Progress 03/01/10 to 08/31/14
Outputs
Target Audience: The target audiences are seafood processors, suppliers of ingredients for Listeria control as well as researchers investigating control measures for Listeria monocytogenes on RTE foods. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided training and professional development opportunities for three post-doctoral fellows that have been partially supported and trained by this project; two of the post-docs that were trained on this project have moved on the faculty positions (Teresa Bergholz has moved to a tenure track faculty position in North Dakota State University, Siyun Wang has moved to a tenure track faculty position at University of British Columbia). In addition, two graduate students and one undergraduate have been partially supported and trained through this project; one of these PhD students (Jihun Kang) will start a post-doc with FDA CFSAN in 01/2015. This project thus has made considerable contributions to the training of future food safety professionals. How have the results been disseminated to communities of interest? As detailed in the publication section above, results from this project have been communicated through two peer-reviewed papers (with one additional paper in preparation and one paper submitted) and through poster presentation at scientific meetings. In addition, results were communicated informally to seafood processors. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Controlling L. monocytogenes has proven difficult for food processors in the seafood industry because this pathogen is commonly found within food processing plants and in raw materials. The impact of this project lies in the fact that our efforts developed (i) rational scientific approaches to develop synergistic combination of growth inhibitors that can effectively reduce L. monocytogenes growth and (ii) provide specific science-based guidance for seafood processors on combinations of bacteriostatic and bacteriocidal antimicrobials to use in smoked seafoods to control L. monocytogenes. Reducing L. monocytogenes growth significantly reduces food safety hazards as the vast majority of human listeriosis cases is caused by products that have allowed growth of L. monocytogenes to considerable numbers. Initial work under this project has created key data that allow for guidance on effective growth inhibitor combinations that can prevent L. monocytogenes growth on smoked seafood. We specially quantified the effects of antimicrobials on growth of 20 L. monocytogenes strains representing diverse genetic backgrounds in BHI broth and on cold-smoked salmon. A key finding from these analyses was that the correlation between BHI and salmon growth parameters was dependent on L. monocytogenes genetic background as described by genetic lineage; thus effective growth inhibitor combinations will need to be evaluated and validated with different L. monocytogenes strains on smoked salmon. Our work will also facilitate selection of appropriate strains for future work that aims to validate growth inhibitors for use in salmon. Strains used in our experiments are freely available to the scientific community to facilitate use of our findings. Our data specifically showed that initial L. monocytogenes density was significantly lowered by addition of nisin with an average reduction of 2.02 +/- 0.99 Log10(CFU/g) on cold smoked salmon, which was similar to that found in BHI broth (1.51+/-0.83 Log10(CFU/ml)). The combination of lactate and diacetate had the greatest increase in lag phase both on cold smoked salmon (7.1+/-3.6 days) and in BHI broth (9.7+/-3.8 days) compared to the controls. Overall, the combination of lactate and diacetate as well as the combination of lactate and nisin had the greatest effect among the inhibitors tested on reducing L. monocytogenes growth, providing specific guidance on growth inhibitor combinations for stakeholders. While both the combination of lactate and diacetate, and lactate and nisin were found to be most effective at preventing L. monocytogenes from reaching numbers that are likely to cause human disease at refrigerator temperatures; the effectiveness of nisin varied considerably and some L. monocytogenes strains showed some resistance to nisin. We thus also determined the antimicrobial resistance mechanisms employed by L. monocytogenes to counteract the effect of nisin as well as other novel antimicrobial compounds to further improve our ability to design affective combination approaches to control L. monocytogenes on smoked seafood including cold smoked