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<OL><li>Ascertain the antimicrobial potential of newly identified chemical and biological antimicrobials designed to preserve microbiological safety of human foods. (i) Conduct studies to identify and quantify antimicrobial activity using in vitro snapshot and kinetic assays. (ii) Conduct studies to characterize or confirm the antimicrobial mechanism(s) of chemical and/or biological preservatives in vitro and in food systems where appropriate.</li>
<li>Develop and characterize novel systems for delivery and incorporation of chemical and biological preservatives in human foods to preserve food safety. (i) Produce lipid-derived micro- and nano-scale antimicrobials encapsulation systems for delivery of plant-derived and microbially-derived antimicrobials on to surfaces of fruit, vegetable, and processed meat systems. (ii) Conduct physico-chemical and rheological analyses of antimicrobial-encapsulating systems to determine storage and process stability of antimicrobial delivery technologies. (iii) Complete in vitro analyses differentiating the antimicrobial activity of naked (i.e. un-encapsulated) versus encapsulated chemical and biological preservatives for differing foodstuffs. (iv) Identify intelligent pairings of antimicrobials for delivery into foods based on compatibility of antimicrobial chemistries and mechanisms of activity. (iv-1) Complete research to identify antimicrobials with similar or compatible water/solvent solubilities to allow for co-delivery of antimicrobials into foods. (iv-2) Complete research determining optimal classes of encapsulating materials for delivery of antimicrobials into foodstuff(s).</li>
<li>Apply innovative food antimicrobial delivery systems to foods to investigate their utility in decontaminating food systems from bacterial foodborne pathogens. (i) Using collaborative model, identify food commodity classes (e.g., fresh or processed meat/poultry, pasteurized dairy, fresh produce) with high affinity/relevance for encapsulated antimicrobials. (ii) Apply/mix antimicrobial systems in foodstuffs following, prior to, or alongside pathogen inoculation procedures to model the antimicrobial activity against a targeted bacterial pathogen of relevance to the U.S. foods industry. (iii) Quantify and publish any enhancement in observed antimicrobial activity of encapsulated antimicrobial preservatives versus non-encapsulated antimicrobial compounds.</li>
<li>Complete research identifying factors/properties of foods inhibiting the successful utilization of antimicrobial encapsulate structures to provide holistic understanding of food safety opportunity of developed antimicrobial technologies. (i) Conduct sensorial research (using collaborating experts) to identify likely drivers/inhibitors of consumer acceptance of antimicrobial-treated food products. (ii) Conduct physico-chemical analyses (using collaborating experts) to identify and quantify physical, rheological, and chemical changes in foods following nano-encapsulated antimicrobials to determine limits to product keeping quality and palatability.Research objectives will be completed utilizing experimental methods conducted under conditions of Biosafety Level (BSL) 1 and 2 containment in research and food production arenas as appropriate to the specific study. Methods for research will be selected from amongst those published in the refereed literature, and will be selected for their capacity to produce needed data towards the answering of study-specific research questions/objectives. These can include spectrophotometric studies, time-course inoculated studies in liquid medium (i.e. in vitro) or in food product(s), studies utilizing non-inoculated food product treated with antimicrobial product, and others. Studies may be completed using known pathogenic microorganisms, pathogen index or indicator organisms, or non-pathogenic microbes characterized as being useful surrogates.Objective 1: Antimicrobial activity of newly developed antimicrobial preservatives will be initially characterized by use of the minimum inhibitory concentration (MIC) and/or minimum microbiocidal concentration (MMC), assays. These assays are well known to many researchers and will be functional in the preliminary identification of antimicrobial utility in a model system or in systems where the impact of a key food component (e.g. excess fat, protein) on antimicrobial activity is of concern. Subsequent assays will engage in time-course type assay, where the change in the population of a targeted microbe is needed to determine the growth inhibition aspects of an antimicrobial. These will allow researchers to determine a "best-possible scenario" for further development and refinement of antimicrobial use in food products. Objective 2: While the development of lipid-derived nano-encapsulates will take place in the investigator's laboratory, using standardized methods for capsule design, further processing of nano-capsules (e.g., polymer coating, infusion into edible coatings) will be completed through a collaborative research process with laboratories possessing necessary processing skills/technologies. Further, in instances where high-resolution analysis of developed nano systems (e.g., microscopy) is required, the investigator will partner with researchers or shared-access laboratories providing training and access to needed equipment/data analysis. Manufacture of nano-encapsulates will utilize