DEVELOPING A NEW CLASS OF ANTIFOULING/NTIMICROBIAL POLYMER COATINGS FOR FOOD-CONTACT SURFACES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: REVISED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013823
• Project No.: NYC-123303
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2021

Project Director
Ma, MI.

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Biological & Environmental Engineering

Non Technical Summary
One of the major causes for food waste and food-borne illness is the contamination of food-contact surfaces at production facilities or during packaging, transportation and storage. In this project, we aim to develop a new class of antifouling/antimicrobial zwitterionic-halamine (Zw-Ha) copolymer coatings to eliminate or mitigate the biofouling of food-contact surfaces.

Animal Health Component

0%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	7410	2020	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
7410 - General technology;

Field Of Science
2020 - Engineering;

Keywords

food safety

food-contact surface

antifouling/antimicrobial

Goals / Objectives
The main objective of this proposal is to develop a new class of antifouling/antimicrobial zwitterionic-halamine (Zw-Ha) copolymers to modify food-contact surfaces. Specifically, we will perform the following lines of research:Aim 1. Synthesize a series of polymerizable monomers that contain N-halamine precursor groups such as hydantionylacrylamide, or zwitterionic groups such as phosphorylcholine, sulfobetaine and carboxybetaine.Aim 2. Develop universal methods for surface-initiated co-polymerization of the monomers synthesized in Aim 1 and modify different types of food-contact surfaces such as stainless steel, glass and plastic.Aim 3. Test the antifouling and antimicrobial properties of the modified surfaces against different proteins and representative bacteria including Staphylococcus aureus and Escherichia coli O157:H7 and evaluate the biofilm control effect of select coated materials.Aim 4. Test microbiological control effect of select coated materials in various food models including dairy, raw meat and fresh produce against related pathogenic or spoilage bacteria in each food category including Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Pseudomonas.

