Preparation and characterization of aminoethyl hydroxypropyl methyl cellulose modified with nisin

Abstract

Nisin grafted aminoethyl hydroxypropyl methyl cellulose (AEHPMC) was prepared by an enzyme-catalyzed reaction in the presence of microbial transglutaminase (MTGase). AEHPMC was synthesized with 2-chloroethylamine hydrochloride (CEH) which was as an intermediate reactant. The parameters, which influenced the NH₂% and the degree of substitution (DS), including reaction time, reaction temperature and the mass ratio of the reactants were investigated. Antioxidant activities of AEHPMC-nisin were evaluated by the scavenging activity of hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. With the DS and concentration increasing of AEHPMC-nisin, the radical scavenging activity increased. The Methylthiazol tetrazolium (MTT) assay indicated that AEHPMC-nisin had low-toxicity to L929 cells. Therefore, the derivative of HPMC may show a promising potential application in biomedical, food and pharmaceutical fields.

Introduction

In recent years, polysaccharides have been widely available biological polymers which is applied in almost every aspect of human life. It has become an important material in medicine, environmental protection, food and pharmaceutical industry [1]. Cellulose is a common natural polysaccharide which is considered as the most abundant natural polymer with low cost and non-toxic [2]. Hydroxypropyl methyl cellulose (HPMC) is one of the most important derivatives of cellulose, which is frequently used in printing technology, food and pharmaceutical industry because of its non-toxic and good mechanical properties [3]. For instance, in pharmaceutical industry, HPMC is usually used for controlled drug-release matrices [3], [4]. In food industry, HPMC is kind to improve the quality of baked products, promote food manufacture development and used as low-fat edible coatings, etc [5], [6], [7].

Nisin is a kind of antibacterial agent produced by Lactococcus lactis which was first discovered in the late 1920s and early 1930s when it was considered as a toxic substance [8], [9], [10], [11]. Along with the more understanding of the properties of nisin, it has been approved as generally recognized as safe for food use by both the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) since 1969 [11]. Structurally, nisin is consists of 34 amino acid polypeptide with a molecular weight of 3500 Da. Its molecular structure includes unusual amino acids and thioether ring which are responsible for the important functional properties [12]. Nisin is also used as a safe preservative and is permitted for use in more than 40 countries [11]. The most striking point of nisin is that it is an important antimicrobial peptide that has an antimicrobial activity against a wide range of Gram-positive bacteria. Nevertheless, it shows little or no antibacterial activity against Gram-negative bacteria, yeasts, or molds [9], [13].

Proteins and polysaccharides are frequently present together can lead to new biomaterials with significantly function properties such as improving the stability, mechanical properties etc. Crosslinking has been the most common approach to get these new biomaterials. Choice of crosslinkers and crosslinking techniques are depend on the type of biopolymer to be crosslinked [14], [15]. Among the numerous chemical crosslinkers, glutaraldehyde is probably the most widely used crosslinking agent because it can react with functional groups in both carbohydrates and proteins [14], [16]. Although glutaraldehyde provides excellent mechanical properties, some evidences have indicated that glutaraldehyde-crosslinked materials have some cytotoxicity [17]. As is often the case, chemical crosslinkers induce toxic side effects or secondary reaction with unwanted products. Hence, green crosslinkers and new crosslinking methods need to be considered when it comes to obtain biomaterials for medical applications. The enzyme-catalyzed reactions are environmentally friendly, high efficient and highly selective which attract more and more researchers [18], [19].

Produced via classical fermentation by microorganism Streptoverticillium moboarense, MTGase is an extracellular enzyme of the class of transferases [20]. It acts in a wide range of pH and temperature and the activation without particular cofactors [21], [22]. The fact that MTGase has been recognised as a safe substance by the Food and Drugs Administration (FDA), makes it very attractive for the food industry [21]. As a biocatalyst, MTGase can catalyze an acyl-transfer reaction by forming covalent cross-links between γ-carboxamide and primary amine [23].

In our previous study, we have prepared collage-chitosan by enzymatic method with good antioxidant activity which promoted L929 cells growth [24]. However, the derivative of HPMC on antioxidant and the enzymatic method combined polysaccharides with proteins is little reported. Therefore, the purpose of this work was to prepare a derivative of HPMC modified with nisin.

In this study, AEHPMC was initially synthesized with CEH. The main purpose of this work was to prepare AEHPMC-nisin by MTGase. The antioxidant activity of AEHPMC-nisin was evaluated by the scavenging activity of hydroxyl radical and DPPH radical. Furthermore, the influence of the derivatives on cells viability was also investigated. This study results may contribute to finding the application of AEHPMC- nisin in food and pharmaceutical industry.