methods including extrusion, reverse-phase solvent evaporation, electrostatic deposition of biopolymers, and electro-spinning, as appropriate. Physico-chemical analyses are varied, and can include size, electrophoretic mobility or surface charge distribution, calorimetric, microscopic, or other analytical procedures. Multiple collaborating laboratories may be contacted in order to complete necessary research studies. Microbiological analysis of keeping quality/safety of food products bearing nano-encapsulated antimicrobials will be completed in similar fashion to those described for completion of Objective 1. Objective 3: In collaboration with institution-located, and with research collaborators, studies will be designed to assess the utility of food antimicrobial systems under conditions approximating those encountered in the foods industry. Food products will be obtained from suitable vendors and inoculated using procedures mirroring the most likely route(s) of cross-contamination that would be expected to occur at the point of food product production, processing/packaging, transport, retail, or in-home preparation. Using appropriate procedures for application of antimicrobial(s) and recovery/enumeration of targeted microorganism(s), the impact of the antimicrobial application procedures will be quantified in terms of the numbers of microbes reduced by the application of the antimicrobial, or the difference in replication of microbes on antimicrobial-treated product versus non-treated controls. Such practices are widely utilized/recognized and can be used to produce industry training/best practices documentation, or assign antimicrobial activity descriptors (e.g., D-value) to a naked or encapsulated antimicrobial.Objective 4: In cooperation with collaborating expert scientists, the impacts on food product palatability and consumer acceptance will be completed where opportunity is presented, to gain holistic understanding of the antimicrobial(s) as food ingredients. Such studies will engage the cooperation of research scientists expert in foods rheology, sensory science, processing, and storage stability. Studies will be completed in laboratories bearing necessary technological capacity and expertise, and will be conducted using products treated under identical conditions to those used for antimicrobial activity assays, but will not incorporate any product intentionally inoculated with known microbial pathogens.</li></OL> </P>
<P>NON-TECHNICAL SUMMARY: Food antimicrobials are diverse in their chemistries, antimicrobial mechanisms of action, environmental sources, and range(s) of microbes inhibited. There is a growing interest amongst U.S. consumers for foods prepared/manufactured with fewer synthetic ingredients. The need for continuing research in the application and evaluation of natural food antimicrobials (chemical and biological) is significant. In addition to the numerous chemical food antimicrobials that are available to foods processors, multiple examples of biological preservatives (biopreservatives) exist that bear federal approval for use in foods that allow preservation of food safety by the antagonism or inhibition of microbial pathogens by the activity of other organisms intentionally applied to the foodstuff. There are two primary classes of biopreservatives: those utilizing microbes that effectively antagonize contaminating pathogens (e.g., fermentative lactobacilli, bacteriophages) and non-microbe containing fermentate of non-pathogenic microorganisms (e.g., bacteriocins, organic acid, etc.). Extensive research has been completed with both types of biopreservatives, and recent changes in U.S. regulations regarding the allowances of phages for direct addition to some foods for pathogen control has introduced opportunities for researchers to explore the utility of these agents as control strategies for food safety preservation. Nonetheless, there remains much left yet unexplored, in particular with respect to the utility of biopreservatives for the control of pathogens either recently identified or those who have recently come under stricter regulatory processes (e.g., the non-O157 Shiga toxin-producing E. coli as adulterants in non-intact beef products). Despite the variety of chemical and biological preservatives available for food protection application, there remain needs for improvement in the utilization of these types of food additives. Food product manufacturers, in light of evolving food safety regulations, must continue to explore and develop means of properly validating the antimicrobial activity of chemical or biological preservatives for the sake of proper adherence to HACCP and other regulatory systems (e.g., preventative controls). Additionally, research must continue in order to further enhance the antimicrobial activity of chemical and biological preservatives in foods. A forward-looking research plan would incorporate elements of all research discussed herein to produce novel application strategies for both chemical and biological antimicrobials to continue working to enhance food safety. This project describes basic and applied researches to investigate and optimize the usefulness of differing food-grade antimicrobial preservatives for the maintenance of food safety and quality. Anticipated outcomes include development of original research manuscripts for publication, extension service training systems for transmission of findings to industry stakeholders, training of graduate research students, and development of novel research grants that will further support outlined researches. </P>
