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Antimicrobial Delivery Systems to Improve Food Safety

Weiss, Jochen
University of Massachusetts
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The objective of this research proposal is to develop innovative delivery systems that increase the activity of food antimicrobials to improve food safety. The central hypothesis is that lack of activity of food antimicrobials can be overcome by encapsulating antimicrobials in delivery systems that are capable of targeting pathogens while decreasing interaction with interfering food components.

Our research objective is to identify the inhibition mechanism and efficacy of three antimicrobial carrier systems: (a) food-emulsifier based nanoparticles that contain encapsulated phytophenols from spice essential oils (thymol, eugenol and carvacrol) (b) colloidal dispersions composed of carrier oils and phytophenols and (c) liposomes manufactured from polar lipids that are able to incorporate polypeptide antimicrobials such as lysozyme and nisin.

Based on our preliminary studies, we suggest that these encapsulation techniques provide a chemically active environment that enhances inhibitory activity and increases stability of antimicrobials in microbiological and model food systems.

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NON-TECHNICAL SUMMARY: Application of nanotechnologies may mprove food safety. Naturally-occuring antimicrobials capable of preventing the growth of pathogenic organisms have generally low activities in foods because of undesirable interactions with food components. In this project we will develop new food preservation strategies based on nanotechnological approaches to produce nanometer sized antimicrobial systems in the form of particles that improve antimicrobial activity in food formulations and food process operations. Three different encapsulation systems have shown promise. These include: (1) natural phenolic compounds encapsulated in surfactant-based micelles for application in liquid/semi-fluid food systems (2) phospholipid liposomes for encapsulation of polypeptide antimicrobials and application in liquid or solid systems and (3) natural phenolic and polypeptide antimicrobials encapsulated in emulsion droplets for delivery in liquid/semif-fluid and solid food systems. We expect that the new systems will have either substantially higher antimicrobial activity or higher stability than free antimicrobials. Because of the small size of capsules, no change in appearance and texture of foods should be observed. This research has the potential to dramatically improves the safety of processed foods and may have counter-bioterrorism as well as military applications.

APPROACH: We plan to test our central hypothesis and accomplish the overall research objective of this application by pursuing the following three research objectives. (1) Determine encapsulation mechanism and inhibitory efficacy of phytophenol antimicrobials in food emulsifier nanoparticles The working hypothesis for this aim is that the efficiency of the encapsulation process is a function of the molecular properties of antimicrobials and food emulsifiers while the inhibition efficacy of encapsulated food antimicrobials depends on the properties of the pathogens (i.e. cell wall/membrane composition) and the properties of nanoparticles such as size and charge. (2) Determine delivery mechanism and inhibition efficacy of phytophenol antimicrobials in colloidal dispersions (UMASS). The working hypothesis for this aim is that the mechanism of delivery of antimicrobials is a function of the colloidal properties of the emulsion such as droplet size, concentration, charge and thickness of droplet interfacial layer. (3) Determine stability of liposomes containing polypeptide antimicrobials (lysozyme and nisin) and their efficacy to inhibit growth of pathogens. The working hypothesis is that the stability of liposomes is a function of bilayer composition and environmental conditions and that the efficiency of the liposomes to prevent growth of pathogens depends on the amount of antimicrobial that can be encapsulated and the properties of phospholipids bilayers.

