Research objective will focus on the optimization of antimicrobial encapsulation efficiency (EE) by alternative methods of antimicrobial loading into liposomal vesicles. Specifically, liposome loading via pH gradient-driven exchange mechanisms and multi-freeze/thaw cycling methodologies will be tested and EE determined via nisin bioassay. Objective II will seek to generate liposomes with broad-spectrum antimicrobial activity via modulating the lipid formulation and co-encapsulation of multiple antimicrobials, active against both Gram-negative and Gram-positive bacterial foodborne pathogens. Capsules that are wrapped in an antimicrobial polymer (chitosan) will be experimented with against model foodborne pathogens. Additionally, co-encapsulates containing both nisin and Gram-negative functional colicins (bacteriocins fermented by Escherichia coli spp) will be inoculated into fluid milk artificially contaminated with pathogens. Objectives three and four will focus on the application of antimicrobial-bearing liposomes to food commodities not previously tested, those being a pasteurized fruit juice (apple) and a ready-to-eat (RTE) processed meat product. Bacterial pathogens will be artificially inoculated in food commodities and liposome-based inhibition determined.
NON-TECHNICAL SUMMARY: Encapsulation of food antimicrobials allows for application of antimicrobials into a broader range of foods as compared to applications of non-encapsulated antimicrobials, thus producing a broader market for antimicrobials and a safer food supply. The purpose of this study is to investigate the efficacy of liposome encapsulation for the entrapment and controlled delivery of the food antimicrobial nisin in food products not normally conducive to nisin application. <P>
APPROACH: Objective 1 (TAMU): Liposomal antimicrobial loading efficiency (EE) will be determined experimentally via spectrophotometric assays by which percent reporter molecule encapsulated will be determined. Subsequent nisin bioassays will be generated whereby nisin or colicin activity as a function of loading methodology will allow for determination of optimized loading technology. Liposome formulation will be carried out according to previously reported methods. <P>Objective 2 (TAMU; UTK): Liposomes bearing antimicrobial(s) will be formulated at Texas A&M University (TAMU) and subsequently coated with the antimicrobial polymer chitosan after shipment to University of Tennessee-Knoxville, Dept. Food Science and Technology (UTK). Physical-chemical and biophysical properties of liposomes (size, -potential, thermotropism, etc.) will be measured using appropriate analytical equipment. Antimicrobial capabilities of coated liposomes against model Gram-negative and Gram-positive pathogens will be determined in vitro by micro-broth dilution checkerboard assay. Cells will be incubated at optimal conditions in the presence of nutritious growth medium and liposomes and changes in experimental vessel optical density (OD) recorded and analyzed for evidence of pathogen growth, stasis, or death. <P>Objective 3 (TAMU): Liposomes will be inoculated into ultra-high temperature (UHT) fluid skim, 1%, and whole milk; milk will be subsequently artificially contaminated with strains of the environmental Gram-positive pathogen L. monocytogenes and survivors will be enumerated on non-selective solid growth medium at different intervals of incubation at temperature-abuse conditions in order to determine the efficacy of antimicrobial encapsulate delivery into a model complex food matrix. Data will consequently be analyzed for potential inhibition of pathogens. <P>Objective 4 (TAMU; UTK): Liposomal antimicrobial will be formulated and inoculated on to polymer casings intended for RTE pork products (e.g. frankfurters). Vesicles will be initially optimized by way of variations in lipid profiles; these optimizations will seek to determine the optimal choice of lipids so as to guarantee heat-driven swelling and rupture of liposomes for the release of entrapped antimicrobial on to the surface of encased RTE meat products. Data gathering and analysis of thermal properties of various liposome formulations will occur at UTK. Samples will be incubated under conditions of storage abuse and survivors enumerated on non-selective growth medium (solidified) and data analyzed for bacterial growth, stasis, or inhibition. Microbiological analyses will occur at TAMU.