salmon. This work specifically showed that the two component regulatory system VirRS and VirRS-mediated regulation of dltABCD are major resistance mechanism used by L. monocytogenes against cell envelope-damaging food antimicrobials, including nisin, lauric arginate, and ε-polylysine (this work has been submitted for publication in 11/2014). These data suggest that antimicrobials that interfere with VirRS and VirRS activation may be able to further enhance the effectiveness of cell envelope-damaging food antimicrobials for L. monocytogenes control. To more broadly advance development and discovery of effective control strategies to inhibit the growth of L. monocytogenes on salmon, we used RNA-sequencing to compare the transcriptome profiles of L. monocytogenes grown at refrigerated temperature on cold smoked salmon and in BHI broth (modified to represent water phase salt concentration and pH typical for cold smoked salmon) in order to identify specific genes and gene expression patterns shown by L. monocytogenes when present on cold smoked salmon. Briefly, L. monocytogenes strain H7858 (FSL F6-0366, lineage I, serotype 4b was grown to stationary-phase and inoculated into (i) 100 ml modified BHI broth with 4.65% waterphase NaCl (pH = 6.1) and (ii) both sides of a 10 ± 0.5g cold-smoked salmon slice. Inoculated BHI broth and vacuum packed salmon slices with initial L. monocytogenes concentration at about 10^6 CFU/ml were incubated at 7 oC for up to 9 days, followed by extraction of total RNA. Our results showed that genes involved in cobalamin biosynthesis, ethanolamine and propanediol utilization, and specific carbohydrate transport systems have significantly higher transcriptional level in L. monocytogenes cells grown on cold smoked salmon as compared to the ones grown in BHI broth. The fact that ethanolamine and propanediol metabolic pathways induced in cold smoked salmon produce acetate and propionic acid as end products, could also suggest that growth inhibitors that include these two organic acids may be able to more effectively inhibit L. monocytogenes growth than currently used growth inhibitors that include acetate and lactate.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Tang, S., M. J. Stasiewicz, M. Wiedmann, K. J. Boor, and T. M. Bergholz. 2013. Efficacy of different antimicrobials on inhibition of Listeria monocytogenes growth in laboratory medium and on cold-smoked salmon. Int. J. Food Microbiol. 165:265-275
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Tang, S., M. J. Stasiewicz, M. Wiedmann, K. J. Boor, T. M. Bergholz. 2013. Correlations of the Effects of Nisin and Organic Acids on Listeria monocytogenes Growth Inhibition in BHI Broth and on Cold Smoked Salmon Are Lineage-dependent. 113th General Meeting, American Society for Microbiology, May 18-21, 2013, Denver, Colorado (poster).
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Progress 02/28/13 to 02/27/14
Outputs
Target Audience: The target audiences that were reached during this year were seafood processors as well as researchers investigating control measures for Listeria monocytogenes on RTE foods. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided training and professional development opportunities for both a post-doctoral fellow that have been partially supported by this project and a graduate student that has been partially supported; this project thus is continuing to make contributions to the training of future food safety professionals that can address the food safety challenges faced by the US seafood industry. How have the results been disseminated to communities of interest? As detailed in the publication section above, results from this project have been communicated through a peer-reviewed paper published in 2013 and through a poster presentation at the 113th General Meeting of the American Society for Microbiology. In addition, results were communicated informally to seafood processors; more formal communication of results to end users will take place towards the end of this project. What do you plan to do during the next reporting period to accomplish the goals? For the next reporting period, we plan to complete the data analysis of the RNA-seq experiments, including Biocyc pathway analyses to identify specific pathways that are differentially regulated when Listeria monocytogenes is present in cold smoked salmon. We also will complete a publication that describes these results and their implications for developing better control strategies for L. monocytogenes.
Impacts
What was accomplished under these goals?
Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a severe illness with symptoms including meningitis, septicemia and spontaneous abortion. It is a leading cause of hospitalization and death due to foodborne illnesses in the United States. Controlling L. monocytogenes has proven difficult for food processors in the seafood industry because this pathogen is commonly found within food processing plants and in raw materials. The impact of this project lies in the fact that our efforts develop rational scientific approaches to develop synergistic combination of growth inhibitors that can effectively reduce L. monocytogenes growth. Reducing L. monocytogenes growth significantly reduces food safety hazards as the vast majority of human listeriosis cases is caused by products that have allowed growth of Listeria monocytogenes to considerable numbers. To rationally develop effective control strategies to inhibit the growth of L. monocytogenes on salmon, it is critical to better understand the mechanism that L. monocytogenes uses to survive and multiple in smoked almonds stored under refrigeration temperatures. Changing gene expression is one of the major strategies that microbes use to sense and respond to stress conditions. Hence, during this reporting period, we used RNA-sequencing to compare the transcriptome profiles of Listeria monocytogenes grown at refrigerated temperature on cold smoked salmon and in modified BHI broth in order to identify specific genes and gene expression patterns shown by L. monocytogenes when present on cold smoked salmon. Briefly, L. monocytogenes strain H7858 (FSL F6-0366, lineage I, serotype 4bwas grown to stationary-phase and inoculated into (i) 100 ml modified BHI broth with 4.65% waterphase NaCl (pH = 6.1) and (ii) both sides of a 10 ± 0.5g cold-smoked salmon slice. Inoculated BHI broth and vacuum packed salmon slices with initial L. monocytogenes concentration at about 10^6 CFU/ml were incubated at 7 C for up to 9 days, followed by extortion of total RNA. A number of technical hurdles had to be overcome to obtain high quality L. monocytogenes RNA from salmon. The final optimized protocol developed included (i) use of RNA Protect reagent (Qiagen, Valencia, CA) to stop the transcription; (ii) separation of fish particles (rich in RNase) from bacterial cells with filter bags, followed by (iii) bacterial cells lysis and RNA extraction using proteinase K, lysozyme, bead-beating, and TriReagent. After mRNA enrichment, we constructed indexed cDNA libraries for sequencing by using ScriptSeq™ Complete Kit (Bacteria) - Low Input and ScriptSeqTM Index PCR Primers. Libraries were sequenced using the Illumina HiSeq system. Sequencing data for four independent replicate RNA samples were analyzed using BWA, SAMtools, baySeq, GOseq, and BioCyc. Using these methods, we have identified 149 differentially expressed genes (Bayesian posterior likelihood > 0.95, fold change > 1.5) between L. monocytogenes growing on cold-smoked salmon and in BHI. Among these, 21 genes are related to cobalamin metabolism and are specifically involved in the oxygen-independent (anaerobic) pathway, 7 genes encode proteins of the PTS system which assures optimal utilization of carbohydrates for bacteria in complex environments, 3 genes and 2 genes are involved in maltose utilization and ethanolamine utilization, respectively. Gene Ontology (GO) enrichment analysis for differentially expressed genes highlighted 13 general biological processes significantly up-regulated during growth on salmon slices, including vitamin metabolic process, cobalamin metabolic process and tetrapyrrole metabolic process. These data provide important initial insights in the mechanisms that L. monocytogenes uses to grow on vacuum-packaged salmon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Tang, S., M. J. Stasiewicz, M. Wiedmann, K. J. Boor, and T. M. Bergholz. 2013. Efficacy of different antimicrobials on inhibition of Listeria monocytogenes growth in laboratory medium and on cold-smoked salmon. Int. J. Food Microbiol. 165:265-275
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Tang, S., M. J. Stasiewicz, M. Wiedmann, K. J. Boor, T. M. Bergholz. 2013. Correlations of the Effects of Nisin and Organic Acids on Listeria monocytogenes Growth Inhibition in BHI Broth and on Cold Smoked Salmon Are Lineage-dependent. 113th General Meeting, American Society for Microbiology, May 18-21, 2013, Denver, Colorado (poster).
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Progress 02/29/12 to 02/27/13
Outputs
Target Audience: The target audiences for the outputs and outcomes generated this year are seafood processors as well as researchers investigating control measures for Listeria monocytogenes on Ready-To-Eat foods. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided training and professional development opportunities for both a post-doctoral fellow that have been partially supported by this project and a graduate student that has been partially supported; this project thus is continuing to make contributions to the training of future food safety professionals that can address the produce safety challenges faced by the US seafood industry. The post-doc supported by the project has recently been appointed to a faculty position in North America, which illustrates the professional development impact that project had. How have the results been disseminated to communities of interest? As detailed in the publication section above, results from this project have been communicated through a poster presentation at the 2012 General Meeting of the American Society for Microbiology. In addition, results were communicated informally to seafood processors; more formal communication of results to end users will take place towards the end of this project. What do you plan to do during the next reporting period to accomplish the goals? For the next reporting period, we plan to optimize combinations of bactericidal and bacteriostatic treatments to identify the most cost-effective treatments against L. monocytogenes on salmon. We will also develop and evaluate RNA-seq based methods to characterize the genome-wide transcriptional response of L. monocytogenes exposed to cold smoked salmon; this will facilitate subsequent studies to characterize the transcriptional response of L. monocytogenes exposed to synergistic combinations of growth inhibitors in cold smoked salmon.
