Rechargeable Antimicrobial Surface Modification of Polyethylene
Total Page：16
File Type：pdf, Size：1020Kb
2042 Journal of Food Protection, Vol. 71, No. 10, 2008, Pages 2042–2047 Copyright ᮊ, International Association for Food Protection Rechargeable Antimicrobial Surface Modification of Polyethylene J. M. GODDARD AND J. H. HOTCHKISS* Department of Food Science, Cornell University, Stocking Hall, Ithaca, New York 14853, USA MS 08-041: Received 22 January 2008/Accepted 17 May 2008 ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/10/2042/2204454/0362-028x-71_10_2042.pdf by guest on 02 October 2021 Polyethylene films were surface modified, to incorporate amine and amide functionalities, and subsequently were evaluated for their ability to recharge the antimicrobial N-halamine structures after contact with sodium hypochlorite, a common food- approved sanitizer. Surfaces were tested for chlorine retention and release, as well as antimicrobial activity against microor- ganisms relevant to food quality and food safety, including Escherichia coli K-12, Pseudomonas fluorescens, Bacillus cereus, and Listeria monocytogenes. N-Halamine functionalized polyethylene exhibited chlorine rechargeability, maintaining 5 to 7 nmol/cm2 N-halamine structures for six successive charges. The N-halamine functionalized films achieved a 4-log reduction for all organisms tested and maintained a greater than 3-log reduction for four successive uses, suggesting that the modified polyethylene films are capable of providing rechargeable antimicrobial activity. The modified films exhibited antimicrobial activity in aqueous suspensions (P Ͻ 0.05) and reduced microbial growth in diluted broth (P Ͻ 0.05), suggesting the potential for biocidal action even in the presence of organic matter. Such a rechargeable antimicrobial surface could supplement existing cleaning and sanitation programs in food processing environments to reduce the adhesion, growth, and subsequent cross- contamination of food pathogens, as well as food spoilage organisms. Foodborne diseases are estimated to cause 76 million which is then free to inactivate microorganisms (38). Al- illnesses annually (28). In addition to the public health con- ternatively, the intact N-halamine might inactivate micro- cern, the financial effect of product recalls (7) and the re- organisms by direct oxidation of biomolecules within the sulting drop in consumer confidence must be taken into microorganism (DNA, RNA, lipids, proteins) (13, 27). The account. Microbial growth also results in economic losses imides, amides, or amines can then be recharged with com- from spoilage (25). Surfaces such as filler nozzles, gasket mercial bleach (sodium hypochlorite) to regain its antimi- materials, conveyor belts, and work tables are prime loca- crobial chlorinated structure. This cycle can be repeated a tions for bacterial adhesion and subsequent contamination number of times. of product. If the food contact surface to which bacteria Textiles and nonwoven fabrics capable of reversibly have adhered were self-sanitizing, it could enhance current and repeatedly forming N-halamine structures have been cleaning and sanitization protocols by providing sustained investigated, as well as polystyrene beads and coatings suit- antimicrobial activity. able for pools and water disinfection applications (3, 5, 8, Commercial antimicrobial surfaces such as AgION 9, 26, 31–37). Similar materials might be applicable in the (AgION Technologies, Inc., Wakefield, Mass.) and Micro- food industry because processing surfaces could become ban (Microban International, Ltd., New York, N.Y.), with recharged after treatment with sodium hypochlorite, a com- silver zeolite and triclosan as the respective active agents, mon approved food contact sanitizing agent (13, 27). Dur- require migration from the surface to be effective. Thus, ing the subsequent processing run, the chlorine could in- the antimicrobial activity of the surface decreases over time activate microorganisms, providing a surface that resists (2, 11, 12). In addition to limited lifetime because of dif- colonization, multiplication, and cross-contamination of fusion of the active compound, silver zeolite– and triclosan- food pathogens and food spoilage organisms. incorporated surfaces have the risk of promoting the de- We have previously developed methodologies for mod- velopment of antimicrobial-resistant organisms (6, 15, 20, ifying the surface chemistry of polyethylene and other 22, 23) and might not be effective in a food-processing polymer surfaces (18, 19). One such surface-modified poly- environment (24, 30). In recent years, a new strategy has ethylene, PE-polyCOOH, was developed by covalently im- been investigated—rechargeable antimicrobial activity. mobilizing polyacrylic acid to oxidized polyethylene via a Chlorination of the nitrogens in imides, amides, and amines polyethylenimine linkage. The hypothesis of this work was results in formation of N-halamine structures (1, 10). In an that the numerous amides and amines present on the surface aqueous environment, N-halamines dissociate to release of PE-polyCOOH films would reversibly charge with chlo- free chlorine (hypochlorous acid and hypochlorite ion), rine, and that chlorine-charged PE-polyCOOH films would possess antimicrobial activity. The specific objectives were * Author for correspondence. Tel: (607) 255-7900; Fax: (607) 254-4868; to assess the chlorine rechargeability of PE-polyCOOH E-mail: [email protected]. films and to evaluate the antimicrobial activity of chlorine- J. Food Prot., Vol. 71, No. 10 RECHARGEABLE ANTIMICROBIAL POLYETHYLENE 2043 FIGURE 1. Structure and chlorination of PE-polyCOOH films. