Bacterial Cellulose-Chitosan paper with antimicrobial and antioxidant activities

The production of paper based bacterial cellulose-chitosan (BC-Ch) nanocomposites was accomplished following two different approaches. In the first, BC paper sheets were produced and then immersed in an aqueous solution of chitosan (BC-ChI); in the second, BC pulp was impregnated with chitosan prior to the production of the paper sheets (BC-ChM). BC-Ch nanocomposites were investigated in terms of physical characteristics, antimicrobial and antioxidant properties, and ability to inhibit the formation of biofilms on their surface. The two types of BC-Ch nanocomposites maintained the hydrophobic character, the air barrier properties, and the high crystallinity of the BC paper. However, BC-ChI showed a surface with a denser fiber network and with smaller pore than BC-ChM. Only 5% of the chitosan leached from the BC-Ch nanocomposites after 96 h of incubation in an aqueous medium, indicating that it was well retained by the BC paper matrix. BC-Ch nanocomposites displayed antimicrobial activity, inhibiting growth and having killing effect against the bacteria S.aureus and P.aeruginosa, and the yeast C.albicans. Moreover, BC-Ch papers showed activity against the formation of biofilm on their surface. The incorporation of chitosan increased the antioxidant activity of the BC paper. Paper based BC-Ch nanocomposites combined the physical properties of BC paper and the antimicrobial, antibiofilm and antioxidant activity of chitosan. INTRODUCTION Bacterial cellulose (BC) is a polysaccharide, synthesized and extruded outside the cell by some microorganisms, especially from the genera Komagataeibacter. The biopolymer is composed of units of glucose lineally linked by → (1 4)–glycosidic bonds. Although identical to cellulose of plant origin in terms of molecular formula, BC presents unique properties that make it superior for many applications. Unlike vegetable cellulose, which is always bound to hemicellulose and lignin requiring subsequent refining treatments, BC is synthesized chemically pure. BC displays a high degree of polymerization and crystallinity, great mechanical strength, and a high waterholding capacity. BC is also biodegradable and biocompatible. Microorganisms produce cellulose as a three-dimensional open porous network of nanofibers, providing a large surface area. Moreover, cellulose contains available hydroxyl groups in its surface that facilitate the possibility of molecular adsorption by the formation of hydrogen bonds and electrostatic interactions. These structural and mechanical features are important for the practical application of BC as the supporting matrix for the preparation of new composite materials. Because of these properties, in recent years there has been an increased interest in commercial applications of bacterial cellulose. Important examples include supports for proteins, cell cultures and microorganisms; products for temporary skin and tissue replacement; material for headphone and loudspeaker membranes, food packing, and edible films. 1,5,6 Chitosan is an amino-polysaccharide obtained by alkaline deacetylation of chitin, which is extracted from marine natural sources such as crustacean shells. Chemically, chitosan is a copolymer composed of (1,4)-glucosamine and N-acetyl-D-glucosamine units. It is biodegradable, nontoxic and possesses reactive amino groups. Moreover, chitosan displays intrinsic antimicrobial activity, which depends on the molecular weight and the degree of


