Source: HARVARD COLLEGE, PRESIDENT & FELLOWS OF submitted to
A NOVEL INTERVENTION NANOTECHNOLOGY FOR FRESH PRODUCE SURFACE DISINFECTION USING ENGINEERED WATER NANOSTRUCTURES.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1000475
Grant No.
2013-67021-21075
Project No.
MASW-2013-01614
Proposal No.
2013-01614
Multistate No.
(N/A)
Program Code
A1511
Project Start Date
Sep 1, 2013
Project End Date
Aug 31, 2017
Grant Year
2013
Project Director
Demokritou, P.
Recipient Organization
HARVARD COLLEGE, PRESIDENT & FELLOWS OF
677 HUNTINGTON AVE
BOSTON,MA 021156028
Performing Department
EH-Molecular+IntegrativePhysio
Non Technical Summary
In the food safety and quality arena, there is a need to develop novel, chemical free, sustainable methodologies that can assist in the battle against foodborne disease while at the same time increase product shelf life. One of the food commodity categories attracting increased interest for stricter safety and quality assurance by regulatory authorities, producers and consumers alike, is the fresh produce category. Fresh produce can be a significant source of pathogenic organisms and are being implicated in serious foodborne disease outbreaks with increasing frequency. Their consumption is increasing worldwide due to the healthier lifestyle trend, also supported by the new US dietary guidelines suggesting for increased fresh produce consumption. Current fresh produce disinfection methods rely heavily on the use of chemicals or irradiation, methods having major shortcomings as well as consumer objections. In this 3 year project we will investigate the effectiveness of a novel, chemical free, nanotechnology-based method for the inactivation of pathogenic and spoilage microorganisms on the surface of fruits and vegetables method using Engineered Water Nanostructures (EWNS). EWNS are synthezedby electro spraying of atmospheric water vapor andit was recently shown by the investigators that they possess unique physico-chemical and biological properties. More importantly, EWNS can interact with and inactivate microorganisms on inanimate surfaces (stainless steel) and in the air.The three specific aims of this project are: Investigate the effect of EWNS on assuring the microbiological safety of "fresh produce" by inactivating surface pathogenic microorganisms Investigate the effect of EWNS on delaying spoilage and decay and extending shelf life of fresh produce by inactivating surface spoilage microorganisms Explore the development of commercial applications of this novel disinfection technology to be employed in key points in the fresh produce production chain, from "Farm to Fork", including the end user. The social, technological, scientific, public health and economical impact will betremendous if this method is proven to be effective. In conclusion, this intervention technology, which does not utilize chemicals or electromagnetic radiation,and with no residues or dangerous byproducts in the final product, could help inassuring the safety and prolonging the shelf life of products consumed raw, such as fruits and vegetables in a sustainable way.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71272992020100%
Goals / Objectives
The three specific aims of this project are to: Investigate the effect of a novel intervention nanotechnology using electrosprayed Engineered Nnanostructures (EWNS) on assuring the microbiological safety of "fresh produce" by inactivating surface pathogenic microorganisms Investigate the effect of EWNS on delaying spoilage and decay and extending shelf life of fresh produce by inactivating surface spoilage microorganisms Explore the development of commercial applications of this novel disinfection technology to be employed in key points in the fresh produce production chain, from "Farm to Fork", including the end user.
Project Methods
The methods for each of the4 parts/themes of the project are as follows Part A: Development and characterization of a generation and exposure system suitable to study the EWNS-microbe interactions on fresh produce. A high volume (up to 15 lpm), high concentration (up to 500,000 #/cc) EWNS aerosol generator will be developed and used in the exposure experiments, constructed with an array of 20 electrospray modules. The generated ozone will be reduced to atmospheric levels by passing the aerosol through the recently developed by our group low particle loss, honey comb glass denuder (Demokritou et al., 2001).The generator will be connected to a custom made polyacrylic environmental chamber which will host the fresh produce. The exposure chamber will be lined with grounded, aluminum panels to minimize particle losses and will be equipped with a stirring fan to ensure homogeneous distribution of the EWNS in the chamber. Aerosol flow and concentration in the EWNS generator output will be monitored in real time during the exposure scenarios, enabling precise control of dosing (Dose will be a function of exposure time and EWNS particle concentration level).EWNSphysico-chemical properties will be fully characterized using state of the art analytical methods (size, ROS content and life expectancy). Emphasis will be given to understand the bacteria inactivation mechanisms and nano-pathogen interactions. Parts B, C, D - Microbiology: Evaluation of disinfection agents involves immersion of the artificially inoculated commodity in a disinfectant solution or exposing it to a special technology, followed by recovery of the surviving population ( Lee S-Y et. al, 2006; Liao et al., 2010; Sapers, 2001). However, these studies do not mimic real life scenarios, so Part C is included, where the EWNS will interact with naturally 'contaminated' samples. Part B: Evaluation of EWNS to inactivate artificially inoculated microorganisms on the surface of fresh produce Samples: Fresh apples, nectarines and tomatoes, purchased from localmarkets will be used in all experiments. Prior to inoculation, samples will be washed with tap water and detergent, sanitized with70% ethanol and rinsed with sterile water to remove native microflora. Inoculums: Significant for fresh produce safety and spoilage, microbes will be evaluated: a) Gram (-) bacteria - E. coli (fecal indicator) and Salmonella spp. (top ranking pathogen), b) Gram (+) bacteria - Listeria spp. and Staphylococcus spp. c) Spoilage organisms - yeasts, molds, soft rot bacteria (Erwinia ) and d) Bacteriophage T4 . Samples will individually be placed in a sterile net and dipped into the bacterial, fungal or viral suspension of known concentration for a specified amount of time then allowed to air dry for 15 min in a laminar hood. The achieved population will be determined to serve as baseline data. Inoculated produce will then be placed in the exposure chamberto interact with EWNS. Time of exposure and EWNS aerosol particle concentration will vary in consequent experiments as part of the "dose-response" investigation. Following treatment, each sample will be microbiologically examined to recover surviving organisms. Recovery of survivors: Each sample will be placed in a sterile bag with 50 ml sterile buffer and manually massaged vigorously for 1 min orsonicated for 15-20 sec. Microbial presence in the suspension liquid will be determined as follows: For the Bacteria: Salmonella, Staphylococcus, Listeria, Erwinia and E. coli: The Total Viable Count will be determined with both culture and staining: i.) The suspension will be cultured with tryptic soy or nutrient agar, by pour or spread plating, incubated for 3d/30°C. Colonies will be counted and results expressed as Colony Forming Units - CFU/ml of suspension liquid; The baseline population will be enumerated from inoculated fruit NOT exposed to EWNSat zero time:a. immediately after inoculation and b. at the end of the EWNS treatmentii.) Fluorescence microscopy will enable the enumeration of any viable but non-culturable microorganisms not detected with culture. For the yeast and molds: Saccharomyces, Penicillium: Similar methods as described above will be used to enumerate the surviving yeast and molds with Potato Dextrose Agar incubated at 25oC/ 5d. Colonies will be counted and results expressed as CFU/ml of suspension. For the Bacteriophages: E.coli T4 phage: The Plaque Assay will be used. The bacterial host suspension is mixed with the bacteriophage suspension and plated onto recommended growth media (Madigan et al, 2010). Following incubation, clearing zones or plaques are counted that indicate the presence of infected bacteria, which in turn indicate the presence of the virus. Part C: Evaluation of EWNS to inactivate naturally occurring microflora on the surface of fresh produce The natural microbiological profile on the surface of the samples will be assessed with 3 fundamental parameters: a) Total Viable Count - hygiene' indicator b) Yeast and Mold Count - spoilage organisms c) Esherichia coli count - fecal indicator. Samples from 2 markets will be individually examined for the parameters mentioned above to establish the 'natural' profile for each batch of commodity: i) Enumeration of the TVC using tryptic soy or nutrient agarat30oC/72hr; ii )Enumeration of the Yeast and Mold countusing PDA at 25oC/5d; and iii) Enumeration of E.coli usingTBX Agar at 44oC/24hr. Colonies are counted and results expressed as CFU/ fruit or vegetable. Mean counts for each parameter will be obtained to establish the microbiological profile for each batch of commodity. In parallel, samples from the same sampling points will be treated with EWNS at multiple exposure and time intervals and each one will be examined for the same microbiological parameters as in non-treated samples to obtain mean counts. The efficacy of EWNS to inactivate the natural species will be calculated by comparing the mean counts for each parameter PRIOR to and AFTERthe exposure. Part D: Investigation of the potential for decay control and shelf life extension Yeasts, molds and soft rot bacteria such as Erwinia carotovora cause spoilage in fresh produce; the ability of EWNS to inactivate them is significant for decay control/shelf life extension. Fresh produce will be placed in clean, disinfected plastic crates. One crate of each commodity will serve as the control (not treated with EWNS) and one crate of each will be treated with EWNS. Exposure time and EWNS concentration levels will vary. Crates will be stored in a cool room/refrigerator at 3+2°C at 60% RH for up to 60 days for shelf life studies. Samples will be examined daily for visual physical changes (mold growth, rotting) and the total count and yeast and mold count will be determined weekly for changes (possible increases) over time. The methodology will be as in Part B (Culture and Microscopy). Feasibility of methodology and means by which results will be analyzed, assessed, or interpreted: Quality assurance: Experiments will be carried out in acc. to Good Laboratory Practice and strict safety guidelines. Microbiological examinationswill be performed in triplicate to minimize uncertainty of measurement. Methodology employed will be standard and validated - ISO based or AOAC. ISO 7218:2007, which covers the general requirements for microbiological examinations, will be followed. Calculations: Colony counting and Calculations will be done acc. to ISO 7218:2007. Calculations for Parts B and C will provide Microbial Log reductions (Log CFU). The reductions will be calculated by comparing baseline counts of fresh produce prior to EWNS exposure (controls) and recovery counts following the exposure. For Part D, shelf life study, microbial counts on the surface of fresh produce, treated and untreated, will be monitored weekly over a 60 day interval. Statistical Analysis will be performedusing the ANOVA software.

