Progress 09/01/04 to 08/31/07
Outputs
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.
Impacts
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.
Publications
- Gaysinsky, S., Davidson, P. M., McClements, D. J., & Weiss, J. (2007). Formulation and Characterization of Phytophenol-Carrying Microemulsions. Food Biophysics, in print.
- Gaysinsky, S., Taylor, T. T., Davidson, P. M., Bruce, B. D., & Weiss, J. (2007). Antimicrobial efficacy of eugenol microemulsions in milk against Listeria monocytogenes and Escherichia coli O157:H7. Journal of Food Protection, in print.
- Gaysinsky, S., & Weiss, J. (2007). Aromatic and Spice Plants: Uses in Food Safety. Stewart Postharvest Solutions, 4, 9-16.
- Taylor, T. T., Bruce, B. D., Weiss, J., & Davidson, P. M. (2007). Listeria monocytogenes and Escherichia coli O157:H7 Inhibition in vitro by Liposome-Encapsulated Nisin and EDTA Journal of Food Safety, in print.
- Taylor, T. T., Davidson, P. M., Bruce, B. D., & Weiss, J. (2007). Antimicrobial Efficacy of Liposomal-Encapsulated Nisin and EDTA Against Listeria monocytogenes and Escherichia coli O157:H7 in Milk. Journal of Food Protection, in print.
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Progress 10/01/05 to 09/30/06
Outputs
We continued investigations into the role of biophysical properties on the activity of food antimicrobials. Results demonstrated the importance of protecting antimicrobials from undesirable interactions with other food components, delivering antimicrobials to the site of action (bacterial surface) and improving the interaction of antimicrobials with the pathogenic target organism. This year, we carried out work on validating efficiency of nano- and microencapsulated food antimicrobials against foodborne pathogens in complex food systems. Our carrier systems included micellar capsules, liposomes, and emulsions containing a wide variety of antimicrobials from strongly hydrophobic compounds such as parabens to phenolics such as eugenol and carvacrol and polypeptides and proteins such as lysozyme and nisin. We had previously shown that the efficiency of the encapsulation process was a function of the molecular properties of antimicrobials and the structure of the carrier
matrix which is determined by the molecular properties of the monomers that it is composed of. We conducted additional experiments demonstrating that the inhibition efficacy of encapsulated food antimicrobials in model microbiological systems depends on the properties of the pathogens (i.e. cell wall/membrane composition) and the overall biophysical properties of the carrier particles (e.g. size, charge, shape) that determine the overall interaction between the two. Of particular importance are the electrostatic interactions, i.e. antimicrobial efficacies can be greatly increased if carrier system and microorganisms carry opposite charges so as to attract each other. We also completed testing of liposomal and micellar systems in a model food system (skim, low fat and full fat milk). We found that encapsulation of nisin in liposomes maintained bacteriostatic activity against Listeria monocytogenes in 2% milk while addition of unencapsulated nisin did not inhibit growth. Addition and
encapsulation of EDTA in liposomes did not induce activity against E. coli, typically observed in model microbiological systems. We attributed this to the interaction of EDTA with the high concentrations of calcium in milk which may have led to EDTA not chelating metal ions in the bacterial cell wall but instead chelating calcium and minerals present in milk. In micellar systems, we demonstrated high activity of eugenol and carvacrol against both E.coli and Listeria monocytogenes in low fat and skim milk, but observed a loss of antimicrobial activity when the systems were added to full fat milk. We attributed the loss of activity to partitioning of the phenolics into the milk fat phase thereby reducing the active concentration of antimicrobial in the aqueous phase. We postulate that modification of the charge of the micellar system might help overcome the delivery of phenolics into the milk fat phase, i.e. we suggest that producing micelles with charges similar to that of the milk fat
droplets may reduce attractive interactions between micelles and milk fat droplets and reduce loss of phenolics into the milk fat phase. This hypothesis will be tested in the remaining time of the grant proposal.
Impacts
Our research has had direct economic and social impacts. The encapsulation of antimicrobials in nano- or microscale carrier systems can greatly increase the activity and in some cases broaden the spectrum of activity against pathogens growing in foods. Thus, the required concentrations of antimicrobials to achieve inhibition of growth decrease when a carrier system is used. The carrier systems can be manufactured form inexpensive, abundantly available materials. Thus the combination of antimicrobial and carrier system can greatly reduces cost for food manufacturers. For some systems, we have demonstrated that required concentrations of antimicrobials can be decreased by >50%, leading to cost savings between 15 - 30% depending on the price of the antimicrobials and the price of raw material for the capsules. Simultaneously, the health and wellbeing of the US consumers can be increased, Our systems broaden the applicability of antimicrobials to a variety of foods were
use of antimicrobials was previously not feasable. With these systems it now becomes possible to build in an additional level of protection against growth of pathogens and spoilage organisms.
Publications
- Taylor, T. M., S. Gaysinksy, P. M. Davidson, B. D. Bruce and J. Weiss (2006). Characterization of Antimicrobial Bearing Liposomes by Zeta-Potential, Vesicle Size and Encapsulation Efficiency. Food Biophysics revision submitted.