Impacts
What was accomplished under these goals?
This project has created key data that allow for the better and more effective growth inhibitor combinations that can prevent Listeria monocytogenes growth on smoked seafood. In this reporting period, we specially quantified the effects of antimicrobials on growth of 20 Listeria monocytogenes strains representing diverse genetic backgrounds in BHI broth and on cold-smoked salmon. Two strains did not show obvious growth for most of the treatments on salmon. Hence, the analyses performed were based on 20 strains for BHI (10 strains in lineage I and 10 strains in lineage II) and 18 strains for salmon (10 strains in lineage I and 8 strains in lineage II). While selected growth parameters were reported in the prior CRIS report, we, in the current reporting period, focused on evaluating the correlation of growth parameters from BHI broth to those from cold smoked salmon. A key finding from these analyses was that the correlation between BHI and salmon growth parameters was dependent on L. monocytogenes genetic background as described by genetic lineage. Multivariate analysis showed positive correlations between a given growth parameter from BHI and the same parameter from salmon. As the correlations varied among lineages and treatments with or without nisin, we split the data into two groups, one with parameters from treatments with organic acid salts only, the other with parameters from treatments with nisin, and analyzed the data by lineage within each group. The coefficient of determination (R2) for each parameter for lineage II strains were higher than those for lineage I strains. This indicates that those growth parameters from BHI had a relatively stronger correlation with those from salmon for lineage II strains. Overall, across antimicrobial treatments, we found that the relatively high R2 values for lineage II strains make it possible to predict the variability of these growth parameters in salmon from data for BHI, while the variability of growth parameters for lineage I strains were relatively unpredictable. For example, for treatments with organic acid salts, R2 for lineage I strains indicates that only 21% of the variance in maximum cell density (Nmax) from salmon was accounted for by change in Nmax for BHI compared to 75% for lineage II. Although the positive correlation coefficients from multivariate analysis and the positive slope parameters from the linear regression both indicate similarities between L. monocytogenes growth characteristics under antimicrobial stress in BHI and on salmon, quantitative prediction of the performance of antimicrobials on salmon was possible for lineage II, but not for lineage I strains of L. monocytogenes. While both the combination of lactate and diacetate, and lactate and nisin were found to be most effective at preventing L. monocytogenes from reaching numbers that are likely to cause human disease at refrigerator temperatures; the effectiveness of nisin varied considerably and some L. monocytogenes strains were highly resistant to nisin. This work has been the basis for new on-going research efforts that focus on characterizing the mechanism of action of different inhibitors, which will further improve our ability to design affective combination approaches to control this foodborne pathogen on smoked seafood including cold smoked salmon. Our finding that results from bacterial growth media could quantitatively predict the variability of growth parameters in salmon for some L. monocytogenes strains (i.e., lineage II strains), but not for lineage I strains, will facilitate selection of appropriate strains for future work that aims to validate growth inhibitors for use in salmon. Strains used in our experiments are freely available to the scientific community to facilitate use of our findings.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Tang, S., M. J. Stasiewicz, T. M. Bergholz, M. Wiedmann, and K. J. Boor. 2012. Comparison of Growth Inhibitor Efficacy Against Listeria monocytogenes in Laboratory Medium and on Cold Smoked Salmon. 112th General Meeting American Society for Microbiology. June 16-19, 2012, San Francisco, CA. (poster)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Kang, J., S. Tang, R. H. Liu, M. Wiedmann, K. J. Boor, T. M. Bergholz, and S. Wang. 2012. Effect of curing method and freeze-thawing on subsequent growth of Listeria monocytogenes on cold-smoked salmon. J. Food Prot. 75: 1619-1626.