Progress 09/01/13 to 08/31/17

Outputs
Target Audience:The target audience(s) reached duringthe entirety of this projectinclude: 1) Industries in the area of microbial control in various 'farm to the fork' applications (BASF, STERIS, Profector, Nanoterra, Nature's Touch); 2) Scientific community working on food safety and/or food microbiology issues (food scientists, nanomaterial scientists); 3) USDA regional research centers working in food safety; 4) Collaborative partnerships between academia and Industry, such as the Center for Produce Safety (CPS); US Department of Defense; 5) General public Efforts included poster and oral presentations at food and nano-related conferences, peer-reviewed publications, patent applications on the developed technologies, seminars in universities and research institutions, as well asinterviews and articles in magazines/newspapers and scientific websites. Furthermore, research outcomes and media articles on the project were posted on Harvard'sCenter for Nanotechnology and Nanotoxicologywebsite (www.hsph.harvard.edu/nano) and other Harvard-related online magazines and journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Career development for postdoctoral trainees:Career development for postdoctoral trainees at Harvard T. H. Chan School of Public Health (HSPH) is supported at the school level through the Office of Faculty Affairs (OFA) and at the department level through multiple activities and in individual labs through mentorship and annual individual development planning. The fellows will have an academic appointment within the Department of Environmental Health at the Harvard School of Public Health. The department's faculty, research staff, and students reflect the multidisciplinary nature of the field and include chemists, engineers, epidemiologists, applied mathematicians, physicians, occupational health nurses, physiologists, cell biologists, molecular biologists, and microbiologists. The department has approximately 115 post doctoral fellowswith 39% of them coming from 25 countries. Postdoctoral fellows joining the departmentbenefit from the committed faculty who are accomplished in their respective fields and collectively publish close to 400 articles in peer-reviewed literature each year. Structures at School and University level:Fellows are welcomed by a structure that supports postdoctoral fellows at the university and school level. Their experience is enhanced by such structures as the Postdoctoral Association, which provides opportunities for HSPH postdoctoral fellows to develop their careers through relevant workshops and seminars, networking at fellow gatherings, and other activities. Additionally, fellows have access to various working groups, as well as the Harvard Center for Workplace Development, which provides professional, organizational, and career development programs that enable them to enhance their management, communication, and leadership skills, and take charge of their careers. More specific courses for fellows are organized through the Office of Faculty Affairs, covering a rangeof topicsincluding:grant preparation workshops, strategic networking, effective teaching, authorship issues, and grant workshops. The school also has in place a post doctoral mentorship program which helps both fellows and facultyachieve their academic and research goals. The fellow will also benefit from academic and other resources afforded by the world-renown centers housed within Harvard, including the HSPH-National Institute of Environmental Health Sciences (NIEHS) Center, the Harvard-EPA Center on Ambient Particle Health Effects, the NIEHS/EPA Center on Children and Environmental Health, Harvard Center for the Environment, and the Center for Nanotechnology and Nanotoxicology. Mentorship Program: Dr. Demokritouwill be responsible for mentoring the post doctoral research fellows. At the beginning of the academic year, the fellow, with input from his mentor, will prepare a detailed annual academic plan stating theirresearch, academic, and professional goals for the year. The academic mentor will approve the plan and be responsible for the evaluation and monitoring of the progress. Monthly meetings will be scheduled to go over the overall progress towards the goals. In addition, biweekly meetings with the rest of the project research team will take place to discuss progress of research for the project.The fellows willalso be encouraged to enhance their skills by attending intensive training courses offered at the Harvard Center for Nanoscale Systems andother programs across the University on various aspects of nanoscience and technology. In summary, the OFA provides trainees and faculty mentors with tools for Individual Development Planning (IDP) thatfosters ongoing and recurring discussions involving evaluation, goal setting, and feedback. The IDP will be used toaddress research and professional progress by benchmarking advancement and identifying barriers to success along the training path. This process allows forevaluation oftrainee performanceand progresswhile assessing issues related to research, training, and mentoring. This project has been particularly usefulfor the development of the research capabilities and the overall personal growth of the members in the group. Postdoctoral fellows were trained on the multidisciplinary approaches encompassing the fields of nanotechnology, food safety,and microbiology. Undergraduate students, visiting students, and graduate students were also involved in every aspect of the project. The work being done was presented at conferenceswith opportunities to improve the presentation skills of fellows. Industry interaction was useful in understanding the real world challenges in order to be able to develop solutions. How have the results been disseminated to communities of interest?The results were communicated to communities of interest via: peer review publications presentations in international and national conferences articles in magazines of general interest presentations at companies with interest in food safety It is worth mentioning that many articles on the transformative nature of this research were published in many online journals and magazines. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the four-year project, we explored the EWNS synthesis, optimized the synthesis platform, assessedthe EWNS properties and the operational parameters that influence them, and assessed their potential to be used as an antimicrobial platform for food safety applications. The aims of the proposed research were: Development of a lab-based system to understand the fundamentals of the EWNS synthesis. Development of an EWNS targeted delivery system for food surface applications Assess the potential of EWNS to interact with and inactivate common food-related bacteria and yeast (E. coli, L. innocua, S. enterica, M. parafortuitum and S. cerevisiae) on fresh produce using inoculation experiments Perform preliminary experiments to assess potential sensory effects of the EWNS-exposed produces The key findings are summarized as follows: EWNS synthesis: A single "needle" EWNS generation system was developed to understand the EWNS synthesis processes. A metal needle containing deionized water (18 MΩ cm-1) is connected to a high-voltage source and held over a grounded electrode. The distance between the needle and the counter electrode can be manually adjusted. During the process, two distinct phenomena take place: i) electrospraying; and ii) ionization of water. In more detail, the strong electric field between the two electrodes causes negative charges to accumulate on the surface of the condensed water at the tip of the needle, leading to the formation of the Taylor cone. As a result, highly charged water droplets form and continue to break into smaller particles as described by the Rayleigh theory. At the same time, the high electric field causes some water molecules to split and can strip off electrons (ionization), resulting in a high number of reactive oxygen species (ROS). The concurrently generated ROS are encapsulated in the forming water droplets to form EWNS. We have developed a lab-based, single needle, EWNS generation system which allows to control critical operational parameters such as the modulation of the applied voltage (V), the distance between the needle and the counter electrode (d), the flow of the water (φ), in order to study the fundamentals of the EWNS synthesis and their formation mechanisms and properties. We were able to fine-tune these operational conditions in order to optimize the EWNS properties and enhance their antimicrobial efficacy as described in our recent publications. It was shown that by adjusting the electric field, flow of water, and other critical operational parameters, EWNS properties (surface charge, size and ROS content) can be controlled and optimized. The single needle EWNS generator system module can generate an aerosol of approximately 40,000 #/cc at 0.5 L/min flow. It is worth noting that the single needle EWNS generator module consumes approximately 5 mW of power, which is low enough to be powered by a battery. Characterization of EWNS properties: The properties of EWNS were characterized using state of the art analytical methods as described in our recent publications. In summary, the optimized EWNS have 22e-/EWNS, loaded with ROS (primarily hydroxyl radicals and superoxides) and an average size in the nano regime (20-30 nm). It was also shown that the high electric charge of EWNS particlesincrease their surface tension, reduceevaporation, and renderthem airborne for hours (over 4 hours in room conditions). It is also worth noting that ozone levels during synthesis are below 60 ppb, which eliminates the need for ozone scrubbers, as these levels are below the EPA allowed (72 ppb for a maximum of 8 hours). Development of a targeted EWNS delivery system: We have developed a "draw through" Electrostatic Precipitation Exposure System (EPES) which takes advantage of the EWNS high surface charge in order to enhance their surface delivery. An electric field can be utilized to guide EWNS to a targeted surface. The EPES consists of an exposure chamber, which houses two parallel metal plates connected to an external high voltage source. The