PROGRESS: 2004/09 TO 2007/08
OUTPUTS: In the course of this projects two Ph.D. students were mentored (Sylvia Gaysinksy and T Mathew Taylor) that completed their Ph.D's in 2007 at the University of Massachusetts and University of Tennessee, respectively. These students conducted the bulk of the research with the advisors (Dr. P.M. Davidson and Dr. J. Weiss) assisting in the analysis of the results, the preparation of the manuscripts, the presentations in the public domain and the thesis. Two Ph.D. thesis were prepared at the respective Universities that summarize the results of the project. Over the course of the three years, results were presented at the Institute of Food Technologists Annual Meeting, the International Association of Food Protection Annual Meeting, the American Oil Chemists' Society, the Institute of Life Sciences North America Meeting, the National Academy of Sciences, the National Research Council (as part of the Food Nanotechnology inquiry). Sylvia Gaysinksy gave 11 presentations, Matt Taylor gave 6 presentations, J. Weiss gave more than 10 presentations on the topic. 12 articles were published in peer-reviewed journals such as Food Biophysics, International Journal of Food Protection, and the Journal of Food Safety. 2 patents were submitted through the University of Massachusetts.
PARTICIPANTS: Academic Supervisors: Jochen Weiss, Associate Professor, University of Massachusetts, Amherst. P. Michael Davidson, Professor, University of Tennessee, Knoxville. Ph.D. Students: Sylvia Gaysinksy (completed Ph.D. in 2007, now works for Sensient Technologies). T. Matthew Taylor (completed Ph.D. in 2006, now works as Assistant Professor at Texas A&M University) Other Training Opportunities: A number of undergraduate reseachers participated in the project. Sylvia Gaysinksy also collaborated with Sarisa Suriyarak, an M.S. student at the University of Massachusetts on the emulsion project. Contacts: The project lead to contacts (and consulting) with Pepsico R&D, Hormel Foods, Coca-Cola, Frito Lay R&D, and Sara Lee.
TARGET AUDIENCES: Target audience for this project is the Food Industry in general. The technology is applicable to a wide variety of food products including beverages, bakery products, meats, fruits and vegetables etc.

IMPACT: 2004/09 TO 2007/08
The objective of this project was to formulate liposomes, emulsions and microemulsions as novel colloidal and nanoscalar carriers of antimicrobials to overcome their low activity and short duration of action in many foods. During the project funding period we developed, characterized and validated the activity of antimicrobial carrying capsules as novel preservation systems for foods. 1. Liposomes as Antimicrobial Carrier Systems. Ability of liposomes to maintain integrity was tested by encapsulation efficiency (EE), zeta potential, and vesicle size. PC, PC/PG 8/2, and PC/PG 6/4 (mol fraction) liposomes retained between ~70-90% EE despite exposure to elevated temperature or extreme pH. Liposome size averaged 100-240 nm. L. monocytogenes inhibition depended slightly upon dose, but was heavily dependent upon phospholipid constituents of liposomes. Near complete inhibition of E. coli O157:H7 with liposomal antimicrobial and chelator at concentrations below those required for unencapsulated antimicrobial and chelator was found. In milk, liposomal nisin was inhibitory to L. monocytogenes strains, and effects on strains were equivalent, regardless of milkfat level. 2. Microemulsions as Antimicrobial Carrier Systems. Eugenol was solubilized into cationic-nonionic (Mirenat-N-T-Maz80K or LAE-TM) and nonionic surfactant mixtures (T-Maz80K-Surfynol485W or TM-S485). Physicochemical characterization included surface tension, particle size, charge and solubilization capacity. The antimicrobial efficiency of cationic-non-ionic micelles was high since LAE alone inhibited the growth of E. coli O157:H7 and Listeria. Micelles inhibited all microbial growth with exception of TM:LAE (5:1) ratio. Addition of eugenol at 3mM inhibited the growth of Listeria and 7 mM inhibited the growth of E. coli O157:H7. When microemulsions were tested in a food system (milk), the antimicrobial efficiency varied depending on the fat level. 3. Emulsions as Antimicrobial Carrier Systems. Emulsions containing eugenol and a carrier lipid were kinetically stable depending on eugenol and lipid mixing ratios. Corn-oil emulsions loaded with eugenol were the most stable and inhibited the growth against E. coli O157:H7 strains depending on loading ratio but failed to inhibit growth of Listeria strains. Specific Impacts/Outcomes: Colloidal carrier systems can prolong activity of a large number of antimicrobials in model microbiological and model food systems. Some carrier systems can enhance the activity of antimicrobials against selected microorganisms and in some cases not only inhibit but inactivate pathogens. Overall, less antimicrobial is needed to retard activity of pathogen if an encapsulation system is used compared with the simple addition of the antimicrobial to the food. Products can be microbially stabilized for a significantly enhanced period. This enables the design of new shelf-stable food products and nanoencapsulation is therefore clearly an enabling technology for the food industry. The research results directly contribute to the enhanced safety and well-being of the US consumer by introducing a new control measure for food pathogens in the market.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Escherichia coli
Natural Toxins
Viruses and Prions
Bacterial Pathogens
Chemical Contaminants