Functional Natural-Based Polymers

Book Preface

Antimicrobial peptides are synthesized by several microorganisms. Nisin is a lantibi-otic peptide, with 34 amino acids, and is secreted by Lactococcus [1–4]. The antimicrobial property of nisin is attributed to pore formation in the cell membrane of microorganisms, with specific binding to the lipidic precursor of the cell wall, attached to the membrane [4,5].
Nisin is considered safe by the World Health Organization (WHO) and the Food and Drug Administration (FDA-United States), being used initially as a food additive [6,7]. Further, its antimicrobial action encourages the clinical use of nisin, whether in topical or systemic therapies, due to its broad-spectrum activity and lower probability of developing microbial resistance [6–8]. The application of nisin extends to several medical areas, from mastitis to oral and gastrointestinal diseases [5,9,10].
Bacterial cellulose (BC) is a polysaccharide that is extracellularly secreted by several mi-croorganisms, such as Agrobacterium, Rhizobium, Escherichia, Sarcina, and Acetobacter [11–13]. Namely, Komagataeibacter xylinus is a non-pathogenic Gram-negative bacterium that can produce significant amounts of cellulose [14]. BC is a linear glucose polymer, formed by a matrix of nanofibers, giving it porous characteristics in a three-dimensional network structure. Although its structure resembles vegetable cellulose, bacterial cellulose presents a high degree of purity, crystallinity, tensile strength, and high-water absorption [15].

Due to its biocompatibility and non-toxicity, the BC application has been directed to medical devices and tissue engineering [16]. Additionally, owing to its structural model, BC has been proposed as an ideal dermal substitute that is capable of inducing the direction of cells for repair in tissue reconstruction [17,18]. BC has been used as wound dressing because it forms a physical barrier against infections. It also allows gas exchange, absorbs exudates, keeps the wound moist to favor tissue reconstitution and it can be easily removed [19].
The incorporation of biomolecules into BC has been studied both by increasing its antibacterial or enzymatic properties and by providing a control release system [20,21]. For instance, nisin immobilization in solid matrices such as bacterial cellulose membranes could control its release [22]. Due to these properties, our research group previously evaluated the antimicrobial and antioxidant activity of nisin loaded into BC [23]. Since the work yielded satisfactory results, our group has provided a supplementary study. For this reason, this study has evaluated BC membranes as carriers for nisin regarding the morphological characteristics, cytotoxicity, and stability of the developed system.
2. Materials and Methods
2.1. Materials
The standard nisin and the bicinchoninic acid kit were purchased from Sigma-Aldrich (São Paulo, Brazil). All other reagents were of analytical grade.
2.2. BC Production and Purification
BC was produced by Komagataeibacter xylinus ATCC 53582, using 24-well plates with Hestrin and Schramm medium. Each well was filled with 1 mL and incubated at 30 ◦C in static conditions for seven days. After that, membranes were immersed in a 2% sulfate dodecyl sodium (SDS) solution under stirring overnight and washed in running water. The bleaching process was carried out with 4% NaOH for one hour until reaching 60 ◦C. The BC membranes were washed in order to remove NaOH and sterilized at 121 ◦C for 15 min [23,24].
2.3. Total Proteins Quantification
Protein concentration was determined by a bicinchoninic acid assay [25]. Bovine serum albumin (BSA) with different concentrations from 0.1 to 1.0 mg/mL was used as the standard protein. Absorbance reading was performed on 96-well microplates with a wavelength of 562 nm, by spectroscopy (Infinite M200 PRO, RCHISTO, Barueri, Brazil). Analyses were performed in triplicate, and the mean of these absorbance values was used to determine protein concentrations.
2.4. Nisin Standard Curve by Agar Diffusion Assay
The standard nisin (Sigma-Aldrich, St. Louis, MO, USA—containing 2.5% nisin, with 1,000,000 IU/g in its composition) solution was prepared by dissolving 1 g of nisin in 10 mL of phosphate buffer solution (PBS-pH 7.0), as in Table 1. The solution was centrifuged at 13,201 g for 10 min at 10 ◦C and the supernatant was collected and filtered in a 0.22 μm membrane (Millipore, Burlington, MA, USA). The nisin standard curve was evaluated by an agar diffusion assay. The nisin bioindicator Lactobacillus sakei ATCC 15521 was used for the agar diffusion assay [6,23].