Project Methods
Aim 1. Synthesize a series of polymerizable monomers that containN-halamine precursor groups such as hydantionylacrylamide, or zwitterionic groups such as phosphorylcholine, sulfobetaine andcarboxybetaine. Polymerizable N-halamine precursor monomers such as hydantionylacrylamide (HA) that contain both vinyl bond and/oranchor moieties (catechol, epoxide, siloxane) can be readily preparedthrough either Bucherer-Berg reaction method from commercialdiacetone acrylamide or substitution reaction methods (alkyl substitution and Gabriel reaction) from commercial5,5-dimethylhydantoin (DMH).12-14 One of my postdoctoralresearchers comes from a group, which has extensive experience insynthesizing a variety of N-halamine monomers; thus we are able tosynthesize desired monomers for different surface coatingtechnologies. Polymerizable zwitterionic monomers such as2-methacryloyloxyethyl phosphorylcholine (MPC), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA), andcarboxybetaine acrylamide (CBAA) are either commercially available orcan be easily prepared ourselves. Our group has extensive experience synthesizing these zwitterionic monomers.Aim 2. Develop universal methods for surface-initiatedco-polymerization of the monomers synthesized in Aim 1 and modify different types of food-contact surfaces such as stainless steel, glassand plastic. We will explore two different strategies to achieve the surface-initiated co-polymerization. First, we will introduce a catecholgroup into an Atom Transfer Radical Polymerization (ATRP) initiatorfollowing a previously published method.15 Copolymers made fromthis initiator can then be dip-coated on a range of different surfacesdue to the highly adhesive catechol group. The second strategy is touse a reactive ATRP initiator,(11-(2-bromo-2-methyl)-propionyloxyl)undecyltrichlorosilane(Br-PUCS) that can form covalent bonds with surface hydroxyl groupspresent on oxidized glass or stainless steel. The zwitterionic andN-halamine precursor monomers can then be directly co-polymerized from the surface.Aim 3. Test the antifouling and antimicrobial properties of themodified surfaces against different proteins and representative bacteria including Staphylococcus aureus and Escherichia coliO157:H7 and evaluate biofilm control effect of select coated materials.Polymer-coated/grafted or untreated substrates will be immersed indifferent protein solutions such as FITC labeled Fibrinogen PBS solution or human plasma to test the protein adsorption. Theadsorption will be quantified by either fluorescence intensity or through using a surface plasmon resonance (SPR) sensor.16 In thescreening stage, a "sandwich" test will be used to quantify the antibacterial efficacy against both Staphylococcus aureusantibacterial efficacy against both Staphylococcus aureus(gram-positive, sphere-shaped) and Escherichia coli O157:H7(gram-negative, rod-shaped) bacteria.11 The "Sandwich" test is anantimicrobial efficacy testing method which measures thecontact-killing effect of solid surface materials. In brief, an aliquot of25 μL of bacterial suspension with a known density (e.g. 4Å~107CFU/mL) will be prepared in a buffer solution and added into thecenter of a 1x1-inch^2 squarecoupon, and a second identical couponwill be placed on the top. A sterile weight will be placed on the top toensure complete contact with the inoculated bacteria. After certaincontact times (e.g., 1,5, 10, 30 min), the coupons will be quenched withan Na2O3S2 solution to remove any oxidative chlorine residuals andvortexed to detach bacteria from the surfaces. Ten-fold serialdilutions will be made for all samples, and each dilution will be platedon selective culture medium agar plates. The plates will be incubated,and bacterial colonies will be enumerated for antimicrobial efficacyanalysis. Antimicrobial efficacy will be determined by comparing the log CFU reduction of bacteria on modified and untreated surfaces. Theantimicrobial activity of the modified-surfaces will be further testedby incubating them in bacterial suspensions; the surviving bacteriaboth in the medium and on the surface will be quantified throughplating. The ability of the surface to control biofilm formation will betested by Scanning Electron Microscopy (SEM) or FluorescenceMicroscopy following a previously reported method.4Aim 4. Test microbiological control effect of select coated materials invarious food models including dairy, raw meat and fresh produceagainst related pathogenic or spoilage bacteria in each food categoryincluding Staphylococcus aureus, Escherichia coli O157:H7, Listeriamonocytogenes, Salmonella spp. and Pseudomonas. Modified surfaceswith optimized antifouling/antimicrobial efficacy developed duringthe first three aims will be selected and challenged with other majorfoodborne pathogenic or spoilage bacteria in the absence and presenceof various food models including dairy, raw meat and fresh producefollowing a previously described method.17 Food liquid orhomogenate will be prepared and inoculated together with relatedbacteria to artificially contaminate the coated materials. After a certainperiod of incubation time under controlled environments,microbiological contamination on the surfaces will be quantified usingstandard environmental microbiological testing methods.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Food microbiology safety research scientists, food hygiene and sanitation product manufacturers, food processors, food safety and quality control managers in food industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained a postdoc research associate, who has developed his skills for experiment design, execution,and report and is dedicated to promotingthe research for improving food safety during food processing. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest (food processing industry) through multiple channelsas planned: 1) conferences and tradeshows: 2018 Fall ACS (American Chemistry Society) meeting, and 2019 SummerIAFP (International Association for Food Protection) annual meeting. Also, results have been published on a peer-reviewed journal that targets food processing industry: ACS Food and Agricultural Chemistry. Also, we have filed a patent throughCornell University.Finally, we have started a company which has seen initial success, providing over10 job opportunities to the local economy. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have accomplished all the research goals. More specifically: Aim 1. Synthesize a series of polymerizable monomers that contain N-halamine precursor groups such ashydantionylacrylamide, or zwitteriobic groups such as phosphorylcholine, sulfobetaine and carboxybetaine. 100% accomplished. A N-halamine precursor monomer hydantionylacrylamide was successfully synthesized in ourlab. A dopamine vinyl monomer was also successfully synthesized. A vinyl monomer containing zwitterionic groups(Sulfobetaine) was selected and tested to prove the feasibility. Aim 2. Develop universal methods for surface-initiated co-polymerization of the monomers synthesized in Aim 1 andmodify different types of food-contact surfaces such as stainless steel, glass and plastic. 100% accomplished. A series of copolymers that containing different ratios of N-halamine functional group(antimicrobial), zwitterionic functional group (antifouling) and dopamine functional group (anchoring) were synthesized.Formulations that are feasible for dip-coating were selected for dip-coating conditions studies. Various materials includingstainless steel, glass and plastics (polypropylene, polyethylene, polyvinylchloride) were tested and proved can be coated with this dip-coating method. Aim 3. Test the antifouling and antimicrobial properties of the modified surfaces against different proteins andrepresentative bacteria including Staphylococcus aureus and Escherichia coli O157:H7 and evaluate the biofilm control effectof select coated materials. 100% accomplished. The antimicrobial effect of zwitterionic-halamine coated stainless steel surface was evaluated. TheN-halamine modified surface can achieve 6-log reductions of gram-negative and gram-positive bacteria within 30 min ofcontact based on in vitro contact kill test. However, the antifouling effect of zwitterionic functional groups was not significant based on the characterization method we used (florescence protein binding test). The biofilm control effect of N-halaminecoating materials was also observed using live/dead florescence staining test. Aim 4. Test biofilm control effect in various food models (dairy, raw meat and fresh produce) with related pathogenic andspoilage bacteria in each food category (Listeria monocytogenes, Salmonella spp. and Pseudomonas). Adjusted and 100% accomplished. The biofilm control effect was tested using Listeria monocytogenes. We adjusted theobjectives for this project based on literature and our experiment confirmation that: 1) it is not necessary to do Salmonella andPseudomonas since N-halamine antimicrobial function is non-specific and broad; 2) This coating is suitable for non-food contact surfaces (zone 2 and 3) so the organic load is not a concern at this stage. For zone 2 and 3, Lm is the biggestconcern as environmental pathogen. In addition, we have developed a new polymer containing halamine and an adhesive group dopamine. This new polymer can be formulated and spray-coated on a range of surfaces. We also started a company based on this new polymer. (www.halomine.com)

