the recovery of L. monocytogenes survivors from the product. For enumeration of L. monocytogenes populations, a 1:10 serial dilution was performed using 0.1% PW. A volume of 0.1 ml was plated from appropriate dilutions onto Oxford Listeria agar (Acumedia, Lansing, MI) plates. Plates were incubated at 35°C for up to 2 days. Listeria monocytogenes populations were manually counted.
Uninoculated products were subjected to the same analysis as previously mentioned to facilitate the recovery of natural flora from the product. Serial dilutions were plated onto 3M Petrifilms™ (3M, St. Paul, MN) and incubated at 35 and 25°C for Aerobic Plate Counts (APC) and Lactic Acid Bacteria (LAB), respectively. Natural flora populations were manually counted following incubation. Counts were converted to log CFU to accommodate the anticipated wide fluctuation common to microbiological data.
Antimicrobial agents were deemed acceptable when less than 2 logs of L. monocytogenes outgrowth was observed throughout the 11 and 13 week target shelf life for large and small diameter products, respectively.
- Small diameter products: all lactate-diacetate formulations retarded the growth of L. monocytogenes. The final L. monocytogenes counts were lower than initial counts at the end of the study.
- Large diameter products: lactate-diacetate formulations were able to suppress growth of L. monocytogenes except on oven roasted chicken breast products containing potassium lactate/sodium diacetate (1.90/0.136). The population of L . monocytogenes increased by approximately 3 logs CFU/100g.
- Lactic acid bacteria counts were below the detection limit (approximately 11 cfu/100 g) throughout the shelf life study.
- Total aerobic bacteria populations were approximately 2.5 log cfu/100 g in general, except for cotto salami that reached 3.5 log cfu/100g.
- The target shelf life of 11 and 13 weeks for large and small diameter products, respectively, was achieved.
- This microbial challenge study suggested the combination of sodium lactate/sodium diacetate, potassium lactate/sodium diacetate, sodium lactatepotassium lactate/sodium diacetate were effective in inhibiting growth of L. monocytogenes to less than 2 logs outgrowth throughout the shelf life.
- In general formulations on small diameter products suppressed the growth of the pathogen more effectively than on large diameter products.
- Sodium-potassium lactate/sodium diacetate combination were as effective in suppressing L. monocytogenes as sodium lactate/sodium diacetate in small diameter products
- Potassium lactate/sodium diacetate combination (1.90/0.136) was not effective in controlling the 2 log outgrowth of L. monocytogenes in oven roasted chicken beyond the 11-week target shelf life. The same level of sodium lactate/sodium diacetate suppressed the growth of L. monocytogenes in spiced luncheon loaf.