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The overall goal of this research is to identify and characterize the mechanisms of growth inhibitor combinations that result in synergistic effects on growth inhibition for Listeria monocytogenes in cold smoked salmon. This information will allow for development of effective, synergistic combinations of growth inhibitors for L. monoyctogenes. <P>
This goal will be attained through the following specific aims: <OL> <LI> Identify synergistic combinations of growth inhibitors for L. monocytogenes in a smoked salmon food matrix<LI>Evaluate the ability of synergistic combinations of growth inhibitors to reduce or prevent L. monocytogenes growth on commercially produced cold smoked salmon<LI>Characterize the genome-wide transcriptional response of L. monocytogenes exposed to synergistic combinations of growth inhibitors in a cold smoked salmon food matrix. </ol> The experiments resulting from these specific aims will quantitatively identify combinations of growth inhibitors that exhibit synergistic effects on L. monocytogenes growth, where the effect of the combination is greater than the sum of each inhibitor alone. Additionally, we will determine the utility of salmon slurry as a proxy to evaluate synergistic combinations of growth inhibitors that can inhibit L. monocytogenes growth on cold smoked fish, which can facilitate broader use of growth inhibitors in the smoked seafood industry.<P>
Furthermore, we will evaluate the impact of synergistic combinations of growth inhibitors on L. monocytogenes gene expression which will improve our understanding of gene expression and physiological variation among L. monocytogenes present in cold smoked salmon.
NON-TECHNICAL SUMMARY: The bacterial pathogen Listeria monocytogenes causes a potentially severe foodborne disease that kills about 500 people annually in the U.S. As initial contamination of food products generally occurs at low levels and as the human infectious dose appears to be high, post-contamination multiplication of L. monocytogenes usually needs to occur for the pathogen to reach high enough levels in food products to cause human disease. Cold smoked seafoods are included among the foods whose consumption has been associated with a high risk of listeriosis. Control of L. monocytogenes represents a considerable challenge for food processors as this pathogen is commonly found in raw materials and in diverse environments, including food processing plants. L. monocytogenes has been isolated from fresh, frozen, and processed seafood, and is of particular concern in cold-smoked fish products, as it is capable of surviving the salting and cold-smoking process. Natural and chemical growth inhibitors have been shown to reduce growth of L. monocytogenes on cold smoked fish products, but need to be validated, including under actual processing conditions. Further research is clearly needed to identify effective growth inhibitor combinations that can be used to control L. monocytogenes in cold smoked fish. The goal of this proposal is to identify combinations of growth inhibitors that have synergistic effects on growth inhibition of L. monocytogenes on cold smoked salmon, ultimately resulting in a safer food product.
<P>APPROACH: Growth of L. monocytogenes strains will be monitored in salmon slurry at 7 degrees C with combinations of the following growth inhibitors: nisin, green tea extract, sodium diacetate, potassium lactate, and nitrite. Growth parameters for each strain in each combination of growth inhibitors will be determined using the Baranyi model and synergistic effects identified by two-way ANOVA. The optimal concentration of synergistic combinations will be determined and then evaluated against a larger, diverse set of L. monocytogenes strains to prove broad efficacy. After synergistic combinations have been identified in salmon slurry, they will be tested for efficacy against L. monocytogenes on cold smoked salmon fillets. L. monocytogenes will be inoculated onto the surface of cold smoked salmon fillets prepared with the synergistic combinations of growth inhibitors and incubated at 7 degrees C. Cell densities will be monitored over time, and growth parameters determined as described for the slurry experiments. ANOVA will be used to determine if growth inhibition occurred in a similar manner in the slurry and on the fillets. The combination of growth inhibitors that has the greatest efficacy in the salmon slurry and on salmon fillets will be chosen for transcriptional profiling experiments. L. monocytogenes RNA will be extracted from cells exposed to salmon slurry (i) without additional inhibitors, (ii) each growth inhibitor of the combination individually, and (iii) the combination of growth inhibitors. Samples will be competitively hybridized on the JCVI L. monocytogenes microarray, and differences in gene expression determined by ANOVA. This data will provide new insights into the L. monocytogenes stress response in foods, and can be used to develop high-throughput screening methods to identify specific stress responses induced by L. monocytogenes in response to different growth inhibitors or food preservation treatments.