bottom plate is always set to positive voltage and the top plate is always set at ground. The deposition efficiency of the EWNS using the EPES was evaluated and it was found to reach up to 99%. It is worth noting that such a targeted EWNS delivery approach increased the inactivation potential due to the higher and faster deposition efficiency, when compared to the "slow" diffusion based delivery approach. Preliminary bacterial Inactivation experiments using the EPES targeted delivery system: To assess the EWNS's antimicrobial potential, we utilized the EPES system and exposed food-related microorganisms (E.coli, L. innocua, S. enterica, M. parafortuitum and S. cerevisiae). Such microorganisms were spot inoculated on the surface of cherry tomatoes. Our data show the robust efficacy of EWNS in inactivating the pathogens reaching inactivation up to 4 logs (99.99% reduction) in 45 mins with just the single needle EWNS generator which generates approximately 40,000 EWNS/cc. More importantly, however, our data also showed that there is a linear dose response of the inactivation as a function of the EWNS dose. The delivered dose can be adjusted either by increasing the EWNS concentration levels or by increasing the exposure time. Mechanisms of inactivation: As part of our investigation, we also explored the mechanism of inactivation using an array of methods. The TEM images showed that the control cells (unexposed) appeared normal and had an intact internal structure and cell membrane, whereas the cells exposed to EWNS appeared to have their cell membrane damaged. In order to quantify the membrane destruction, we performed a "live-dead" assay using confocal microscopy that also verified the EWNSability to destroy the EWNS membrane. Further, the molecular mechanism of inactivation was assessed using a lipid peroxidation assay. The bacteria exposed to the EWNSshowed a high concentration of lipid peroxide, a result of the lipid membrane oxidation from the ROS. However, when Vitamin C, a known antioxidant, is added in the bacteria, there is no lipid peroxidation. This indicates that the ROS presence is one of the primary mechanisms of inactivation. Preliminary sensory evaluation and shelf-life extension assessment: Two groups of cherry tomatoes, one exposed to 80,000 #/cc for two hours while the other was kept under room conditions, were evaluated by an independent panel of 16 individuals regarding the overall quality, texture, aroma, color, and skin integrity. Overall, the comparison between the treated and control tomatoes showed no significant differences, indicating that the treatment is not inducing any sensory effects on the tomatoes. This is a very important finding as the food industry is in need of technologies that do not compromise sensory characteristics of products. Also, we quantitatively evaluatedthe effect of EWNS on the tomato firmness (external appearance parameter) and the internal pH (internal quality parameter) of tomatoes that have been exposed to the EWNS aerosol for 18 days, 4 hours each day. At the end of the experiment, the EWNS-exposed tomatoes retained 47% more firmness compared to the control group (unexposed) while the internal pH showed no difference (unpublished data). These results collectively show that exposure to the EWNS does not affect the overall appearance and sensory quality of the food. Further, the quantitativeassessment showed that there is a positive effect on shelf life: exposed tomatoes retained almost 50% more of their firmness, an extended shelf life indicator, while the internal qualities, such as pH, remained unchanged.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Pyrgiotakis G, McDevitt J, Gao Y, Branco A, Eleftheriadou M, Lemos B, Nardell E, Demokritou, P. Mycobacteria inactivation using Engineered Water Nanostructures (EWNS). Nanomed Nanotech Biol Med. 2014 Aug;10(6):1175-83.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Pyrgiotakis G, Mitchell R, Vasanthakumar A, Gao Y, DeAraujo A, Eleftheriadou M, Demokritou P. A Novel, Chemical free Intervention Nanotechnology For Fresh Produce Surface Disinfection Using Engineered Water Nanostructures. Nanotech 2014, Washington, DC. June 15-18, 2014.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Pyrgiotakis G, Vasanthakumar A, Gao Y, Eleftheriadou M, Toledo E, DeAraujo A, McDevitt J, Han T, Mainelis G, Mitchell R, Demokritou P. Inactivation of foodborne microorganisms using Engineered Water Nanostructures (EWNS). Environ Sci Technol. 2015 Mar;49(6):373745.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Demokritou, P. Nanotechnology to the Rescue: Food Pathogen Inactivation using Engineered Water Nanostructures. Nanoscale Science & Engineering for Agriculture & Food Systems Gordon Research Conference, Bentley University, Waltham, MA. June 7-12, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Vedantam, P. Engineered Water Nanostructures (EWNS): A chemical free, nanotechnology based method for inactivation of foodborne microorganisms. Nanoscale Science & Engineering for Agriculture & Food Systems Gordon Research Conference, Bentley University, Waltham, MA. June 7-12, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Demokritou, P. Nanotechnology to the Rescue: Food Pathogen Inactivation using Engineered Water Nanostructures. International Food Technology Conference, Chicago IL. July 11-14, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pyrgiotakis, G. Nanotechnology-based Approach for the Inactivation of Foodborne Microorganisms. International Association for Food Protection, Portland, OR. July 24-28, 2015.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pyrgiotakis G, Vedantam P, Cirenza C, McDevitt J, Eleftheriadou M, Leonard SS, Demokritou P. Optimization of a nanotechnology based antimicrobial platform for food safety applications sing Engineered Water Nanostructures (EWNS). Sci Rep. 2016 Feb;6:21073.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Eleftheriadou M, Pyrgiotakis G, Demokritou P. Nanotechnology to the rescue: using nano-enabled approaches in microbiological food safety and quality. Curr Opin Biotechnol. 2017 Apr;44:87-93.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Cirenza C.; Ventadam P.; Pyrgiotakis G.; Eleftheriadou M.; Demokritou P.;. Engineered Water Nanostructures (EWNS): A chemical free, nanotechnology based method for inactivation of foodborne microorganisms. Sustainable Nanotechnology Organization Conference, Portland, OR. November 7-10, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pyrgiotakis G, Cirenza C, Ventadam P, Eleftheriadou M, Demokritou P. EWNS as an intervention technology at the battle against foodborne infections. International Association for Food Protection, MA. July 25-28, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Pyrgiotakis G, Cirenza C, Eleftheriadou M, Demokritou P. Nanotechnology to the Rescue: Foodborne pathogens inactivation using the EWNS platform. International Congress of Nanotoxicology, Boston, MA. June 1-4, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou P. A Novel Chemical Free, Nanotechnology Based Platform for Food Pathogen Inactivation. BASF Agricultural Division, Durham, NC. April 6, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou P. Nanotechnology to the Rescue. Chicago, MI. July 11-14, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Pyrgiotakis G, Cirenza C, Eleftheriadou M, Demokritou P. Foodborne pathogens inactivation using the EWNS platform. USDA/NIFA Grantees meeting, State College, PA. June 5-6, 2016.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Vaze N, Jiang Y, Mena L, Zhang Y, Bello D, Leonard SS, Morris AM, Eleftheriadou M, Pyrgiotakis G, Demokritou P. An integrated electrolysis  electrospray  ionization antimicrobial platform using Engineered Water Nanostructures (EWNS) for food safety applications. Food Control. 2017; in press.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Vaze N, Pyrgiotakis G, Eleftheriadou M, Demokritou P. An antimicrobial targeted and precision delivery platform using Engineered Water Nanostructures (EWNS) for food safety applications. Validation of Nonthermal Technologies Symposium, Cornell University, Ithaca, NY. October 29-31, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform for Food Pathogen Inactivation using Engineered Water Nanostructures. Nanyang Technological University, Singapore. October 3, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. The Connecticut Agricultural Experiment Station, New Haven, Connecticut. February 17, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. Duke University, Durham, North Carolina. February 22, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. Eidgen�ssische Technische Hochschule Z�rich, Department of Mechanical and Process Engineering, Zurich, Switzerland. March 10, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Pyrgiotakis G, Eleftheriadou M, Melo A, Cirenza C, McDevitt J, Demokritou P. A chemical free, antimicrobial platform using Engineered Water Nanostructures. 8th International Nanotoxicology Congress, Boston, MA. June 2-4, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Eleftheriadou M, Pyrgiotakis G, Cirenza C, Mello A, Vedantam P, McDevitt J, Demokritou P. A Novel, Nanotechnology-Based Antimicrobial Platform using Engineered Water Nanostructures (EWNS) for Food Safety Applications. ASM Microbe 2016, Boston, MA. June 16-20, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pyrgiotakis G, Vedantam P, Cirenza C, McDevitt J, Eleftheriadou M, Leonard S, Demokritou P. Optimization of a nanotechnology based antimicrobial platform for food safety applications using Engineered Water Nanostructures (EWNS). Sci Rep. 2016 Feb;6(21073):1-12.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Eleftheriadou M, Pyrgiotakis G, Demokritou P. Nanotechnology to the rescue: using nano-enabled approaches in microbiological food safety and quality. Curr Opin Biotechnol. 2017 Apr;44:8793.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform for Food Pathogen Inactivation using Engineered Water Nanostructures. Sustainable Packaging Symposium, University of Massachusetts, Lowell, Massachusetts. November 3, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. Brown University, Providence, Rhode Island. November 18, 2016.