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Progress 03/01/11 to 02/28/12
Outputs
OUTPUTS: The presence of the food-borne pathogen Listeria monocytogenes on cold-smoked salmon is a major concern for the seafood industry. Understanding processing and post-processing handling factors that affect the ability of this pathogen to grow on cold-smoked salmon is critical for developing effective control strategies. In this study, we investigated the effect of curing method and freeze-thawing of cold-smoked salmon on (i) physicochemical properties and (ii) subsequent growth of genetically diverse strains of L. monocytogenes (inoculated after freeze-thawing) and endogenous lactic acid bacteria. The majority of the measured physicochemical properties were unaffected by freezing and thawing. Based on the results obtained from this study, we focused on evaluating growth inhibitors against L. monocytogenes on wet-cured, frozen-thawed cold smoked salmon, as that combination of factors provided the most permissive growth conditions for L. monocytogenes. Eighteen strains of L. monocytogenes were grown in brain heart infusion (BHI) broth, as well as on the surface of commercially produced wet-cured cold smoked salmon slices at 7C. BHI broth and cold smoked salmon were supplemented with potassium lactate (PL) (2% water phase (w.p.)), sodium diacetate (SDA) (0.14% w.p.), nisin (NI) (50ppm), three binary combinations of two different inhibitors at the same levels, or without inhibitors as the control. Growth parameters (initial cell density (N0), lag phase (λ), maximum growth rate (μmax), maximum cell density (Nmax)) were then calculated from plate counts of L. monocytogenes over time. PARTICIPANTS: Individuals who worked on this project include Kathryn Boor, Project Director, Teresa Bergholz, research associate, Siyun Wang, postdoctoral researcher, Jihun Kang, Ph.D. student in Food Science, and Silin Tang, Ph.D. student in Food Science. TARGET AUDIENCES: The target audiences for the outputs and outcomes generated this year are seafood processors as well as researchers investigating control measures for Listeria monocytogenes on RTE foods. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Overall, wet-cured cold-smoked salmon had a higher pH, water activity, and moisture, as well as lower fat, water-phase salt, and phenolic content, compared to dry-cured cold-smoked salmon. The curing method and freeze-thawing did not affect growth of endogenous lactic acid bacteria. Freeze-thawing cold-smoked salmon prior to inoculation led to pronounced growth of L. monocytogenes at 7C. The increase in cell density between day 0 and day 30 was significantly (p = 0.0078) greater for cold-smoked salmon that was frozen and thawed prior to inoculation compared to non-frozen cold-smoked salmon. On dry-cured, freeze-thawed cold-smoked salmon, L. monocytogenes had a lag phase ranging from 3.7+/-0.1 to 11.2+/-1.4 days compared to salmon that was wet-cured and freeze-thawed, on which L. monocytogenes began to grow within 24 h. Variation in growth among L. monocytogenes strains was also observed, indicating the significance of assessing multiple strains. Further efforts to understand the impact of processing and post-processing handling steps of cold-smoked salmon on the growth of genetically diverse L. monocytogenes would contribute to improved challenge study designs and data. This, in turn, will likely lead to more reliable and unbiased risk assessments and control measures. In a comparison of growth inhibitors, singly and in combination, we found that growth parameters of L. monocytogenes were significantly influenced by the combinations of growth inhibitors. Initial cell density was significantly lowered by addition of nisin with an average reduction of 1.91 +/- 1.14 Log10(CFU/g) on cold smoked salmon, which was similar to that found in BHI broth (1.48+/-0.88 Log10(CFU/ml)). The combination of lactate and diacetate had the greatest increase in lag phase both on cold smoked salmon (9.71+/-4.96 days) and in BHI broth (9.25+/-4.49 days) compared to the controls. Synergistic reductions in Nmax on cold smoked salmon were not predicted by results in BHI broth, where the combination of lactate and nisin had a synergistic reduction of Nmax in BHI broth only, and the combination of lactate and diacetate led to synergistic reduction of Nmax on cold smoked salmon only. Overall, the combination of lactate and diacetate as well as the combination of lactate and nisin had the greatest effect among the inhibitors tested on reducing L. monocytogenes growth. The effect of lactate was enhanced by nisin on lowering initial cell density and Nmax, and enhanced by diacetate on extending lag phase and lowering Nmax. These data suggest that results from the lab medium can underestimate the effects of inhibitors on growth but could be used to rapidly identify effective combinations for further examination on cold smoked salmon.
