Antimicrobial N-halamine modified chitosan films

Abstract

The inherent antimicrobial properties and biodegradability of chitosan make it an ideal candidate for antimicrobial materials. In this study, N-halamine precursor 3-glycidyl-5,5-dimethylhydantoin (GH) was synthesized and bonded onto chitosan by a ring opening reaction between chitosan and GH. The chitosan film modified with the N-halamine precursor could be rendered biocidal after exposure to a dilute household bleach solution. Syntheses routes, characterization data, and antimicrobial test results are presented. The chlorinated films with 2.60 × 10¹⁸ atoms/cm² of active chlorine were challenged with Staphylococcus aureus (ATCC 6538) and Escherichia coli O157:H7 (ATCC 43895) and showed good efficacy against these two bacterial species with log reductions of 7.4 and 7.5 within 10 and 5 min of contact time, respectively. These films may serve as potential materials for food packaging and biomedical applications.

Introduction

Recently, there has been a growing interest in the development of antimicrobial films in various applications including biomedical surface coatings and food packaging. The antimicrobial packaging films have shown great potential to control growth of food borne pathogens, including Listeria monocytogenes, Escherichia coli O157: H7 and Salmonella typhimurium (Cagri, Ustunol, & Ryser, 2004). Extensive research work has been performed to develop antimicrobial packaging to inactivate the pathogens and spoilage microorganisms on the surface of food (de Moura et al., 2012, Tankhiwale and Bajpai, 2012). One of the simplest and most feasible methods of producing antimicrobial films is to load antimicrobial agents, such as nanoparticles (Au, Ag, Cu, ZnO, TiO₂) (Fayaz et al., 2009, Li et al., 2009, Sikong et al., 2010), essential oils (Kuorwel, Cran, Sonneveld, Miltz, & Bigger, 2011), bacteria originated antibacterial proteins (bacteriocins) (Seydim & Sarikus, 2006), enzymes (Buonocore et al., 2003), fruit extracts (Du et al., 2008), and chitosan (Aider, 2010, Tripathi et al., 2010) onto films. However, the release of these additives from the films, especially water-soluble compounds and metal nanoparticles, may raise issues regarding adverse environmental problems and their safe use in food products. The addition of these compounds might also affect the film properties and food qualities.

Chitosan, the linear and partly acetylated (1-4)-2-amino-2-deoxy-β-d-glucan (Muzzarelli et al., 2012), is a biopolymer obtained from chitin. It is the second most abundant polysaccharide in nature after cellulose, and has received great attention because of its non-toxicity, biocompatibility, versatility, biodegradability, and antimicrobial properties (Xu, Xin, Li, Huang, & Zhou, 2010). Because chitosan has intrinsic antimicrobial properties and good film-forming ability, it has been used in antimicrobial films and coatings to inhibit the growth of not only Gram-positive and Gram-negative bacteria, but also yeasts and molds (Chen, Yeh, & Chiang, 1996). However, the antimicrobial activity of chitosan is only moderate which may limit its many applications. In order to improve its antibacterial activity, numerous modifications of chitosan have been reported such as N-carboxybutylation (Muzzarelli et al., 1990, Muzzarelli et al., 1994), O-carboxymethylation (Chen & Park, 2003), quaternization (Sajomsang, Ruktanonchai, Gonil, & Warin, 2010), sugar-modification (Sajomsang, Gonil, & Tantayanon, 2009), N-alkylation (Yang, Chou, & Li, 2005), and chitosan/nanoparticle complexes (Li, Deng, Deng, Liu, & Li, 2010).

For over three decades, N-halamine compounds have gained growing attention as antimicrobial agents due to their efficacies against a broad-spectrum of microorganisms, long-term stabilities, non-toxicity to humans, and regenerabilities upon exposure to aqueous free chlorine solutions (Worley et al., 2003, Kocer et al., 2011). N-halamines refer to compounds that contain amine, amide, and imide halamine bonds, which have the capability of rapid and total inactivation of various microorganisms without causing the microorganisms to develop resistance to them. Furthermore, N-halamine biocides are capable of killing microorganisms directly without the release of free chlorine into the system (Barnes et al., 2006). These N-halamines could be incorporated into or attached onto textiles, medical devices, and other solid surfaces related to health care (Worley, Chen, Wang, & Wu, 2005). The introduction of the N-halamines in textiles by covalent bonding between the precursor moieties and host polymers has been extensively explored in these laboratories and elsewhere over the past decade (Liang et al., 2007, Luo and Sun, 2008). N-halamine moieties have been applied onto cotton by using grafting techniques or other coating methods to produce antimicrobial cellulose (Sun & Sun, 2001), nylon (Lin, Cammarata, & Worley, 2001), and polyester (PET) (Ren et al., 2008).

However, little research has been done on the modifications of chitosan by covalently incorporating N-halamine moieties to produce biocidal chitosan. Recently, Cao and Sun (2008) reported that antimicrobial chitosan was prepared by exposure of chitosan to dilute sodium hypochlorite. The N-halamine chitosan provided total kill of 10⁸–10⁹ CFU/mL of E. coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) in 10 and 60 min, respectively. The physical state of chitosan is a crucial factor affecting antimicrobial activity, and extensive works have been done for chitosan in water solution. Little attention has been paid to the investigation of inactivation of microorganisms by chitosan in the solid state, such as for films and fibers. In this study, an N-halamine precursor 3-glycidyl-5,5-dimethylhydantoin was synthesized and attached to chitosan by a ring-opening reaction. The newly synthesized N-halamine chitosan derivative was characterized by FT-IR, NMR, DTG, and TGA. The chitosan derivative was dissolved in acetic acid solution and coated onto polyester transparency slides and then rendered antimicrobial upon chlorination. The chlorinated films were evaluated for biocidal efficacies against both Gram-negative bacteria E. coli O157:H7 and Gram-positive bacteria S. aureus.