Progress 09/01/16 to 08/31/17

Outputs
Target Audience:The target audience(s) reached duringthis grant period include: 1) Industries in the area of microbial control in various 'farm to the fork' applications (BASF, STERIS, Profector, Nanoterra, Nature's Touch); 2) Scientific community working on food safety and/or food microbiology issues (Food Scientists, Nanomaterial Scientists); 3) USDA regional research centers working in food safety; 4) Collaborative partnerships between academia and Industry, such as the Center for Produce Safety (CPSC); US Department of Defense; 5) General public Efforts included poster and oral presentations at food and nano-related conferences, peer-reviewed publications, patent applications on the developed technologies, seminars in universities and research institutions, as well as interviews and articles in magazines/newspapers and scientific websites. Furthermore, research outcomes and media articles on the project were posted on Harvard's Center for Nanotechnology and Nanotoxicologywebsite (www.hsph.harvard.edu/nano) and other Harvard-related online magazines and journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Career development for postdoctoral trainees:Career development for postdoctoral trainees at Harvard T. H. Chan School of Public Health (HSPH) is supported at the school level through the Office of Faculty Affairs (OFA) and at the department level through multiple activities and in individual labs through mentorship and annual individual development planning. The fellows will have an academic appointment within the Department of Environmental Health at the Harvard School of Public Health. The department's faculty, research staff, and students reflect the multidisciplinary nature of the field and include chemists, engineers, epidemiologists, applied mathematicians, physicians, occupational health nurses, physiologists, cell biologists, molecular biologists, and microbiologists. The department has approximately 115 post doctoral fellowswith 39% of them coming from 25 countries. Postdoctoral fellows joining the department benefit from the committed faculty who are accomplished in their respective fields and collectively publish close to 400 articles in peer-reviewed literature each year. Structures at School and University level: Fellows are welcomed by a structure that supports postdoctoral fellows at the university and school level. Their experience is enhanced by such structures as the Postdoctoral Association, which provides opportunities for HSPH postdoctoral fellows to develop their careers through relevant workshops and seminars, networking at fellow gatherings, and other activities. Additionally, fellows have access to various working groups, as well as the Harvard Center for Workplace Development, which provides professional, organizational, and career development programs that enable them to enhance their management, communication, and leadership skills, and take charge of their careers. More specific courses for fellows are organized through the Office of Faculty Affairs, covering a range of topicsincluding:grant preparation workshops, strategic networking, effective teaching, authorship issues, and grant workshops. The school also has in place a post doctoral mentorship program which helps both fellows and faculty achieve their academic and research goals. The fellow will also benefit from academic and other resources afforded by the world-renown centers housed within Harvard, including the HSPH-National Institute of Environmental Health Sciences (NIEHS) Center, the Harvard-EPA Center on Ambient Particle Health Effects, the NIEHS/EPA Center on Children and Environmental Health, Harvard Center for the Environment, and the Center for Nanotechnology and Nanotoxicology. Mentorship Program: Dr. Demokritouwill be responsible for mentoring the post doctoral research fellows. At the beginning of the academic year, the fellow, with input from his mentor, will prepare a detailed annual academic plan stating their research, academic, and professional goals for the year. The academic mentor will approve the plan and be responsible for the evaluation and monitoring of the progress. Monthly meetings will be scheduled to go over the overall progress towards the goals. In addition, biweekly meetings with the rest of the project research team will take place to discuss progress of research for the project. The fellows will also be encouraged to enhance their skills by attending intensive training courses offered at the Harvard Center for Nanoscale Systems andother programs across the University on various aspects of nanoscience and technology. In summary, the OFA provides trainees and faculty mentors with tools for Individual Development Planning (IDP) thatfosters ongoing and recurring discussions involving evaluation, goal setting, and feedback. The IDP will be used toaddress research and professional progress by benchmarking advancement and identifying barriers to success along the training path. This process allows forevaluation oftrainee performanceand progresswhile assessing issues related to research, training, and mentoring. How have the results been disseminated to communities of interest?The results were communicated to communities of interest via 1) peer-reviewed publications,2) presentations in international and national conferences,3) articles in magazines of general interest, and4) presentations to food and beverage companies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Development of iEWNS nano-sanitizer platform:The EWNS synthesis apparatus was further utilized to develop a novel platform for the delivery of antimicrobial nano-sanitizers. An aqueous solution containing an active ingredient 'i' was added to the deionized water and passed through EWNS generation setup. The particles thus created were termed as iEWNS, (the 'i' denoting the active ingredient used). With this platform, various 'nature inspired' antimicrobials were delivered to microorganisms on surfaces. These antimicrobials included: electrolyzed water (used in the food industry), hydrogen peroxide (used in cellular processes and leaves no toxic residue), citric acid (found in lemon and other juices) and lysozyme (Chicken egg white). We also evaluated combinations of these antimicrobials to determine if there are synergistic effects. These particles were characterized for their physicochemical properties. Physicochemical Characterization of iEWNS particles:These iEWNS particles were characterized for their physico-chemical properties using methods described in our publications. The particles varied by size, ranging from 10 nm to 50 nm, and were loaded with electrons (10 to 80eper particle). The particles were also analyzed for the presence of ROS. Electron Spin Resonance (ESR) results indicated the presence of short lived species such as Hydroxyl (OH•) And Superoxide (O2-) in the EWNS particles. The rEWNS particles produced with electrolyzed water showed an enhancement in the levels of these short lived species. The quantification of these ROS was carried out using Trolox Equivalent Antioxidant Capacity assay. The results indicate that the there was a 2 fold increase in the concentration of total ROS produced. Also detected wasthe presence of H2O2being produced in the rEWNS particles. Further speciation of the short-lived species in rEWNS particles was carried out using ESR. These results confirmed the presence of specific ROS in the rENWS. Recently, the other iEWNS particles were also evaluated using the Trolox method, where particles of hydrogen peroxide (h1EWNS) were found to have the highest levels of ROS, including short lived species produced during the ionization process. Levels of active ingredients used, such as citric acid were determined using LC-MS/MS and protein assay methods. These particles were used to treat the microorganisms on surfaces in a targeted manner for maximum inactivation effect while delivering minute quantities. Direct targeted treatment of microorganisms with iEWNS:In earlier published work, we had utilized the EPES system, which utilizes a separate electric field in order to target the EWNS particles onto the surface of interest. Here, we further modified the setup and utilized the electric field generating the particles itself to target them on to the target surface. This direct targeted approach can lead to a more precise delivery of the active ingredient being utilized to produce the iEWNS. We utilized this approach to treat produce such as berries and cherry tomatoes with the enhanced EWNS particles. This approach is more suitable for commercial upscaling of the system with a multiple needle approach. Inactivation of microorganisms using nano-sanitizers:Inactivation studies were performed with food related microorganisms, such asE. coliand L. innocua, as well as other pathogens of interest, such as Acinetobacter baumannii and Influenza H1N1/PR/8. The rEWNS particles, produced with electrolyzed water, produced a 2-fold increase in the inactivation rate over EWNS, generated with DI water alone. These rEWNS were also utilized to treat Blackberries. Their effect on both the natural flora of the berries and inoculatedE. coliwas evaluated. The results showed