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The long-term goal of our research project is to develop novel and effective strategies to prevent the growth of biofilms on food and food processing surfaces to improve consumer and workers health and safety. To achieve this long-term goal, we intend to develop a better understanding of the complex mechanisms that govern the interaction of cleaners (surfactants) and sanitizers/antimicrobials with biofilms. Ultimately, we would like to formulate comprehensive biophysical models that are capable of describing the mode of action of surfactants and sanitizers/antimicrobials against pathogens growing in biofilms in terms of the genetic, biochemical and biophysical properties of the biofilm. The objective of this particular grant proposal, is to improve control measures for foodborne microbial pathogens growing in biofilms by delivering surfactants and disinfectants/antimicrobials simultaneously by selecting compatible combinations of the two classes of compounds that spontaneously form water-soluble swollen micelles. The central hypothesis for the proposed research is that encapsulation of antimicrobial compounds in surfactant micelles may improve diffusion or convection-driven transport of biofilm-inhibiting or inactivating substances into biofilms and potentially weaken adhesion forces that keep cells attached to each other and/or to substrate surfaces. These systems may aid in both inactivation and removal. Furthermore, the use of nonionic (uncharged) surfactants suitable to deliver potent hydrophobic antimicrobials that are typically insoluble in water may help overcome the common problem of inactivation of active compounds in the top layer of biofilms. We suggest that nonionic capsules would neither be repelled not attracted (which would lead to irreversible binding and subsequent build-up of a repulsive layer) by positively or negatively charged cell surfaces and exopolymers that comprise the top layer of the biofilm. In this project, we propose to encapsulate lipophilic antimicrobials using a micellar system. In particular, nonionic micelle-encapsulated antimicrobials are chemically benign and their activity stems solely from the disruption of the integrity of bacterial cell membranes rather than a chemical reaction that is found in traditional sanitizers such as chlorine compounds or QUATS. Problems associated with corrosion and evaporation of traditional sanitizers could be prevented as well. Encapsulation of volatile compounds in micelles has shown to lead to a decrease in vapor pressure thereby depressing evaporation. In contrast to traditional sanitizers that can have limitations upon usage temperature and pH, our preliminary work over the past 2 years has shown that antimicrobial micelles are both pH (2 - 12) and temperature stable (70 - 90C), indicating that swollen micelles could be dispersed in hot water which might further improve their efficiency to remove biofilms or used in combination with an acid or a base. Most importantly, our systems can be manufactured from GRAS compounds and could therefore be used to inactivate and remove biofilms from food as well as processing surfaces.
NON-TECHNICAL SUMMARY: Surface growth of pathogens on food and food processing equipment leading to the formation of biofilms poses an extremely serious problem in the food and agriultural industry. The purpose of this study is to develop antimicrobial/carrier combinations that can effectively destroy and remove pathogenic biofilms. In the process, we expect to learn much more about the mechanism of action of disinfectants and cleaners to combat biofilms. <P>APPROACH: In order to test our central hypothesis and accomplish the overall research objective of this application, we propose to complete the following three tasks: 1. Identify effective surfactant-antimicrobial combinations to achieve the highest cellular destruction in model colony biofilms. The working hypothesis for this aim is that the efficiency of antimicrobial - surfactant combinations is a function of the composition of capsules (type of antimicrobial and surfactant and surfactant to antimicrobial loading ratio), the overall concentration of capsules, and the biophysical properties of capsules (size and charge). While we have verified the antimicrobial efficiency of surfynol - eugenol and surfynol - carvacrol combinations, the goal is to screen a much wider range of suitable surfactant - antimicrobial combinations including uncharged, positively and negatively charged micelles and determine both their encapsulation efficiencies and antimicrobial activity to inactivate colony biofilms. Results will yield first insights into the role of size and electrostatic charge of micelles on antimicrobial activity. 2. Determine the efficacy of antimicrobial micelles against CBR grown biofilms and identify mechanism of inactivation and/or growth. Systems identified in Task #1, that exhibited antimicrobial activity will be carried forward to the CBR biofilm studies for validation and for mechanistic studies. In this task, we try to answer the question of whether activity of our systems is based solely on inactivation of cells, on inactivation and removal or on removal only. While presence of antimicrobials will interfere with bacterial cell wall integrity, the simultaneous adsorption of surfactants monomers from micellar solutions may reduce the surface free energy and lead to detachment of single cells or cell clusters. To gain insights into the mechanism of interaction of micellar-encapsulated antimicrobials with biofilms, we propose to determine the fate of micelles by fluorescence and confocal microscopy and determine changes in the microstructure and mechanical strength of the biofilm after treatment by atomic force microscopy. The results of this study are intended to lead to a first formulation of a biophysical model that correlates physicochemical properties of the capsules with inactivation efficiency. 3. Identify the effect of environmental conditions on inactivation efficiency of surfactant/antimicrobial combinations. Finally, we intend to test the applicability of our systems under a variety of environmental conditions that are typically encountered in the food industry. Specifically, we intend to determine the efficiency of removal and inactivation at elevated temperatures, in the presence of potentially interfering organic matter such as food and under the influence of a superimposed flow. The results of this part of the study will yield recommendation to food processors as to the optimal operating conditions that should be chosen in a cleaning operation using surfactant-encapsulated antimicrobials.<P>
