The goal of this project is to identify combinations of food-approved antimicrobials that are bactericidal to L. monocytogenes in processed poultry and meat products, including uncured, unsmoked turkey (<5% fat) and cured, smoked wieners (30% fat). Use of several ingredients that have antimicrobial activity will allow lower usage levels to prevent adverse effects on sensory and functional properties of the food. The project will be divided into two phases. Phase 1 will screen multiple combinations of antimicrobials in wiener or turkey slurries for listericidal activity. Use of 25% slurries provides an easy means to screen for antilisterial activity for multiple combinations of antimicrobials. Slurries have an advantage over media for these studies because they include food components that may have a neutralizing effect on certain antimicrobials. However, although brine content and pH are adjusted to mimic that of finished product, levels of fat, moisture, phenols, and nitrite, may not be the same as that found in finished product. Therefore, combinations identified as useful in slurries may be more or less effective than found in commercially acceptable RTE products. Pending results from the slurry studies, Phase 2 will verify if proposed combinations maintain their bactericidal activity when used in cooked turkey and beef/pork wiener products manufactured under commercial conditions. The application of combinations of GRAS antimicrobials to inactivate L. monocytogenes in these products could ensure their safety and prevent additional outbreaks and recalls.
During the first year of the project, 65 treatments were evaluated using single, double or triple-combinations of antimicrobials in both wiener and turkey slurries (pH adjusted to 6.2 after addition of antimicrobials) stored at 4 and 10 C. Several single antimicrobials and combinations demonstrated observable inhibition of L. monocytogenes compared with the control without antimicrobials. Statistical analysis of data has not been completed to date.<p>
Populations of L. monocytogenes increased an average 1 to 2- log, o CFU/g in control slurries without antimicrobials stored at 4 C for 1 week and an additional 1-2 log] 0 CFU/g at 2 weeks, after which populations remained constant at 8-log, o CFU/g during the duration of the study.
L. monocytogenes grew in most slurries stored at 10 C with the exception of a delay of growth by 100 ppm nisin, 50 ppm nisin+0.3% sodium diacetate, 0.25% propionate or benzoate, and in wiener slurries with combinations of >0.2% antimycotic agents (sorbate, benzoate, and/or propionate) or combinations of 0.1% propionate or benzoate and 0.5% Lem-O-Fos.<p>
All three of the antimycotic agents (sorbate, benzoate, and propionate) tested appreciably delayed growth of the pathogen in both wiener and turkey slurries stored at 4 C (Fig. 1). L. monocytogenes did not grow in wiener or turkey slurries supplemented with 0.25% potassium sorbate or propionic acid. Populations of the pathogens decreased in wiener slurries supplemented with 0.25% benzoic acid, but remained constant in turkey slurries containing the same level. Inconsistent results were observed for slurries supplemented with 0.1 % antimycotic agents, but generally delayed growth for 2-3 weeks compared with the control.
Combining any two or three antimycotics to provide at least 0.15% combined sorbate, propionate and benzoate inhibited growth of L. monocytogenes for 4 weeks storage at 4 C in both turkey and wiener slurries. Similarly, the combination of 0.05% propionate and 0.05% benzoate also prevented listerial growth in both slurry types. The addition of 0.5% Lem-O-Fos to 0.1% propionate, sorbate, or benzoate prevented listerial growth in wiener slurries for the 4 week storage at 4 C, but supported growth at 3 weeks in turkey slurries.<p>
Future Research: Several factors may influence the efficacy of antimicrobials against L. monocytogenes in processed meat products including moisture and salt content, nitrite, phenols from smoke, partitioning of non-water soluble antimicrobials into the fat phase, or binding of antimicrobials to muscle proteins. Although brine content and pH were adjusted to mimic that of finished product, levels of fat, moisture, phenols, phosphates, and nitrites may not be the same as that found in finished product. Therefore, combinations identified as useful in slurries may be more or less effective than found in commercially acceptable RTE products. Pending feedback from manufacturers on the results from Phase 1, formulations for RTE turkey and wieners will be manufactured with select combinations of antimicrobials and challenged with L. monocytogenes to verify efficacy in product.
Although the addition of the bacteriocin, nisin, can have potent bactericidal effects on L. monocytogenes, resistant strains of the pathogen are able to recover and grow in products stored at refrigeration temperatures during normal shelf-life times. The addition of Optiform, diacetate, or LemO-Fos can delay recovery. However, discussions with manufacturers suggest that enzymes present in the raw batter may inactivate nisin before cooking. Therefore, we suggest that nisin not be tested in Phase 2 of this study.<p>
Of all the classes of antimicrobials tested in this study, the antimycotic agents sorbate, propionate, and benzoate demonstrated the most consistent inhibitory activity (Fig. 1). The addition of 0.25% benzoic acid to slurries was bactericidal for product with nitrite, but bacteriostatic in product without nitrite. Supplementing slurries with 0.1% sodium diacetate or 0.5% Lern-O-Fos did not enhance the antimicrobial activity of potassium sorbate or propionic acid in either meat type compared with the use of 0.1% ofthe antimycotic agent alone; however, the addition of 0.5% Lem-O-Fos did provide an additional margin of safety to wiener and turkey slurries formulated with 0.1 % benzoic acid. Data suggests that when used in combination with each other, lower levels of individual antimycotic agents can inhibit growth of L. monocytogenes if the total concentration of the agents are >0.15%, or if 0.05% benzoic acid is used in combination with 0.05% propionic acid or potassium sorbate.<p>
The water solubility of sorbic acid, propionic acid, and benzoic acid differs from their sodium or potassium salt forms, which may determine the efficacy of these compounds in high-fat vs. low-fat ready-to-eat meat products. Additional trials in media are in progress to compare the relative antilisterial activity of the various forms of these agents (added on a percentage basis) in media formulated with and without added fat.<p>
Pending statistical analysis of the data and recommendations by the meat industry, Phase 2 of this study will evaluate the ability of two test formulations (plus control) for both cured and uncured ready-to-eat meat products.