a 97% (1.5-log) inactivation of the total viable count, a 99% (2-log) reduction in the yeast and mold count and a 2.5-log reduction of the inoculatedE.coliafter 45 minutes of exposure, without any visual changes to the fruit. h1EWNS produced with 1% hydrogen peroxide produced 5 log reduction inE. coliin 5 minutes. Other microorganisms were tested with these h1EWNS and similar 5 log reductions were achieved in L. innocua, A. baumannii and Influenza H1N1/PR/8. l0.1EWNS nanostructures produced with 0.1% Lysozyme also were equally effective againstE. coli, with 5 log reductions in 5 minutes. Citric acid nanostructures produced similar results inE. coliin 15 minutes. A synergistic combination of 1% hydrogen peroxide and 1% citric acid was tested withE. coliand it produced a rapid inactivation rate of 5 logs in 2 minutes. These breakthrough results represent a highly significant reduction in treatment time, making the iEWNS technology an attractive option for produce treatment in real time. Mechanisms of iEWNS action:The mechanism of the action of iEWNS was evaluated through TEM studies.E. colicells treated with each iEWNS with a lethal dose were analyzed. The TEM images for controls show intact cellular membranes. The treated cells indicate varying degrees of damage to the cell wall. The mechanisms of iEWNS observed were similar to those observed for their active ingredients. Hydrogen peroxide nanostructures produced rupture of the cell membrane and the subsequent leakage of cellular components can be observed from the image. Lysozyme nanostructures produced severe membrane damage through hydrolysis of peptidoglycans. Citric acid nanostructures did not produce major membrane damage, further supporting the hypothesis of citric acid action through acidic action and respiratory effects, rather than membrane hydrolysis. The mixture of hydrogen peroxide and citric acid affected the cells in various ways, the major mechanism being stretching of the membrane by the action of hydrogen peroxide (possibly facilitating the entry of citric acid).

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform for Food Pathogen Inactivation using Engineered Water Nanostructures. Nanyang Technological University, Singapore. October 3, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform for Food Pathogen Inactivation using Engineered Water Nanostructures. Sustainable Packaging Symposium, University of Massachusetts, Lowell, Massachusetts. November 3, 2016.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Vaze ND, Jiang Y, Mena L, Zhang Y, Bello D, Leonard SS, Morris AM, Eleftheriadou M, Pyrgiotakis G, Demokritou P. An Integrated Electrolysis  Electrospray  Ionization Antimicrobial Platform Using Engineered Water Nanostructures (EWNS) for Food Safety Applications. Food Control. 2017; in press.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. Brown University, Providence, Rhode Island. November 18, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. The Connecticut Agricultural Experiment Station, New Haven, Connecticut. February 17, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. Duke University, Durham, North Carolina. February 22, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Demokritou, P. Nanotechnology to the Rescue: A Novel, Chemical Free, Antimicrobial Platform using Engineered Water Nanostructures. Eidgen�ssische Technische Hochschule Z�rich, Department of Mechanical and Process Engineering, Zurich, Switzerland. March 10, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Eleftheriadou M, Pyrgiotakis G, Cirenza C, Mello A, Vedantam P, McDevitt J, Demokritou P. A Novel, Nanotechnology-Based Antimicrobial Platform using Engineered Water Nanostructures (EWNS) for Food Safety Applications. ASM Microbe 2016, Boston, MA. June 16-20, 2016.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Eleftheriadou M, Pyrgiotakis G, Demokritou P. Nanotechnology to the rescue: using nano-enabled approaches in microbiological food safety and quality. Curr Opin Biotechnol. 2017 Apr;44:8793.


Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The results were communicated to communities of interest via 1) peer review publications; 2) presentaions in inernational and national conferences; 3) articles in magazines of general interest. It is worth mentioning that many articles on the transofrmative nature of this research were published in many online journals and magazine Target Audience The target audience(s) reached during the current reporting period include:1) Industries in the area of microbial control in various "farm to the fork" applications (BASF, STERIS, Profector, Nanoterra); 2) Scientific community working on food Safety and /or Microbiology issues; ( Food Scientists, Nano-Material scientists ); 3) USDA regional Research Centers working in food safety; 4) USA Department of Defence; 5) Lay public More specifically, efforts included presentations in Food and Nano related conferences, peer reviewed publications, seminars in Universities and research institutions and interviews and articles in magazines/newspapers and scientific websites. Furthermore, research outcomes and media articles on the project were posted on Harvard Centers website (www.hsph.harvard.edu/nano) and other Harvard related online magazines and journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A total of 7 Faculty, a great number of students and post doctoral fellows participaed in the research activities this year 3. How have the results been disseminated to communities of interest? The results were communicated to communities of interest via 1) peer review publications; 2) presentaions in inernational and national conferences; 3) articles in magazines of general interest; 4) Presentations at companies with interest in food safety. It is worth mentioning that many articles on the transofrmative nature of this research were published in many online journals and magazines What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project hasthe following aims: 1) Development of a lab based system to understand the fundamentals of the EWNS synthesis; 2) Development of an EWNS targeted delivery system for food surface applications; 3) Assess the potential of EWNS to interact and inactivate common food-related bacteria and yeast (E. coli, L. innocua, S. enterica, M. parafortuitum and S. cerevisiae) on fresh produce using inoculation experiments; 4) Preform preliminary experiments to assess potential sensory effects of the EWNS exposed produces. The key findings and accomplishmentsfor this period are summarized as follows: We have developed a lab based, single needle, EWNS generation system which allows to control critical operational parameters such as the modulation of the applied voltage (V), the distance between the needle and the counter electrode (d), the flow of the water (φ), in order to study the fundamentals of the EWNS synthesis and their formation mechanisms and properties. We were able to fine-tune these operational conditions in order to optimize the EWNS properties and enhance their antimicrobial efficacy as described in our recent publications. It was shown that by adjusting the electric field, flow of water and other critical operational parameters, EWNS properties (surface charge, size and ROS content) can be controlled and optimized. The current single needle EWNS generator system module can generate an aerosol of approximately 50,000 #/cc at 0.5 L/min flow. It is worth noting that the single needle EWNS generator module consumes approximately 5 mW of power which is low enough to be powered by a battery. Characterization of EWNS properties: The properties of EWNS were characterized using state of the art analytical methods as described in our recent publications Development of a targeted EWNS delivery system: We have developed a "draw through" Electrostatic Precipitation Exposure System (EPES) which takes advantage of the EWNS high surface charge in order to enhance their surface delivery. An electric field can be utilized to guide EWNS to a targeted surface. The EPES consists of an exposure chamber, which houses two parallel metal plates connected to an external high voltage source. The bottom plate is always set to positive voltage and the top plate is always set at ground. The deposition efficiency of the EWNS using the EPES was evaluated and it was found to reach up to 99%. It is worth noting that such a targeted EWNS delivery approach increased the inactivation potential due to the higher and faster deposition efficiency, when compared to the slow"diffusion based delivery approach. Preliminary bacterial Inactivation experiments using the EPES targeted delivery system: To assess the EWNS's antimicrobial poitential, we utilized the EPES system and exposed food-related microorganisms (E.coli, L. innocua, S. enterica, M. parafortuitum and S. cerevisiae). Such microorganisms were spot inoculated on the surface of cherry tomatoes. Our data show the robust efficacy of EWNS in inactivating the pathogens reaching inactivation up to 4 logs (99.99% reduction) in 45 mins with just the single needle EWNS generator which generates approximately 50,000 EWNS/cc (figure 5a). More importantly, however, our data also showed that there is a linear dose response of the inactivation as a function of the EWNS dose. The delivered dose can be adjusted either by increasing the EWNS concentration levels or by increasing the exposure time. Mechanisms of inactivation: As part of our investigation, we also explored the mechanism of inactivation using an array of methods. The TEM images showed that the control cells (unexposed) appeared normal and had an intact internal structure and cell membrane, whereas the cells exposed to EWNS appeared to have their cell membrane damaged.