Article pubs.acs.org/JAFC
Antimicrobial Coatings with Dual Cationic and N‑Halamine Character: Characterization and Biocidal Efficacy Luis J. Bastarrachea and Julie M. Goddard* Department of Food Science, University of Massachusetts, 102 Holdsworth Way, Amherst, Massachusetts 01003, United States S Supporting Information *
ABSTRACT: A method to prepare an antimicrobial coating for food-handling materials is reported. Alternating layers of branched polyethylenimine and styrene maleic anhydride copolymer were applied onto the surface of polypropylene. The resulting coatings had low surface energy and presented enhanced antimicrobial character due to the presence of both cationic and N-halamine forming structures. In its unchlorinated form, the coating inactivated Listeria monocytogenes by ∼3 logarithmic cycles. In the form of N-halamines >5 logarithmic cycles were reached. Microbial inactivation kinetics showed a Weibullian behavior when the coating was unchlorinated and a sigmoidal behavior when chlorinated. Microscopy confirmed that the reduction in the microbial load was due to biocidal effects of the coating and not bacterial adhesion onto the modified surface. The modified surface was able to be repeatedly rechlorinated. Such rechargeable antimicrobial coatings may support improving food safety by reducing cross-contamination of microorganisms from food-processing equipment. KEYWORDS: antimicrobial coatings, N-halamines, cationic polymers, inactivation kinetics, bacterial adhesion
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and polyamines.17,18 They are believed to impart antimicrobial activity by disrupting cell membrane functions mainly through ionic exchange.19−21 A hurdle to the utilization of cationic polymer-based antimicrobial coatings is their risk of fouling by organic molecules.22 Previous studies have explored the possibility of incorporating N-halamines and a cationic antimicrobial within the same system, with promising results.23−25 A coating with a dual antimicrobial nature containing N-halamines and cationic moieties could integrate the rechargeable nature of the Nhalamine while retaining the antimicrobial character after chlorine dissociation due to its cationic nature. Additionally, antimicrobial coatings composed by polymers may represent a lower risk of residual toxicity, because the antimicrobial character remains on the surface and is not expected to delaminate.10,26 We hypothesized that tailoring the chemistry of the coating could enable retention of the N-halamine and cationic antimicrobial character while reducing the risk of bacterial adhesion. The objective of the present study was to create a coating with dual antimicrobial activities composed of branched polyethylenimine (PEI) and styrene maleic anhydride copolymer (SMA), characterize it, and demonstrate its effectiveness in both N-halamine and unchlorinated cationic form after application onto polypropylene (PP, a plastic polymer widely used for food contact surfaces27) against the common food pathogen Listeria monocytogenes.
INTRODUCTION Contamination of materials used in healthcare and the food industries (in different types of materials found in processing appliances, food-handling accessories, conveyor belts, etc.) by pathogenic microorganisms remains a significant challenge to public health.1−6 A number of antimicrobial coatings have therefore been proposed in an effort to reduce infections resulting from such cross-contamination.4−8 Incorporation of small-molecule biocides (e.g., antibiotics, metal nanoparticles) into a coating is effective; however, reliance on migration of an antimicrobial agent for activity has two significant drawbacks. Materials inherently lose antimicrobial activity over time, and migration of small-molecule antimicrobial agents may promote development of microbial resistance.8,9 Polycationic and Nhalamine-based coatings have been explored to overcome these challenges. N-Halamines constitute a diverse class of antimicrobial compounds. They are generally organic substances characterized by the presence of nitrogen atoms, which are normally in the form of amines, amides, and imides. These nitrogenous functional groups are able to form covalent bonds with halogens (N−X) such as bromine, iodine, and chlorine. NHalamines exert antimicrobial activity toward a wide range of microorganisms by releasing their halogen through a mechanism believed to include