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Food microbiology safety research scientists, food hygiene and sanitation product manufactures, food safety and qualitycontrol managers in food industry. Changes/Problems:We did some adjustment for Aim 4: the biofilm control effect was tested using Listeria monocytogenes. We adjusted the objectives for this project based on literature and our experiment confirmation that: 1) it is not necessary to do Salmonella and Pseudomonas since N-halamine antimicrobial function is non-specific and broad; 2) This coating is suitable for non-food contact surfaces (zone 2 and 3) so the organic load is not a concern at this stage. For zone 2 and 3, Lm is the biggest concern as environmental pathogen. What opportunities for training and professional development has the project provided?This project has trained a postdoc research associate, who has developed his skills for experiment design, execution, andreportand is dedicated to promote the research for improving food safety during food processing. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest (food processing industry) through multiple channels asplanned: 1) conferences and tradeshows: 2018 Fall ACS (American Chemistry Society) meeting, and 2019 Summer IAFP(International Association for Food Protection) annual meeting. Also, results have been published on a peer-reviewed journalthat targets food processing industry: ACS Food and Agricultural Chemistry. Also, we have filed a patent through CornellUniversity, which has been successfully licensed out to a startup company for commercializing this technology to serve foodindustry. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major activities completed Synthesized and characterized all chemical compounds (monomers and polymers) for the halamine-zwitterionic coating; Optimized the coating conditions for dip-coating methods (e.g. concentration, treating time, temperature, etc.) and characterized the coatings; Performed the antimicrobial tests and antifouling test and evaluated the biofilm control effect of coating on stainless steel; Specific objectives met We already achieved Aim 1, 2 and 3 in the proposal. Specific response and results can be found in the following sections: Aim 1. Synthesize a series of polymerizable monomers that contain N-halamine precursor groups such as hydantionylacrylamide, or zwitteriobic groups such as phosphorylcholine, sulfobetaine and carboxybetaine. 100% accomplished. A N-halamine precursor monomer hydantionylacrylamide was successfully synthesized in our lab. A dopamine vinyl monomer was also successfully synthesized. A vinyl monomer containing zwitterionic groups (Sulfobetaine) was selected and tested to prove the feasibility. Aim 2. Develop universal methods for surface-initiated co-polymerization of the monomers synthesized in Aim 1 and modify different types of food-contact surfaces such as stainless steel, glass and plastic. 100% accomplished. A series of copolymers that containing different ratios of N-halamine functional group (antimicrobial), zwitterionic functional group (antifouling) and dopamine functional group (anchoring) were synthesized. Formulations that are feasible for dip-coating were selected for dip-coating conditions studies. Various materials including stainless steel, glass and plastics (polypropylene, polyethylene, polyvinylchloride) were tested and proved can be coated with this dip-coating method. Aim 3. Test the antifouling and antimicrobial properties of the modified surfaces against different proteins and representative bacteria including Staphylococcus aureus and Escherichia coli O157:H7 and evaluate the biofilm control effect of select coated materials. 100% accomplished. The antimicrobial effect of zwitterionic-halamine coated stainless steel surface was evaluated. The N-halamine modified surface can achieve 6-log reductions of gram-negative and gram-positive bacteria within 30 min of contact based on in vitro contact kill test. However, the antifouling effect of zwitterionic functional groups was not significant based on the characterization method we used (florescence protein binding test). The biofilm control effect of N-halamine coating materials was also observed using live/dead florescence staining test. Aim 4. Test biofilm control effect in various food models (dairy, raw meat and fresh produce) with related pathogenic and spoilage bacteria in each food category (Listeria monocytogenes, Salmonella spp. and Pseudomonas). Adjusted and 100% accomplished. The biofilm control effect was tested using Listeria monocytogenes. We adjusted the objectives for this project based on literature and our experiment confirmation that: 1) it is not necessary to do Salmonella and Pseudomonas since N-halamine antimicrobial function is non-specific and broad; 2) This coating is suitable for non-food contact surfaces (zone 2 and 3) so the organic load is not a concern at this stage. For zone 2 and 3, Lm is the biggest concern as environmental pathogen. Significant results achieved, including major findings, developments or conclusions (both positive and negative); and The most encouraging development achieved in current research was to prove the concept of using dip-coating methods to achieve functional and relative durable coatings on various food-contact substrate materials. Although the coating is thin (~50 nm), it was sufficient to provide potent antimicrobial killing effect and biofilm controlling function. Also, we proved the Lm biofilm prevention effect of our new coating on real food equipment with the same sampling method that is used in the food processing plant. This will create the opportunity to further applications in food processing environments. Key outcomes or other accomplishments realized Key outcomes are: a. developed the formulations and conditions for synthesize halamine/dopamine/zwitterionic copolymers; b. developed the formulations and conditions for creating thin-film coating on various substrate materials that are used in food processing environments; c. proved the antimicrobial and biofilm controlling functions of proposed coating; d. proved the efficacy of biofilm controlling effect of this coating on food equipment against Lm.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Qiao M, Liu Q, Yong Y, Pardo Y, Worobo R, Liu Z, Jiang S, Ma M. 2018. Scalable and Rechargeable Antimicrobial Coating for Food Safety Applications, Journal of Agricultural and Food Chemistry., 66, 11441.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Food microbiology safety research scientists, food hygiene and sanitation product manufactures, food safety and quality control managers in food industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoc working on this project has opportunity to publish papers, present results, and write applications for federal grant and university fund, all these activities enhanced personal development. How have the results been disseminated to communities of interest?First, we have published our results on a food science journal, Agricultural and Food Chemistry. Second, we have also presented our results in conferences that are highly related to target audience, including American Chemical Society (ACS) and American Society for Agricultural and Biological Engineers (ASABE). What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will test microbiological control effect of select coated materials in various food models including dairy, raw meat and fresh produce against related pathogenic or spoilage bacteria in each food category including Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Pseudomonas.