[56] In order to quantify the membrane destruction we performed a "live-dead" assay using confocal microscopy that also verified the EWNS ability to destroy the EWNS membrane. Further, the molecular mechanism of inactivation was assessed using a lipid peroxidation assay. The bacteria exposed to the EWNS, showed a high concentration of lipid peroxide a result of the lipid membrane oxidation from the ROS. However, when Vitamin C, a known antioxidant, is added in the bacteria there is no lipid peroxidation. This indicates that the ROS presence is one of the primary mechanisms of inactivation. Preliminary sensory evaluation and shelf-life extension assessment: Two groups of cherry tomatoes, one exposed to 80,000 #/cc for two hours while the other was kept under room conditions, were evaluated by an independent panel of 16 individuals regarding the overall quality, texture, aroma, color and skin integrity. Overall the comparison between the treated and control tomatoes showed no significant differences, indicating that the treatment is not inducing any sensory effects on the tomatoes. This is a very important finding as the food industry is in need of technologies that do not compromise sensory characteristics of products. Also, we evaluated quantitatively the effect of EWNS on the tomato firmness (external appearance parameter) and the internal pH (internal quality parameter) of tomatoes that have been exposed to the EWNS aerosol for 18 days, 4 hours each day. At the end of the experiment, the EWNS exposed tomatoes retained 47% more firmness compared to the control group (unexposed) while the internal pH showed no difference (unpublished data). These results collectively show that exposure to the EWNS does not affect the overall appearance and sensory quality of the food. Further the quantitatively assessment showed that there is a positive effect on shelf life: exposed tomatoes retained almost 50% more of their firmness, an extended shelf life indicator, whilethe internal qualities such as pH remained unchanged.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pyrgiotakis, G.; Vedantam, P.; Cirenza, C.; McDevitt, J.; Eleftheriadou, M.; Leonard, S. S.; Demokritou, P. Optimization of a Nanotechnology Based Antimicrobial Platform for Food Safety Applications Using Engineered Water Nanostructures (EWNS). Sci. Rep. 2016, 6, 21073.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Eleftheriadou, M.; Pyrgiotakis, G.; Demokritou, P. Nanotechnology to the rescue: using nano-enabled approaches in microbiological food safety and quality. Current Opinion in Biotechnology, accepted, 2016
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Cirenza C.; Ventadam P.; Pyrgiotakis G.; Eleftheriadou M.; Demokritou P.;. Engineered Water Nanostructures (EWNS): A chemical free, nanotechnology based method for inactivation of foodborne microorganisms. Sustainable Nanotechnology Organization Conference, Portland, OR. November 7-10, 2015.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Pyrgiotakis G.;Cirenza C.; Ventadam P.; Eleftheriadou M.; Demokritou P.;. EWNS as an intervention technology at the battle against foodborne infections. International Association for Food Protection, MA. July 25-28, 2015.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Pyrgiotakis G.;Cirenza C.; Eleftheriadou M.; Demokritou P.;. Nanotechnology to the Rescue: Foodborne pathogens inactivation using the EWNS platform. International Congress of Nanotoxicology, Boston, MA. June 1-4, 2016.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Pyrgiotakis G.;Cirenza C.; Eleftheriadou M.; Demokritou P.;. Foodborne pathogens inactivation using the EWNS platform. USDA/NIFA Grantees meeting, State Collage, PA. June 5-6, 2016.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Demokritou P.; A Novel Chemical Free, Nanotechnology Based Platform for Food Pathogen Inactivation, BASF Agricultural Division, Durham, NC. April 6, 2016
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Demokritou P. Nanotechnology to the Rescue. Chicago, MI. July 11-14, 2016


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The target audience(s) reached during the current reporting period include:1) Industries in the area of microbial control in various "farm to the fork" applications (BASF, STERIS, Profector, Nanoterra); 2) Scientific community working on food Safety and /or Microbiology issues; ( Food Scientists, Nano-Material scientists); 3) USDA regional Research Centers working in food safety; 4) USA Department of Defence; 5) Lay public Efforts included presentations in Food and Nano related conferences, peer reviewed publications, seminars in Universities and research institutions and interviews and articles in magazines/newspapers and scientific websites. Furthermore, research outcomes and media articles on the project were posted onHarvard Centers website (www.hsph.harvard.edu/nano) and other Harvard related online magazines and journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A total of7 Faculty, students and post doctoral fellows participaed in the research activities this year 2. We plan to continue engaging students and faculty in the research activities this coming year How have the results been disseminated to communities of interest?The results were communicated to communities of interest via 1) peer review publications; 2) presentaions in inernational and national conferences; 3) articles in magazines of general interest. It is worth mentioning that many articles on the transofrmative nature of this research were published in many online journals and magazine What do you plan to do during the next reporting period to accomplish the goals?Goal 2: Shelf life extension studies: Protocols have been developed and tested with organic tomatoes using measurable attributes such as:1.pH value, 2. tomato firmness ( penetrometer) 3. Visual check for mold development and or skin collapse/leaking and 4. Microbial surface measurements Total Viable count and Yeast and Mold count). These experiments are expected to be completed within the next 3 months. Goal 3: Explore the development of commercial applications of this novel disinfection technology to be employed in key points in the fresh produce production chain, from "Farm to Fork", including the end user. We have started a pilot project with Profector, an electrospray company exploring the scale up of a high throughput EWNS generation system capable of generating a high volume (10 lpm), high concentration EWNS aerosol (up to 500,000#/cc) which can be used for commercial applications. This project is expected to continue for the next 6 months

Impacts
What was accomplished under these goals? Goals1 & 2: Investigate the effect of a novel intervention nanotechnology using electrosprayed Engineered Nanostructures (EWNS) on assuring the microbiological safety of "fresh produce" by inactivatingmicroorganisms Alab based EWNS generation system was developed to enable control of critical synthesis parameters for optimizing EWNS physico-chemical properties and maximize their antimicrobial efficacy. Characterization of EWNS properties was performed using state of the art analytical methods. Their microbial inactivation potential wasevaluated with the use of representative foodborne microorganisms: Escherichia coli, Salmonella enterica, Listeria innocua, Mycobacterium parafortuitum, and Saccharomyces cerevisiae inoculated onto the surface of organic tomatoes. Removal rates were more than 5 logs/hr for most of the microorganisms at an EWNS aerosol dose of 40,000 #/cm3. Electron Microscopy imaging and the superoxide dismutase assay revealed that the EWNS could destroy the microbial cell membrane leading to inactivation. The results from this second year investigation indicate that EWNS properties can be fine-tuned during synthesis resulting in a multifold increase of their antimicrobial inactivation efficacy. The optimized EWNS were also tested in the inactivation of the naturally occurring microflora on tomatoes. The EWNS were successful to remove up to 3 logs of the microflora. More importantly, EWNS can be a powerful tool, which can be used across the farm to the fork continuum to enhance food safety. In more detail: Optimization of EWNS properties- Physicochemical characterization of optimized EWNS Several combinations of the voltage and the distance between the needle and the counter electrode, were evaluated. Among them two were found to be the most stable and reproducible and were selected for the complete property investigation and comparison withthe properties of the previously, unoptimized EWNS (baseline EWNS). The measured ionization electric current was between of 2 - 6 μΑ and the voltage