Publications
- No publications reported this period
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Progress 03/01/10 to 02/28/11
Outputs
OUTPUTS: The overall goal of this project is to identify growth inhibitor combinations with synergistic effects on L. monocytogenes growth on cold smoked salmon, and to characterize the mechanisms of action of these inhibitors. As part of this project, we have quantified growth of L. monocytogenes in salmon slurry with growth inhibitors. The inhibitors tested include 0.14% water phase (w.p.) sodium diacetate, 2% w.p. potassium lactate, and 50ppm nisin, both individually and in combination. Plate counts over time were used to calculate growth parameters for strain H7858 in the presence of the inhibitors. Our results indicate that nisin, alone as well as in combination with lactate or diacetate, reduced initial cell density by ~0.5 log10CFU/g compared to the control without inhibitors. The combination of lactate and diacetate led to the greatest increase in lag phase, which was 6 days longer than the control. Nisin in combination either with diacetate or lactate increased the length of lag phase by ~ 3 days compared to diacetate or lactate alone. The combinations of lactate and diacetate, as well as nisin and lactate, were found to have synergistic effects on increasing lag phase. To characterize the mechanism of action of inhibitors by determining how L. monocytogenes responds to the inhibitor at the cellular level, we will measure changes in L. monocytogenes gene expression that occur in the presence of the inhibitor(s). Isolation of high quality L. monocytogenes RNA from cells inoculated onto smoked salmon was a technical hurdle that needed to be addressed in order to begin these experiments. We have developed and validated a protocol to isolate high quality RNA from L. monocytogenes inoculated onto smoked salmon slices using a phenol:chloroform extraction. 108 CFU/g L. monocytogenes cells were surface inoculated onto 25g cold smoked salmon at 7C. Inoculated salmon slices were transferred to 25mL RNA protect and vortexed. The supernantant containing cells that were removed from the salmon surface was then used for RNA extraction using TRIreagent and a bead beater. Following DNAse treatment and a final phenol:chloroform extraction, RNA quality was evaluated on a Bioanalyzer, and the RNA integrity was >7, indicating good quality. This RNA extraction procedure will be used to isolate RNA from L. monocytogenes inoculated onto salmon slices with and without inhibitors, and RNA sequencing will be used to quantify differences in the transcriptome. PARTICIPANTS: Individuals who worked on this project include Kathryn Boor, Project Director, Teresa Bergholz, research associate, Siyun Wang, postdoctoral researcher, and Silin Tang, Ph.D. student in Food Science. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: We have modified the method that we will use to evaluate changes in gene expression. Our original plan was to monitor changes in gene expression of L. monocytogenes in a smoked salmon slurry using microarrays. Researchers on this project have now gained the skills necessary to utilize RNA sequencing to evaluate gene expression changes, and we will use RNA sequencing instead of microarrays. RNA sequencing has several advantages over microarrays - it is not dependent on hybridization to known probe sequences, so different strains can be evaluated, and it provides an increased level of detail, including transcription start and stop sites.
Impacts
The major outcome of this project over the last year is the development and validation of the RNA extraction protocol. This method will allow us to evaluate how L. monocytogenes modifies its transcriptome in response to inhibitors and other stresses when it is grown on an RTE food (i.e. smoked salmon), and provide more insights into the mechanisms this organism uses to manage these stresses when on an actual food product. The combinations of lactate and diacetate, as well as the combinations of nisin and lactate, were found to have synergistic effects on reducing L. monocytogenes growth, and will be the focus of further investigation.
Publications
- No publications reported this period
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