- Gaysinsky, S., P. M. Davidson and J. Weiss (2006). Antimicrobial Activity of Lauric Arginate and Benzoic Acid against Listeria monocytogenes and Escherichia coli O157:H7. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
- Gaysinsky, S., D. J. McClements and J. Weiss (2006). Emulsions as antimicrobial delivery systems: Influence of essential oil concentration on emulsion stability. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
- Taylor, T. M., P. M. Davidson, B. D. Bruce, S. Gaysinsky and J. Weiss (2006). Characterization of antimicrobial-bearing liposomes by zeta-potential, vesicle size, and encapsulation efficiency. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
- Taylor, T. M., P. M. Davidson, B. D. Bruce and J. Weiss (2006). Inhibition of Listeria monocytogenes by liposome-encapsulated nisin in skim, lowfat, and whole milk. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
- Taylor, T. M., P. M. Davidson, B. D. Bruce and J. Weiss (2006). Enhanced Listeria monocytogenes and Escherichia coli O157:H7 in vitro inhibition by liposome-encapsulated antimicrobial and chelator. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
- Weiss, J., D. Rosales and S. Gaysinsky (2006). Formation of Mixed Micelles Improves Antimicrobial Activity of Lauric Arginate against Listeria monocytogenes and Escherichia coli O157:H7 at Elevated pH Annual Meeting of the International Association of Food Protection, Calgary, Canada.
- Weiss, J., D. Rosales and D. J. McClements (2006). Improving pH and salt stability of lauric arginate (Mirenat-N) for food applications. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
- Weiss, J. (2005). Nanotechnological Approaches to Food Antimicrobial Delivery and Beyond. WorldNutra 2005, Anaheim, CA.
- Weiss, J. (2006). Advances in nanostructured design of encapsulation systems for use in foods. Annual Meeting of the Institute of Food Technologists, Orlando, FL.
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Progress 10/01/04 to 09/30/05
Outputs
The objective of this project is to develop novel encapsulation methods to improve activity of antimicrobials in food and food related systems. We successfully completed studies on encapsulation mechanism and inhibitory efficacy of phytophenol antimicrobials in food emulsifier nanoparticles. The working hypothesis for this aim was 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. Our investigations comfirmed this hypothesis. Different combinations of surfactant/phytophenol antimicrobials yielded significantly different activity in model microbiological systems. The investigated system Eugenol-Surfynol 465 had the highest observed activity and successfully inhibited E.coli and Lusteria
monocytogenes. Recent testing in model food system (skim and 2% milk) were very promising and indicated that activity while slightly lower than in model microbiological system is still maintained. In the second set of studies we determined the stability of liposomes containing polypeptide antimicrobials (lysozyme and nisin) and their efficacy to inhibit growth of pathogens. Our working hypothesis was 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. This hypothesis has so far been confirmed. Stability of liposomes was strongly influenced by the choice of phospholipids which also influenced the activity of these compounds in model microbiological systems against Listeria monocytogenes and Escherichi coli O157:H7.
Impacts
Nanostructured encapsulation systems can greatly improve activity of food antimicrobials in model microbiological and food systems thereby reducing concentrations needed for bacteriostatic and/or bacteriocical activity and limiting potential flavor impacts of antimicrobials on foods. Nanoencapsulated food antimicrobials may be used in the future as a substantial part of a hurdle concept to ensure food safety of a large variety of products
Publications
- Gaysinksy, S., P. M. Davidson, B. D. Bruce & J. Weiss (2005). "Growth Inhibition of Escherichia coli O157:H7 and Listeria monocytogenes by Carvacrol and Eugenol Encapsulated in Surfactant Micelles." Journal of Food Protection, 68(5).
- Gaysinksy, S., P. M. Davidson, B. D. Bruce & J. Weiss (2005). "Stability and Antimicrobial Efficiency of Eugenol Encapsulated in Surfactant Micelles as Affected by Temperature and pH." Journal of Food Protection, 68(7), 1359-1366.
- Taylor, T. M., P. M. Davidson, B. D. Bruce & J. Weiss (2005). "Liposomal Nanocapsules in Food Science and Agriculture." Critical Reviews in Food Science and Technology, in print.
- Taylor, T. M., P. M. Davidson, B. D. Bruce & J. Weiss (2005). "Ultrasonic Spectroscopy and Differential Scanning Calorimetry of Liposomal Encapsulated Nisin." Journal of Agricultural and Food Chemistry, in print.
- Were, L. M., B. D. Bruce, P. M. Davidson & J. Weiss (2004). "Encapsulation of nisin and lysozyme in liposomes enhances efficacy against Listeria monocytogenes." Journal of Food Protection, 67(5), 922-927.
- Were, L. M., B. D. Bruce, P. M. Davidson & J. Weiss (2004). "Size, stability and entrapment efficiency of phospholipid nanocapsules containing polypeptide antimicrobials." Journal of Agricultural and Food Chemistry, 51(27), 8073-8079.
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