PROGRESS: 2007/07 TO 2008/06<BR>
OUTPUTS: One Graduate M.S. student is currently being educated and slated to receive his M.S. degree from the University of Massachusetts with a focus on Food Microbiology by Summer of 2009. In the course of the project, two Post-Doctoral Workers are being trained in fundamental food microbiology principles as it applies to the growth of biofilms as well as in specialized analytical techniques that include zeta potential analysis, light scattering, fluorescence spectroscopy and confocal microscopy. Results of this study have been presented at a variety of meetings, such as the Annual Meetings of the Institute of Food Technologists and the International Association of Food protection. Results have been submitted for publication to the Food Biophysics and the Journal of Food Protection or are being prepared for submission to Applied Environmental Microbiology. Results of the study were also presented to the food industry through a series of consulting arrangements. The Department of Food Science has a Strategic Research Alliance which has 25 industrial members that are annually briefed on the progress of the research. We are currently in consultation with DuPont, who is interested in a commercialization of our systems. The project lead to a strengthening of international relations with the University of Murcia, Spain where one of our coworker and collaborator; Dr. Dario Perez-Conesa is located. <BR>
PARTICIPANTS: New participants include: (1) Lucie Hauff, ENSBANA, Dijon, France - Visiting Researcher and B.S. Student (2) Chris Aurand, M.S. Student, University of Massachusetts, Amherst, MA 01003 (3) Liwen Chen, Ph.D., Post-Doctoral Researcher, University of Massachusetts, Amherst, MA 01003 <BR>
TARGET AUDIENCES: The target audience includes the food industry in general but in particular food process engineers and plant personnel wishing to improve and develop better sanitation protocols to improve food safety and food quality through application of novel and better working antimicrobial systems. Audience outside the food industry includes the pharmaceutical and medical industries where persistent growth of biofilms poses a hazard, and the cosmetics and personnel care industries. <BR>
PROJECT MODIFICATIONS: Please not that the Principal Director is moving to Germany to take on a new position at the University of Hohenheim as of the next reporting period. We are currently in the process of requesting a transfer of P.D. to Dr. Lynne McLandsborough. Dr. Jochen Weiss (the current P.I.) will stay on as a collaborator and advisor on the project. We do not forsee any changes in the project objectives nor will it influence the completion date of the project.
<P>IMPACT: 2007/07 TO 2008/06<BR>
The overall objective of this project was to develop an effective remediation method for food pathogen biofilms using micellar encapsulated antimicrobials. This reporting period, we achieved the following objectives: We successfully formulated micellar encapsulated antimicrobial systems using eugenol as a model antimicrobial compound using nonionic and ionic surfactant mixtures. The use of surfactant mixtures allowed us to (a) increase the concentration of active compound in the micellar systems and (b) modify the charge that the micelles carry. We demonstrated that the charge of micelles plays a key role in their ability to penetrate the biofilm. Micelles containing high negative charges were less able to inactivate cells in the biofilm. Confocal microscopy demonstrated that the micelles were repelled from the surface of biofilms and thus less able to penetrate into the biofilms. Hence cells that were located deeper in the biofilm were no longer effected by the application of the antimicrobial micellar system. Conversly, micelles that were predominantly nonionic were able to penetrate the entire biofilm thereby inactivating a large number of cells embedded in the exopolymeric substances. This has important consequences for the formulation of effective micellar antimicrobial remediation systems. Negatively charged surfactants leading to a repulsion should thus be avoided, while nonionic surfactants are preferred We gained key new insights into the defense mechanism that biofilms employ to maintain viability in the presence of sanitizers and disinfectants. Adsorption of these compounds to the surface of the biofilms can lead to subsequent charge repulsion, rendering a subsequent treatment ineffective. Thus, carrier systems able to penetrate the biofilm evenly via diffusion or convection are the most promising systems to improve food safety and quality by controlling growth of biofilms in process equipment and/or food. We developed a new method to visualize cell death in both Gram positive and Gram negative organisms. Comparing the traditionally used BacLight stain with the more recently developed CTC stain indicated that the BacLight stain is not suitable to accurately visualize live/dead cells in biofilms. On the other hand, staining of biofilms with the CTC fluorescent stain system allows for accurate determination of live/dead cell ratios via confocal fluorescence imaging or fluorescence spectroscopy. This is an important result that will improve the rapid characterization of sanitizer or disinfectant efficiencies when treating biofilms.