cell membrane disruption and inner cell molecules oxidation.10 The most remarkable characteristic of N-halamines is their ability to be recharged with halogens for many cycles, providing continuous antimicrobial activity.11−13 Even though the effectiveness of N-halamine-derived coatings has been extensively demonstrated, they are generally ineffective against microorganisms in their unchlorinated state, and their need for continuous rechlorination can affect their chemical integrity due to the high pH and oxidative reactivity of bleach solutions.9,14 Cationic polymers represent another well-studied class of antimicrobial substances that include polylysine,15 chitosan,16 © 2015 American Chemical Society
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MATERIALS AND METHODS
Materials. Polypropylene pellets (PP, isotactic) and styrene maleic anhydride copolymer (SMA, MW 6000 Da) were from Scientific Received: Revised: Accepted: Published: 4243
January 23, 2015 April 1, 2015 April 6, 2015 April 14, 2015 DOI: 10.1021/acs.jafc.5b00445 J. Agric. Food Chem. 2015, 63, 4243−4251
Article
Journal of Agricultural and Food Chemistry
Figure 1. Process for application of the reported antimicrobial coating. Polymer Products (Ontario, NY, USA). Isopropanol, acetone, methanol, and glycerol were from Fisher Scientific (Pittsburgh, PA, USA). Branched polyethylenimine (PEI, MW 25000), 2-ethoxy-1ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, MW 247.3), and 4-(2hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) were from Sigma-Aldrich (St. Louis, MO, USA). 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES, MW 217.2) was from GenScript Inc. (Piscataway, NJ, USA). The dye Orange (II) (Cert) (AO7), the sodium hypochlorite solution (5% chlorine), ethylene glycol, and absolute ethanol were from Acros Organics (Fair Lawn, NJ, USA). N,N-Diethyl-p-phenylenediamine DPD total chlorine reagent powder (DPD) was from Hach Co. (Loveland, CO, USA). Tryptic soy broth (TSB), tryptic soy agar (TSA), and neutralizing buffer were from Difco, Becton Dickinson (Sparks, MD, USA). Antimicrobial Coating Application onto Polypropylene Coupons. Preparation of PP Coupons. PP pellets were cleaned by sonication first with isopropanol, then with acetone, and finally with deionized (DI) water (two cycles of 10 min were applied for each solvent). Cleaned PP pellets were left to dry overnight under anhydrous calcium sulfate (RH < 20%) and then hot pressed at 170 °C with a load force of 9000 lb. Coupons of 2 × 2 cm were cut from the obtained films (thickness = 0.5 ± 0.1 mm) and cleaned and dried under the same conditions applied to the PP pellets. PP Surface Activation. The 2 × 2 cm coupons were UV-ozoneirradiated from one side with a Jelight Co. model 42 UVO Cleaner (Irvine, CA, USA) for 15 min to create carboxylic acid groups on their surfaces28,29 (this step will be referred as UV-O3). To create surface anhydride groups (and make the PP surface reactive toward the primary amines of PEI through nucleophilic attack), UV-O3 coupons were shaken for 2 h at room temperature in a 0.1 mM solution of EEDQ in 50 mM MES buffer (pH 5.5).30 Before mixing with the MES buffer, the necessary amount of EEDQ was dissolved in a volume of methanol equal to 5 logarithmic reductions. The inactivation kinetics showed a different behavior between modified PP and chlorinated modified PP (Figure 6B). The inactivation kinetics
of unchlorinated modified PP exhibited a Weibullian behavior (the cationic form, eq 3, R2 = 0.84), whereas chlorinated modified PP showed a sigmoidal behavior (N-halamine form, eq 4, R2 = 0.84). Cationic polymers have been reported to inactivate microorganisms in previous studies, and the possible explanations provided for their action have been an ion exchange process between the positive charges of the polymers and the cell membrane (causing its deterioration), the generation of reactive oxygen species that can provoke oxidative stress and damage genetic information and other biomolecules, and interruption of the electron transport chain, vital for energy generation.19−21 In previous works, N-halamines11,28 and cationic polymers50 have exhibited inactivation kinetics similar to the ones observed here. In the cited works, inactivation kinetics given by N-halamines exhibits an initially slow biocidal effect, which is followed by a drastic and abrupt reduction in the microbial population. In contrast, according