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/10/2042/2204454/0362-028x-71_10_2042.pdf by guest on 02 October 2021 charged PE-polyCOOH films against a range of microor- which was prepared by soaking glassware in a 100 ppm of NaOCl ganisms relevant in food safety and food spoilage. aqueous solution for 1 h, followed by rinsing in copious water. The amount of chlorine available from the PE-polyCOOH-Cl MATERIALS AND METHODS films was determined by a modification of the DPD assay. DPD reagent was prepared by dissolving one packet of DPD total chlo- Additive free, low-density polyethylene 640I (PE, 100 m) rine reagent powder in 1 ml of reagent-grade deionized water. was kindly donated by Dow Chemical Company (Midland, Films were submerged in 2 ml of reagent-grade deionized water Mich.). Polyacrylic acid (molecular weight [MW], 450,000) was to which 50 l of the DPD reagent was added. After 2 min of purchased from Scientific Polymer Products (Ontario, N.Y.). Chro- shaking at room temperature, absorbances were read at 512 nm, mium trioxide (anhydrous), sodium hypochlorite (5%), N,N-dieth- and the concentration of free chlorine was determined by com- yl-p-phenylenediamine (DPD) total chlorine reagent, and toluidine parison to a standard curve of sodium hypochlorite in reagent- blue O were purchased from Fisher Scientific (Fair Lawn, N.J.). grade deionized water. PE, PE-COOH, and PE-polyNH films sub- Acid orange 7, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide, 2 jected to chlorine charging (15 min of shaking in 3,000 ppm of and N-hydroxysuccinimide (NHS) were purchased from Sigma- NaOCl), as well as unchlorinated films at each step of modifica- Aldrich (St. Louis, Mo.). Reagent-grade deionized water was pur- tion, served as controls. chased from RICCA Chemical Company (Arlington, Tex.). Foam Force LP, an alkaline NaOCl-based cleaner used in dairy saniti- Antimicrobial activity of chlorinated polyethylene. To zation, was purchased from Ecolab, Inc. (St. Paul, Minn.). All grow pure cultures, isolates were streaked onto TSA plates and other reagents were obtained from commercial lab supply stores incubated at 30 or 37ЊC for 24 to 48 h until isolated colonies and were reagent grade or better. appeared. TSB was inoculated with a single colony and incubated Escherichia coli K-12 (ATCC 29425) was obtained from the overnight at 30 or 37ЊC with shaking. Overnight cultures were American Type Culture Collection (Manassas, Va.). All other iso- adjusted to 1 to 5 ϫ 108 CFU/ml to match McFarland Standard lates were obtained from the Cornell University Food Science 0.5 to 1 (Remel, Lenexa, Kans.). Of the 1 ϫ 108 to 5 ϫ 108 CFU/ Food Safety Laboratory culture collection, where they were stored ml culture, 250 l was added to 25 ml of diluent to reach a final Ϫ Њ at 80 C until use. Pseudomonas fluorescens (FSL D3-283) was concentration of 106 to 107 CFU/ml. Two sterile diluents (water isolated from the valve of a dairy plant (14); Listeria monocyto- or 10% TSB) were tested to assess the effectiveness of the films genes (FSL F2-944) was isolated from a swab of a meat cutting in different media. Serial dilutions were performed (in neutralizing table (29); Bacillus cereus (FSL H3-272) was isolated from a buffer then phosphate-buffered saline [PBS] as described below) commercial milk plant filler spout. Optimal growth temperatures on initial aqueous inoculum to determine starting concentration. Њ were determined to be 30 C for P. fluorescens and B. cereus and Twelve pieces of film (1 by 1 cm2) were placed in a sterile Њ 37 C for E. coli and L. monocytogenes. Tryptic soy broth (TSB), culture tube, to which 1 ml of inoculum was added. The nature Bacto agar, and Difco neutralizing buffer were obtained from Bec- of the film surfaces in conjunction with the rotation of the tubes ton Dickinson (Sparks, Md.). Tryptic soy agar (TSA) was pre- ensured a distribution of liquid between the films and prevented pared by dissolving 30 g TSB, 15 g Bacto agar, and 20 g sodium stacking. After 15 min to 8 h rotating incubation at 30 or 37ЊC, chloride in 1 liter of ultrapure deionized water. All reagents used 50 l of bacterial suspension was diluted in 450 l of neutralizing Њ for microbiological testing were autoclaved at 121 C for 25 min. buffer, forming an initial 1:10 dilution, followed by serial dilutions Polyethylene surface modification. PE films were cleaned in PBS. One hundred microliters of each dilution was spread onto Њ and modified as previously described (19). Briefly, they were ox- TSA plates, which were then incubated at 30 or 37 C.