<P>APPROACH: Research objectives will be completed utilizing experimental methods conducted under conditions of Biosafety Level (BSL) 1 and 2 containment in research and food production arenas as appropriate to the specific study. Methods for research will be selected from amongst those published in the refereed literature, and will be selected for their capacity to produce needed data towards the answering of study-specific research questions/objectives. These can include spectrophotometric studies, time-course inoculated studies in liquid medium (i.e. in vitro) or in food product(s), studies utilizing non-inoculated food product treated with antimicrobial product, and others. Studies may be completed using known pathogenic microorganisms, pathogen index or indicator organisms, or non-pathogenic microbes characterized as being useful surrogates.Objective 1: Antimicrobial activity of newly developed antimicrobial preservatives will be initially characterized by use of the minimum inhibitory concentration (MIC) and/or minimum microbiocidal concentration (MMC), assays. These assays are well known to many researchers and will be functional in the preliminary identification of antimicrobial utility in a model system or in systems where the impact of a key food component (e.g. excess fat, protein) on antimicrobial activity is of concern. Subsequent assays will engage in time-course type assay, where the change in the population of a targeted microbe is needed to determine the growth inhibition aspects of an antimicrobial. These will allow researchers to determine a "best-possible scenario" for further development and refinement of antimicrobial use in food products.Objective 2: While the development of lipid-derived nano-encapsulates will take place in the investigator's laboratory, using standardized methods for capsule design, further processing of nano-capsules (e.g., polymer coating, infusion into edible coatings) will be completed through a collaborative research process with laboratories possessing necessary processing skills/technologies. Further, in instances where high-resolution analysis of developed nano systems (e.g., microscopy) is required, the investigator will partner with researchers or shared-access laboratories providing training and access to needed equipment/data analysis. Manufacture of nano-encapsulates will utilize methods including extrusion, reverse-phase solvent evaporation, electrostatic deposition of biopolymers, and electro-spinning, as appropriate. Physico-chemical analyses are varied, and can include size, electrophoretic mobility or surface charge distribution, calorimetric, microscopic, or other analytical procedures. Multiple collaborating laboratories may be contacted in order to complete necessary research studies. Microbiological analysis of keeping quality/safety of food products bearing nano-encapsulated antimicrobials will be completed in similar fashion to those described for completion of Objective 1.Objective 3: In collaboration with institution-located, and with research collaborators, studies will be designed to assess the utility of food antimicrobial systems under conditions approximating those encountered in the foods industry. Food products will be obtained from suitable vendors and inoculated using procedures mirroring the most likely route(s) of cross-contamination that would be expected to occur at the point of food product production, processing/packaging, transport, retail, or in-home preparation. Using appropriate procedures for application of antimicrobial(s) and recovery/enumeration of targeted microorganism(s), the impact of the antimicrobial application procedures will be quantified in terms of the numbers of microbes reduced by the application of the antimicrobial, or the difference in replication of microbes on antimicrobial-treated product versus non-treated controls. Such practices are widely utilized/recognized and can be used to produce industry training/best practices documentation, or assign antimicrobial activity descriptors (e.g., D-value) to a naked or encapsulated antimicrobial.Objective 4: In cooperation with collaborating expert scientists, the impacts on food product palatability and consumer acceptance will be completed where opportunity is presented, to gain holistic understanding of the antimicrobial(s) as food ingredients. Such studies will engage the cooperation of research scientists expert in foods rheology, sensory science, processing, and storage stability. Studies will be completed in laboratories bearing necessary technological capacity and expertise, and will be conducted using products treated under identical conditions to those used for antimicrobial activity assays, but will not incorporate any product intentionally inoculated with known microbial pathogens.In all researches, studies will be designed with the assistance of researchers with expertise in experimental design and biostatistics. All data gathered will be subjected to statistical procedures deemed appropriate by biostatistics specialists. All laboratory personnel will undergo institution-required employee biosafety and laboratory safety training, and all investigator-supervised personnel will be required to submit all gathered original data to the investigator upon their departure from the institution. </P>