to the cited reference in the case of cationic polymers, the inactivation kinetics shows initially a drastic and fast decrease in the microbial population, which is followed by a plateau or by an abrupt decrease in the speed of inactivation. Scanning Electron Microscopy (SEM). To confirm that reductions in bacterial populations were due to inactivation by the antimicrobial coating and not a result of adhesion onto its surface due to its cationic nature, electron microscopy was performed on coupons following antimicrobial activity assays (Supporting Information, Figure S1). No visual evidence of attached bacteria was found on native or coated PP. Previous works have shown that PP exhibits a low level of surface attachment of L. monocytogenes as compared to other materials’ surfaces. In the same mentioned studies, adhesion of L. monocytogenes cells on surfaces has not been possible to link with materials properties such as roughness and hydrophobicity,27 which makes this phenomenon unpredictable on the basis of surface properties. The results obtained confirm that, under the test conditions and at the inoculum level used, 4249
DOI: 10.1021/acs.jafc.5b00445 J. Agric. Food Chem. 2015, 63, 4243−4251
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microscopy; AO7, Acid Orange 7; DPD, N,N-diethyl-pphenylenediamine; TSB, tryptic soy broth; TSA, tryptic soy agar; SEM, scanning electron microscopy
the reduction in microbial load exerted by modified PP and chlorinated modified PP is not caused by cell adhesion on the coupons’ surfaces. Chlorine Rechargeability of the Antimicrobial Coating. Modified PP was exposed to 10 cycles of chlorination to demonstrate the ability of the coating to regenerate Nhalamines (Supporting Information, Figure S2). Some of the bonds that hold together the layers of PEI and SMA exhibited hydrolysis after the 10 rechlorinations according to the ATRFTIR spectra. The absorbance increases in the 3400−3200 cm−1 range (Supporting Information, Figure S2A) and in the 1740−1720 cm−1 range (Supporting Information, Figure S2B), which are characteristic of the CO and CO vibrations of carboxylic acids, respectively, and an absorbance reduction at ∼1650 cm−1 (Supporting Information, Figure S2B, CO vibration of amides) confirm the hydrolysis of amide and imide bonds. As can be observed (Supporting Information, Figure S2C), an initial increase in chlorine absorption is exhibited, followed by stabilization. From the SEM images (Supporting Information, Figure S1D), it can be seen that a single chlorination causes a change in the surface pattern. However, as the antimicrobial evaluation confirmed, the biocidal efficacy was retained after 10 rechlorinations (Figure 6A). Similar results have been reported in studies involving rechlorination of polymeric N-halamines.29
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ASSOCIATED CONTENT
S Supporting Information *
A table of XPS analysis results, atomic percentages, and deconvolution of high-resolution bands of O 1s and N 1s; figures showing results from SEM analysis and the N-halamine content of the modified PP coupons during rechlorination and the ATR-FTIR spectra before and after the chlorine rechargeability evaluation. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*(J.M.G.) Phone: (413) 545-2275. Fax: (413) 545-1262. Email:
[email protected]. Funding
This material is based upon work supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under Project 2011-65210-20059. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. Lynne A. McLandsborough for assistance in the antimicrobial evaluations, Prof. D. Julian McClements for the use of his Drop Shape Analyzer DSA100; Prof. Thomas McCarthy and Jacob Hirsch for use of their Physical Electronics Quantum 2000; and Dr. Sekar T. Dhanasekaran for training and assistance in the AFM analysis.
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ABBREVIATIONS USED PP, polypropylene; PEI, branched polypropylene; SMA, styrene maleic anhydride copolymer; DI, deionized; UV-O3, ultravioletozone; EEDQ, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquioline; MES, 2-(N-morpholino)ethanesulfonic acid; ATR-FTIR, attenuated total reflectance Fourier transform infrared spectroscopy; XPS, X-ray photoelectron spectroscopy; AFM, atomic force 4250
DOI: 10.1021/acs.jafc.5b00445 J. Agric. Food Chem. 2015, 63, 4243−4251
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