was between -3.8 and -6.5 kV, resulting in energy consumption less than 50 mW for this single needle EWNS generation module. During the EWNS production, the ozone levels were very low, never exceeding the 60 ppb levels. Electric Field: The strength of the electric field is related to the voltage and the distance between the needle, the counter electrode and the overall geometry. The maximum value of the electric field at tip of the needle can be used as a reference of the electric field strength. The field was calculated to be 2×105 V/m, and 4.7×105 V/m for the [-6.5 kV, 4.0 cm] and [-3.8 kV, 0.5 cm] scenarios respectively. This is expected since the voltage distance ratio is significantly higher for the second case. EWNS Size: The average EWNS diameter was found to be 27.36 nm and 19.33 nm respectively for the [-6.5 kV, 4.0 cm] and [-3.8 kV, 0.5 cm] scenarios. The geometric standard deviation of the distribution was 1.41 and 1.45 respectively for the [-6.5 kV, 4.0 cm] and [-3.8 kV, 0.5 cm] scenarios, which is indicative of a narrow size distribution. Both the average size and the geometric standard deviation are very close to the baseline EWNS(25 nm and 1.41 respectively). EWNS electric charge: The data represent the average measurement of 30 number Concentration (#/cm3) and Current (I) concurrent measurements. The analysis showed that the average charge per EWNS is (22 ± 6) e and (44 ± 6) e- respectively for the [-6.5 kV, 4.0 cm] and [-3.8 kV, 0.5 cm] respectively.Compared to the baseline EWNS, the optimized EWNS have significantly higher surface charge (two times for the [-6.5 kV, 4.0 cm] and four times higher for the [-3.8 kV, 0.5 cm] scenarios). EWNS number concentration: Based on the results, the particle number for the [-6.5 kV, 4.0 cm] scenario is significantly higher (23913 #/cm3) compared to the [-3.8 kV, 0.5 cm] scenario (8,765 #/cm3). The EWNS number concentration was monitored for up to 4 hrs where the EWNS generation stability showed similar particle number concentration levels for both scenarios. Due to the significantly higher particle number concentration yield the [-6.5 kV, 4.0 cm] scenario was selected for the inactivation experiments and ROS characterization (see below). ROS Investigation: The number of the EWNS reacted with the spin trap was calculated at 7.5×104 EWNS/s which is similar to the one previously reported for the baseline EWNS. The ESR spectrum clearly indicates the presence of two ROS species, with O2- being the dominant species and OH• present in smaller amounts. The direct comparison of the peak intensity indicates that the optimized EWNS have significantly higher ROS content compared to the baseline EWNS. EWNS deposition in EPES: The deposition even for the low voltage of 3 kV is reaching nearly 100% for both cases of the EWNS. Generally, the 3 kV is enough to reach 100% deposition regardless of the variations on the surface charge. The baseline EWNS had only 56% deposition efficiency due to the lower electric charge (10e per EWNS on average). Microbial Inactivation The inactivation of food related microorganisms inoculated on the tomato surface and the corresponding logs/hr reductions following exposure to approximately 40,000 #/cm3 EWNS for 45 min were obtained for the [-6.5 kV, 4.0 cm] scenario. E. coli showed a significant 3.82 log reduction of 5.09±0.052 Log/hr. Similar significant levels of inactivation were also seen for L. innocua (3.81 log reduction corresponding to a 5.08±0.046 logs/hr reduction). At the same conditions, Salmonella enterica, however, exhibited lower log reductions (2.18 log reduction resulting in a 2.90±0.359 logs/hr. Both S. cerevisiae and M. parafortuitum showed only a 1 log reduction at the 45 min mark resulting to 1.333±0.698 logs/hr log reduction. Mechanism of microorganism inactivation: Electron microscopy: Electron micrographs depict the inactivation of E. coli, S. enterica and L. innocua by EWNS as indicated by the damaged outer membrane. Meanwhile the thick glycogen layer is well observed in the control S. enterica cells and the exposed cells clearly show damage at various sites of the outer membrane. Enzymatic Assays: The activity of the SOD and the catalase enzymes were obtained. The SOD activity was significantly (P<0.0001) more compared to the control E. coli at 0 and 45 minutes. On the contrary the catalase assay does not show any significant difference between the exposed and the control E. coli bacteria. This is in agreementwith the dominant presence of superoxides compared to hydroxyl radicals presented in the EWNS property characterization results above Natural Flora Experiments The Effect of EWNS on the inactivation of numbers and types of surface microflora on organic and conventionally grown tomatoes were obtained. These numbers reflect the culturable bacteria counts. On average, conventionally grown tomatoeshad a total load of 3.82 102 CFU/fruit (1.72 x 102 CFU/fruit of TVC and 2.1 X102 CFU/fruit of Fungi). The exposure to the EWNS was able to achieve a 90.50 % inactivation of the surface bacteria, while the fungi counts showed a 31.62 % inactivation. No E.coli was found on the surface of the conventionally grown tomatoes. Organic tomatoes showed higher numbers of TVC (1.85 x 105 CFU/fruit). The Fungi count was 2.67 x 102 CFU/fruit. Unlike conventional tomatoes, organic tomatoes carried a large load of E. coli of 1.78 x 105 CFU/fruit. The inactivation achieved was 99.97% for the bacteria while for the fungi was 88.37%. The microbial load distribution of the two types oftomato (organically grown vs conventional)was also assessed.The conventionally grown tomatoes consist of 1/3 fungi withthe restbeingbacterial species. On the contrary, the organically grown tomatoes have more than 97% bacteria and aminimumfungi load. Shelf life extension studies: See section below

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: G. Pyrgiotakis, R. Mitchell, A. Vasanthakumar, Y. Gao, A. Dearaujo, M. Eleftheriadou, P. Demokritou; Inactivation of foodborne microorganisms using Engineered Water Nanostructures (EWNS), Environ. Sci. Technol., 2015, 49, 37373745.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: G. Pyrgiotakis, P. Vedantam, Caroline Cirenza, J. McDevitt, M. Eleftheriadou, S. Lenard, P. Demokritou; Optimization of a nanotechnology based antimicrobial platform for food applications using Engineered Water Nanostructures (EWNS), Submitted  Nanoscale
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: P. Vedantam, G. Pyrgiotakis, Caroline Cirenza, J. McDevitt, M. Eleftheriadou, P. Demokritou; A nanotechnology-based, fresh produce, antimicrobial platform for natural flora inactivation and shelf life extension using Engineered Water Nanostructures (EWNS), in submission
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Nanotechnology to the Rescue: Food Pathogen Inactivation using Engineered Water Nanostructures, Philip Demokritou, Nanoscale Science & Engineering for Agriculture & Food Systems Gordon Research Conference, Bentley University, Waltham, MA, June 7-12 - invited presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Engineered Water Nanostructures (EWNS): A chemical free, nanotechnology based method for inactivation of foodborne microorganisms, Pallavi Vedantam, Nanoscale Science & Engineering for Agriculture & Food Systems Gordon Research Conference, Bentley University, Waltham, MA, June 7-12 - Poster presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Nanotechnology to the Rescue: Food Pathogen Inactivation using Engineered Water Nanostructures, Philip Demokritou, International Food Technology Conference, Chicago IL, July 11-14, 2015  invited Podium Presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Nanotechnology-based Approach for the Inactivation of Foodborne Microorganisms. Georgios Pyrgiotakis, International Association for Food Protection, Portland, OR, July24-28, 2015, - invited Podium Presentation


Progress 09/01/13 to 08/31/14

Outputs
Target Audience: The target audience(s) reached during the current reporting period include:1) Industries in thearea of microbial control in various " farm to the fork" applications (BASF, STERIS,Panasonic, Nanoterra);2) Scientific community working on food Microbiology issues; 3 Lay public Efforts included presentations in Food and Nano related conferences, peer reviewed publications, lectures in Universities and interviews and articles in magazines and scientific websites and media ( Economist, Chemical world , etc) . Furthermore, research outcomes and media articles on the project were posted on our Centers website (www.hsph.harvard.edu/nano). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A total of 15 Faculty, students and post doctoral fellowsparticipaed in the research activities this year 1. We plan tocontinue engaging students and faculty in the research activities this coming year How have the results been disseminated to communities of interest? The results were communicated to communities of interest via 1) peer reviewed publications; 2) presentaions in inernational and national conferences; 3) articles in magazines of general interest. It is worth mentioning that an article on the transofrmative nature of this research was published in the Economist in June 2014 (http://www.economist.com/news/technology-quarterly/21603236-water-particles-could-provide-powerful-airborne-shield-against-nasty-bacteria) What do you plan to do during the next reporting period to accomplish the goals? We plan to continue to assess the inactivation potential of EWNS on food borne bacteria using the developed and optimized protocols and delivery methods developed in year 1. In more detail: 1)Test the efficacy of the EWNS in inactivating a representative set of microorganisms (pathogens, surrogates/indicators and spoilage organisms) inoculated on the surface of fresh produce. Derive dose response relationships for indicative bacteria 2)Test the efficacy of EWNS in inactivating naturally occurring microflora on the surface of fresh produce-Real life Scenario (Months 15-19) 3)Test the efficacy of EWNs on delaying spoilage and decay and providing shelf-life extension – Shelf life Studies (Months 20-25)

