Developing Bacteriophage Biomaterials for Applications in Food Safet

Bacteriophage therapy has several significant benefits over other antimicrobial approaches to control foodborne pathogens in food. Phages are host specific and can produce 10-1000 viral progeny per successful infection of a host cell. These advantages allow for specific targeting of the pathogen while leaving the food microbiota intact and regenerate themselves instead of depleting. However, phages have limited stability outside of an aqueous environment of neutral pH and ambient temperatures. Additionally, delivery to the food matrix is also a challenge as phages are large particles that are diffusion limited, and therefore, in order to effectively eliminate foodborne pathogens, the phages must be evenly distributed at high numbers.;The central approach of this work is to evaluate phage biomaterial formulations to address phage stability and delivery to foods. The specific aims of this work are to: (a) immobilize phages on cellulose membranes via physical adsorption, protein-ligand binding, and electrostatic interactions; (b) to encapsulate phages in whey protein isolate (WPI) films and dip coatings for direct application to foods; and (c) evaluate the mass transfer mechanism for phage release from WPI films. Escherichia coli DH5-alpha and BL21 with T4 and T7 phage, respectively, were used as surrogate organisms for E. coli O157:H7 and its phage. The phage biomaterial formulations were evaluated for phage loading on the material, the phage stability while in the material, phage release from the material, the antimicrobial efficacy of the phage material, and the phage distribution within the material. For phage loading, stability, and release, active phage particles were measured. For antimicrobial efficacy, the change in bacterial counts were measured. For the phage distribution, the phages were fluorescently labeled and imaged using a confocal microscope. For evaluating phage mass transfer from biomaterial formulations, a simple diffusion equation was numerically solved and probed for an effective diffusion coefficient based on fit with experimental data.;The results of this research demonstrate that phages overall benefit from being combined with biomaterial formulations. In cellulose membranes, phages gain improved loading and release from an electrostatic immobilization approach as compared to physical adsorption and protein-ligand binding. Both physical adsorption and protein-ligand binding loaded very little phage. Phages immobilized by electrostatic binding were evenly distributed across the fiber surfaces. In a separate approach, phages were encapsulated in WPI films and dip coatings. In WPI films, phages remained stable over several weeks at ambient and refrigerated temperatures, and the WPI films released phages over several hours in water and on leaf surfaces. Fluorescence imaging of the films revealed phage aggregates distributed randomly throughout the film matrix. In a similar vein, phages in WPI dip coatings were evaluated on sliced cucumbers, cut apples, and whole cherry tomatoes to evaluate phage loading, stability, antimicrobial activity, and gastric stability on diverse food surfaces. Sliced cucumbers, cut apples, and whole cherry tomatoes represent high moisture, low pH, and low moisture surfaces, respectively. Phage WPI dip coatings improved phage loading, stability, and antimicrobial efficacy in general over the surface types. However, phages, either in WPI dip coatings or control coatings, did not survive gastric digestion. Lastly, phage release from WPI films was evaluated against a numerically solved diffusion model. Phages from two types of WPI films, 1:1 and 2:1 protein to glycerol, were reported to have different effective diffusion coefficients.;Overall, the results of this research demonstrate the adaptability of phage therapy to biomaterials and biomaterials to addressing phage limitations. These approaches can enable new methods to controlling bacterial foodborne pathogens in foods.