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Despite strict regulations to control presence of human pathogens in our food supply, there is an increasing incidence of disease resulting from food contaminated with human pathogens. Food safety issues with fresh produce and cut fruits are particularly significant as these products are handled heavily during harvesting and distribution, but minimally processed prior to consumption. To address these challenges we propose to develop novel and sustainable solutions based on integration of multifunctional food grade nanofibers with bacteriophages and food grade enzymes for controlling specific pathogens and disrupting biofilms on surface of fresh produce.<P> The specific objectives of this research are to: <ol> <LI> Develop food grade nanofibers with model biocontrol agents; <LI> Develop antimicrobial coatings based on encapsulation of phages in edible films and compare their antimicrobial properties with nanofiber-phage complexes;<LI> Design and develop food grade nanofibers linked with biofilm extracellular polysaccharide degrading enzymes and evaluate synergy between nanofiber bound enzymes and nanofiber-phage complexes in treating biofilms on food materials. </ol>The key expected outcome includes: <ol> <LI> Novel food grade nanomaterials with enhanced antimicrobial properties (specificity for target pathogens, improved performance of biocontrol agents) to maintain safety of minimally processed food for an extended period of time;<LI> Synergistic combination of nanomaterials, bacteriophages and enzymes to effectively treat biofilms on surfaces of food products;<LI> Edible antimicrobial material formulations that can be integrated with current packing materials and edible antimicrobial sprays. This research develops antimicrobial nanofiber-phage strategies to improve food safety and provides training for students.
Non-Technical Summary: Food safety continues to be one of the most significant challenges in our food supply. Safety issues with fresh produce and cut fruits are especially significant as these products are minimally processed and undergo multiple handling steps prior to consumption. To address these challenges, we propose to develop a novel and a sustainable biocontrol approach based on integration of nanoscale materials with bacteriophages and enzymes. It is expected that the combination of biological control agents (bacteriophages and enzymes) with advanced design of materials will lead to improved stability, sustained release and enhancement in activity of these biological control agents, resulting in more effective control on food borne pathogens. Bacteriophages, nature's selective antimicrobials, have unique advantages over conventional antimicrobials because of their high specificity for targeting bacteria and multiplying ability upon infecting the target bacteria. This research is aimed at integrating bacteriophages with nanoscale food grade materials to enhance stability of bacteriophages under ambient conditions and on food surfaces and improve anti-microbial activity by effective control of local concentration of bacteriophages on fresh produce. On food products, food borne pathogens often organize to form multi-species biofilms that can limit the access and activity of antimicrobial agents. This significant unmet need is addressed by investigating the synergy among nanoscale food grade materials, bacteriophages and biofilm extracellular matrix degrading enzymes to disperse biofilms and limit food borne pathogens. The overall plan is aimed at developing novel materials integrated with biological control agents to enhance the stability and efficacy of biological control agents for controlling food borne pathogens. This research will have a direct impact on technology development, its evaluation in model food systems and potential translation to food processing and packaging environment to develop sustainable biological control methods to improve food safety. <P> Approach: Aim 1: Design, synthesis and analysis of food grade nanofibers and nanofibrous membranes functionalized with model biocontrol agents. Nanofibers and nanofibrous membranes will be synthesized and fabricated from food grade polymer using wet chemical methods. Phages will be bound by physisorption, chemical conjugation, electrostatic interaction and physical encapsulation methods. Nanofiber and nanofibrous membrane bound phages will be characterized using a combination of microscopic and chemical analyses (SEM, TEM, FTIR etc.), microbiological, fluorescence imaging and toxicological analysis. Aim 2: Develop antimicrobial coatings based on encapsulation of phages in edible films and compare antimicrobial properties of nanofiber-phage complexes and edible films using model fresh produce and cut fruit surfaces inoculated with target pathogens. This aim is to compare performance of nanofiber-phage complexes with phages encapsulated in thin edible films. Performance will be compared based on a combination of antimicrobial efficacy and antimicrobial control for an extended period of time. Antimicrobial activity of selected composition from Aim 1 will be compared with thin edible films in various model systems including bacteria in solution, on solid surfaces e.g. glass, metal etc., fresh produce surface, cut fruits etc. A combination of microbial culture test (colony forming units) and non-invasive measurements using luciferase gene expression will be made. Aim 3: Design, synthesis and analysis of food grade nanofibers linked with biofilm extracellular polysaccharide (EPS) degrading enzymes and to evaluate synergy between enzymes linked nanofibers and nanofiber-phage complexes in treating biofilms on food materials. This aim is to develop multifunctional nanofibers based on a combination of bacteriophages and biofilm extracellular polysaccharide degrading enzymes. Nanofibers formulation selected in Aim 1 will be modified with biofilm EPS degrading enzymes and combined with nanofiber phage complexes to develop multifunctional nanofibers and nanofibrous membranes. The synergy between phages and biofilms degrading enzymes will be evaluated using model biofilms on stainless steel surface and lettuce leaves. These studies will generate knowledge needed to design and develop novel phage-based nanomaterials with enhanced antimicrobial properties (improved specificity as compared to conventional antimicrobials and activity of biocontrol agents). This project will also lead to novel materials that integrate biocontrol agents with enzymes to disrupt biofilms on surface of minimally processed food materials.