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The overall hypothesis for the proposed research is that aerosolizing liquid sanitizer in nano particle size smaller than 200 nm (the width of Salmonella Typhimurium LT2 was observed to be ~ 200 nm) will overcome the physical barrier of occluded air formed on produce surface and ensure the contact of sanitizer and microbial cell; hence, improve the efficacy of aqueous based sanitizers. This hypothesis is formulated based on scientific evidence and supportive preliminary results detailed in a later section that confirm the extensive work of our group to understand the mechanistic phenomena on the interaction of produce surface characteristic and survival of microbial cells aginst chemical aqueous based intervention methods.
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To test our overall hypothesis and accomplish the specific objective of this application by pursuing the following four specific objectives: <OL> <LI> Study the effect of produce surface characteristics on the internalization of different foodborne pathogenic bacteria The working hypothesis for this objective is that the degree of penetration of pathogenic bacteria and the rate of agglomeration is influenced by the microbial cell size, wettability of liquid inoculum and produce surface characteristics. Electron microscopy techniques such as Confocal Scanning Laser Microscopy (CSLM) and Scanning Electron Microscopy (SEM) will provide accurate measurement of cell internalization. <LI>Evaluate influence of physicochemical characteristic of liquid sanitizer that affect droplet size and stability of aerosolized aqueous sanitizers The working hypothesis for this objective is that physicochemical characteristic of liquid sanitizers such as viscosity and surface tension will influence droplet size formation and stability of aerosolized aqueous sanitizer, thus reducing effectiveness of dose delivery inside the produce surface. <LI>Evaluation of different droplet size penetration and rate of deposition inside different product surface. The working hypothesis for this objective is that by obtaining experimental data of the degree of drop penetration and deposition inside produce surface will be possible to validate mathematical models in order to predict effective dose delivery of aqueous sanitizer inside deep location below the produce surface and assure the destruction of internalized microbial cells. <LI>Evaluation of bactericidal effect of different droplet size of liquid base sanitizer The working hypothesis of this objective is that we will able to generate a stable and adequate droplet size carrying sufficient concentration of sanitizer to assure the reduction of large amount (~ 5 log10 CFU) of microbial cells internalized inside different surface characteristics.
NON-TECHNICAL SUMMARY: The rationale that underlies the proposed research is that understanding the phenomena of microbial internalization inside different produce surface morphology will enable to design and validate methods to improve sanitizer delivery to deep location or to remove viable cells of enteric pathogens. In addition, adapting and validating emerging technologies like nano-aerosolizations will lead to develop efficient intervention methods and ensure the safety of fresh produce without compromising their quality. Fruits and vegetables play an important role in our diet and health by providing essential vitamins, minerals, and fibers. In recent years there has been an increase in consumption of fruits and vegetables that has been associated with an increase in the presence of human pathogens found on fresh fruits and vegetables from both imported and domestic fresh produce. In an effort to reduce these concerns, this study proposes to improve and characterize nano-aerosolization technique to reduce the initial contamination of Escherichia coli, Salmonella enterica, and Listeria monocytogenes, in fruits and vegetables. Ultra (nano)-aerosolization (nebulization) technique of liquid based sanitizer will be used as model systems. Physicochemical properties, including viscosity, surface tension, and density of liquid sanitizer will be characterized to evaluate their influence in droplet size formation and stability. Application of nebulized liquid sanitizer in terms of antimicrobial effectiveness will be evaluated at ambient and refrigerated conditions. <P>
APPROACH: OBJECTIVE 1: Study the effect of produce surface characteristics on the internalization of different foodborne pathogenic bacteria. Internalization and attachment of microbial cells will be conducted based in Microscopy observations. Wettability of liquid solutions will be directly measured as surface tension with a semiautomatic tensiometer Model 21 Tensiomat (Fisher Scientific, Fair Lawn, N.J.). A contact angle analyzer (First Ten Angstroms, Portsmounth, VA) that included a video camera coupled to image analysis will be used to measure contact angles of two standard liquids (water and diiodomethane) formed on the produce surface. Surface tension of the produce will be indirectly calculated from the geometric mean equation using contact angles that standard liquids formed on the solid surface. <P>OBJECTIVE 2: Evaluate influence of physicochemical characteristic of liquid sanitizer that affect droplet size and stability of aerosolized aqueous sanitizers. Stability and concentration of aerosolized sanitizers affected by the drop size and their physicochemical characteristic will be assessed by sampling and quantification analysis of condensed aerosolized sanitizer. Lactic acid will be quantified by HPLC and the output curves detected will be compared with a standard curve previously obtained with appropriated standards. Hydrogen peroxide will be quantified using a colorimetric method with 4-aminoantipyrine and phenol in the presence of peroxidase <P>OBJECTIVE 3: Evaluation of different droplet size penetration and rate of deposition inside different product surface. Evaluation of different droplet size penetration on different product surface will be carried out incorporating a fluorescent dye compounds such as Rhodamine B that emit strong red color signals at 590 nm. Generation of different drop size, application period time and amount of dye absorption will be used to validate lagrangian or Eularian models for prediction of adequate dose delivery of sanitizer. <P>OBJECTIVE 4: Evaluation of bactericidal effect of different droplet size of liquid base sanitizer. Produce surface (e.g. cantaloupe, strawberry, curly parsley, spinach, lettuce, orange and tomato stem scar) previously inoculated with individual or cocktail of rifampicin resistant microorganism (Rif+ E. coli O157:H7 and Rif+ Salmonella serotypes Agona, Montevideo, and Typhimurium LT2) will be treated with the most stable and suitable sanitizer at different droplet size in the range of 3 to 100 nm, generated by an electro spray Model 3480 (TSI Inc., St. Luis, MN). Samples of 100 ul from appropriate serial dilution in 0.1% of peptone water will be spread plated on differential media lactose-sulfite-phenol red-rifampicin agar (LSPR, 80 ug/ml of rifampicin). These plates will be incubated at 37C for 24 h. Rif+ E. coli O157:H7 will produce yellow colonies and the Rif+ Salmonella sp. colonies will develop a black center surrounded by a pink halo. Both colonies will be reported separately as CFU/ ml.