Impacts
What was accomplished under these goals? 1. Major activities completed Accomplished major goals for chemical synthesization, coating optimization on food machine materials, and microbiological testing. Disseminated results to related parties through publishing results in peer-reviewed journals, presenting results in conference meetings, and filing a patent. Submitted a USDA foundational program grant application based on results from this study. 2. Specific objectives met We already achieved Aim 1, 2 and 3 in the proposal. Specific response and results can be found in the following sections: Aim 1. Synthesize a series of polymerizable monomers that contain N-halamine precursor groups such as hydantionylacrylamide, or zwitteriobic groups such as phosphorylcholine, sulfobetaine and carboxybetaine. Accomplished. A N-halamine precursor monomer hydantionylacrylamide was successfully synthesized in our lab. A dopamine vinyl monomer was also successfully synthesized. Aim 2. Develop universal methods for surface-initiated co-polymerization of the monomers synthesized in Aim 1 and modify different types of food-contact surfaces such as stainless steel, glass and plastic. Accomplished. A series of copolymers that containing different ratios of N-halamine functional group (antimicrobial) and dopamine functional group (anchoring) were synthesized. Formulations that are feasible for dip-coating were selected for dip-coating conditions studies. Various materials including stainless steel, glass and plastics (polypropylene, polyethylene, polyvinylchloride) were tested and proved can be coated with this dip-coating method. Aim 3. Test the antifouling and antimicrobial properties of the modified surfaces against different proteins and representative bacteria including Staphylococcus aureus and Escherichia coli O157:H7 and evaluate the biofilm control effect of select coated materials. Accomplished. The antimicrobial effect of halamine-dopamine coated stainless steel surface was evaluated. The N-halamine modified surface can achieve 6-log reductions of gram-negative and gram-positive bacteria within 30 min of contact based on in vitro contact kill test. The biofilm control effect of N-halamine coating materials was also observed using live/dead florescence staining test. Aim 4. Test microbiological control effect of select coated materials in various food models including dairy, raw meat and fresh produce against related pathogenic or spoilage bacteria in each food category including Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Pseudomonas. Not fully accomplished. We will work more on this aim in this year. 3. Significant results achieved, including major findings, developments or conclusions (both positive and negative) The most encouraging development achieved in current research was to prove the concept of developing an easy-to-apply coating system on food processing machine. This will create the opportunity to further applications in food processing environments. 4. Key outcomes or other accomplishments realized Key outcomes are: a. developed the formulations and conditions for synthesize halamine/dopamine copolymers; b. developed the formulations and conditions for creating thin-film coating on various substrate materials that are used in food processing environments; c. proved the antimicrobial and biofilm controlling functions of proposed coating.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Qiao M, Liu Q, Yong Y, Pardo Y, Worobo R, Liu Z, Jiang S, and Ma M. 2018. Scalable and rechargeable antimicrobial coating for food safety applications. Journal of Agricultural and Food Chemistry Oct 8, 2018 DOI: 10.1021/acs.jafc.8b03864 (Featured on Cover)