Impacts
What was accomplished under these goals? 1. Impact: The societal and economic burden of foodborne related diseases is huge and the food industry is in need of new chemical free approaches to reduce risks. The aim of this study is to investigate the potential application of a recently developed by our group nanotechnology based, chemical free platform using Engineered water nanostructures (EWNS) made by the electro-spraying of deionized water to inactivate bacteria on fresh produce. 2. Materials and Methods 2.1 EWNS synthesis and characterization: The EWNS synthesis and physicochemical characterization has been described in great in our previously pubications (Pyrgiotakis et al., 2012; 2014a; 2014b). A high volume, high concentration, EWNS generation system was developed and used in the exposure experiments of this study. 2.2 EWNS Exposure approaches: The assessment of EWNS inactivation was performed under two distinct exposure approaches: 1) Diffusion - The pathogen inoculated surfaces were exposed in an atmosphere containing the EWNS aerosol and the nanoparticleswere allowed to reach and interact with the surface via diffusion; 2) A" draw through" electrostatic precipitation exposure system (EPES) was developed and used to take advantage of the EWNS high surface charge to enhance interactions and deposition of EWNS on surfaces. 2.3 EPES Characterization experiments:The EWNS deposition efficiency and particle losses in the EPES system was evaluated prior to its use in bacteria inactivation experiments using real time particle monitoring equipment. The deposition efficiency was assessed for varied flow rates through the chamber (0.5, 1, 2 l/min) and voltages (500 V, 1,000 V and 3,000 V). 2.4 Microbiological Procedures: The following Microbiological procedures/protocols were developed and optimized for the EWNS-pathogen investigation experiments 2.4.1 Test Microorganisms: Escherichia coli (ATCC # 27325), a known fecal indicator and a surrogate to enteropathogenic strains; Salmonella enterica (ATCC # 53647), a pathogen and surrogate to other pathogenic Salmonella strains, and Listeria innocua (ATCC # 33090), an environmental indicator and surrogate to pathogenic Listeria monocytogenes. 2.4.2 Bacterial inoculum preparation: For the stainless steel experiments, the E. coli test suspension was prepared by concentrating (X10) the overnight culture in phosphate buffer saline (PBS). A high concentration of E.coli (~1010 colony forming units (CFU)/mL) was used since E. coli was particularly sensitive to air-drying on the stainless steel surface. Two different surfaces were selected for inoculation due to the high relevance to the food industry - stainless steel, and tomatoes.. At each time point, the stainless steel coupons or tomatoes were removed from the t exposure chambers and processed to recover surviving bacteria. 2.5 Bacteria inactivation experiments: In the bacteria inactivation experiments the targeted surfaces were inoculated and exposed to the two different exposure approaches. Following exposure to EWNS the bacteria were recovered and counted to assess the removal compared to the controls (Non EWNS exposed bacteria). 2.6 Statistical analysis: All the experiments were done in triplicates and the standard deviation of the concentration was used as the measurement error. Log-reductions of bacterial concentrations were computed for a given condition 2.7 Sensory Evaluation Experiments: The effect of EWNS exposures on the overall quality and appeal of the tomatoes including their color, texture, skin integrity and aroma was assessed. 2.8 Mechanism of bacteria inactivation: The hypothesis that ROS encapsulated in the EWNS particles is the primary mechanism of microbial inactivation, by damaging of the outer cell membrane was assessed quantitatively using a lipid peroxidation (LPO) assay and qualitatively via Electron Microscopy imaging. 3. Results and Discussion 3.1 EWNS Deposition efficiency of the EPES system: Particle losses were found to be in the range of 5%. Overall, the deposition rate depending on the condition (flow through chamber and voltage) varies from 40% to 60% with optimum conditions at 3kV voltage and 2 L/min flow rate. 3.2 Bacteria recovery from inoculated surfaces: The assay developed in our study was simple, yet provided reproducible results for recovery of bacteria from the stainless steel surface. The loss observed in the control treatment consistently remained within 0.5 log (compared to time zero concentration) for E.coli. 3.3 Inactivation of bacteria on stainless steel and tomato surfaces using EWNS 3.3.1 EWNS Diffusion exposure approach Stainless steel surface: All three bacteria tested here showed between 1.8 and 0.6 log removal, as compared to control, after exposure to a EWNS treatment (EWNS exposure levels 24,000 #/cc), at exposure timesbetween 45 to 90 mins. More specifically, E. coliat 45 minutes of exposure, the results show an approximate 1.8 log removal. This represents a rate of inactivation 30 times as fast as the control. Listeria exposed to EWNS was reduced with an average 0.8 logs removal in 90 minutes, as compared to the control. Finally, similar results were obtained for Salmonella exposed to EWNS which was reduced by 0.6 logs in 90 minutes, as compared to the control Bacteria Inactivation on tomato surface: E. coli was able to survive air drying much better when inoculated on tomato surfaces compared to stainless steel surfaces. After 90 minutes EWNS exposure (at 24,000 particles/cc)the results show an approximate 1 log removal, as compared to the control. Similarly for Listeria, an average 0.7 logs removal in 90 minutes was found. Finally, similar results were observed with, Salmonella with an average 0.5 logs removal, as compared to the control, in 90 minutes of EWNS exposure. 3.2.2 Inactivation of bacteriaon tomatoes using the EPES system: For the E. coli case and 50,000 #/cm3 EWNS exposure levels without the electric field (control) the removal rate was at the same level as the no EWNS exposure control. In contrast, the inoculated tomatoes exposed to the EWNS aerosol underthe electric field conditions showed a 2.3 logs removal (1.4 logs compared to the control). For listeria the exposure was done at two doses. A 1.2 logs removal compared to the control for the 23,000 #/cm3 and 0.5 12,000 #/cm3 exposure levels were found for listeria. 3.3 Mechanism of bacteria inactivation: Dataconfirmed thatROS encapsulated in the EWNS particles is the primary mechanism of microbial inactivation, by damaging of the outer cell membrane. This was proven quantitatively with a lipid peroxidation (LPO) assay (Pyrgiotakis et al., 2014b) and qualitatively via Electron Microscopy imaging. 3.4 Sensory Evaluation Experiments: Overall the comparison between the treated and control tomatoes showed no significant difference, indicating that the treatment is not inducing any sensory effects on the tomatoes. 4. Conclusions: Overall, the first year data on the inactivation of food-borne bacteria on food production and fresh produce surfaces demonstrated promising results. The EWNS were effective in inactivating three microorganisms particularly important in the food industry: E. coli, Salmonella and Listeria. The inactivation potential was further increased when the delivery of the EWNS was better targeted with the implementation of an electric precipitator concept. This resulted in increased inactivation (x 3 times) as compared to the simple diffusion method. In future studies, our efforts will be focused on further increasing the inactivation by either enhancing the delivery to surfaces or by increasing the EWNS concentration number.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Pyrgiotakis, G. McDevitt, J. Gao, Y., Branco, A., Eleftheriadou, M., Lemos, B., Nardell E. and Demokritou, P.  Inactivation of Mycobacteria using Engineered Water Nanostructures (EWNS). Nanomedicine: Nanotechnology, Biology and Medicine, 2014, 10, 1175-1183
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: A Novel, Chemical free Intervention Nanotechnology For Fresh Produce Surface Disinfection Using Engineered Water Nanostructures G. Pyrgiotakis, R. Mitchell, A. Vasanthakumar, Y. Gao, A. Dearaujo, M. Eleftheriadou, P. Demokritou, Nanotech 2014, Washington, DC, Jun 15-18, 2014