INTRODUCTION
Bacterial cellulose (BC) is a polysaccharide, synthesized and extruded outside the cell by some microorganisms, especially from the genera Komagataeibacter. The biopolymer is composed of units of glucose lineally linked by → (1 4)-glycosidic bonds. Although identical to cellulose of plant origin in terms of molecular formula, BC presents unique properties that make it superior for many applications. Unlike vegetable cellulose, which is always bound to hemicellulose and lignin requiring subsequent refining treatments, BC is synthesized chemically pure. BC displays a high degree of polymerization and crystallinity, great mechanical strength, and a high waterholding capacity. 1 BC is also biodegradable and biocompatible. 2 Microorganisms produce cellulose as a three-dimensional open porous network of nanofibers, providing a large surface area. Moreover, cellulose contains available hydroxyl groups in its surface that facilitate the possibility of molecular adsorption by the formation of hydrogen bonds and electrostatic interactions. These structural and mechanical features are important for the practical application of BC as the supporting matrix for the preparation of new composite materials. 3,4 Because of these properties, in recent years there has been an increased interest in commercial applications of bacterial cellulose. Important examples include supports for proteins, cell cultures and microorganisms; products for temporary skin and tissue replacement; material for headphone and loudspeaker membranes, food packing, and edible films. 1,5,6 Chitosan is an amino-polysaccharide obtained by alkaline deacetylation of chitin, which is extracted from marine natural sources such as crustacean shells. Chemically, chitosan is a copolymer composed of (1,4)-glucosamine and N-acetyl-D-glucosamine units. It is biodegradable, nontoxic and possesses reactive amino groups. Moreover, chitosan displays intrinsic antimicrobial activity, which depends on the molecular weight and the degree of deacetylation of the polymer as well as on the type of microorganism. 7 Also, chitosan is considered a secondary antioxidant because it can chelate the metal ions involved in the catalysis of an oxidant reaction due to the presence of active hydroxyl and amino groups in the polymer chains. 8 Owning to its properties, chitosan is considered a versatile material that participates in multiple applications, which include the formation of biodegradable films, blends, coatings, composites and nanocomposites; as a flocculating agent in wastewater treatment; in the generation of chitosan-based membranes for water purification; as an additive for food packages or food preservation; and in wound dressing. 4,[9][10][11][12][13][14] The blending of polymers to produce composites materials with new properties has received extensive attention as a strategy for supporting new applications. 4,[15][16][17] The combination of chitosan with BC has been successfully described for biomedical and packaging applications. 4,9,10 In those works, the matrix of BC was in form of native never-dried membrane or in form of film. Recently, the production of paper from BC pulp has been reported. 18 BC paper sheets combine the attributes of BC nanofibers with the stiffness and physical properties of paper, showing remarkable mechanical characteristics and barrier properties to water and oil. 18 Some studies have shown how chitosan-coated vegetable paper has impaired paper properties, such as its resistance to water or steam transfer. 11,19 However, to our knowledge, no work has yet been reported regarding the combination of BC paper and chitosan.
The aim of this work was to compare two different procedures to blend BC with chitosan in order to obtain a novel nanocomposite based on BC paper and investigate its physical characteristics and its antimicrobial, antioxidant, and antibiofilm properties. The study is framed in the research area of bioactive papers with potential applicability in the design of biomedical devices and in the field of packaging of food and high value goods.

Microbial strains
The cellulose producing strain was Komagataeibacter xylinus CECT 7351. Antimicrobial activity was tested against Staphylococcus aureus CECT 234, Pseudomonas aeruginosa PAO1 CR32 and Candida albicans CECT 1001. Strains were obtained from the Spanish Type Culture Collection (CECT).

Production of Bacterial Cellulose
To produce bacterial cellulose, Komagataeibacter xylinus was grown on the Hestrin and Schramm (HS) medium, containing 20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract, 1.15 g/L citric acid, 6.8 g/L Na2HPO4, pH 6. 20 Suspensions of K. xylinus were used to inoculate 10 cm-Petri dishes containing 40 mL of HS medium that were statically incubated at 25-28 °C for 7 days. After incubation, bacterial cellulose pellicles generated in the air/liquid interface of the culture media were harvested, rinsed with water, and incubated in NaOH (1%) at 70 ºC overnight to remove the bacteria. Finally, the BC pellicles were thoroughly washed in deionized water until the pH reached neutrality. To obtain the BC pulp, pellicles were mechanically cut into small pieces (1 cm 2 approximately) and disrupted with a homogenizer (CAT Unidrive X1000 Homogenizer, Germany) at 20000 rpm for 10 m.

Preparation of Bacterial Cellulose/Chitosan nanocomposites
To obtain the composites of BC paper containing chitosan, two approaches were followed: the formation of BC paper followed by the impregnation by immersion of the paper sheets with chitosan; and the impregnation in mass of the BC pulp with chitosan followed by the formation of paper sheets.
Impregnations were carried out with the water soluble derivative Methyl Glycol Chitosan (FUJIFILM Wako Pure Chemical Corporation, MW: (375,2)n) with a ratio of 0.3 mg of chitosan / mg of dry bacteria cellulose.
The impregnation of the previously produced BC paper was done by immersing pieces of 1 cm 2 of paper in a 3 mg/mL of an aqueous solution at pH 6 and incubating overnight at room temperature. After incubation, the paper sheets were washed with deionized water to remove poorly attached chitosan. To perform the impregnation in mass, BC pulp and chitosan at 3mg/mL (final concentration) were mechanically mixed in a blender (proBlend6 3D, Philips-Spain) until homogenization (500 rpm, 5 m). The mixture was incubated overnight at room temperature and, after incubation, washed with deionized water. The pulp of BC impregnated with chitosan was then used to produce paper sheets.
Paper sheets were produced following the ISO-5269:2004 standard method using a Rapid- Then, the weight of the swollen pieces was measured.
Water absorption capacity was calculated as follows:

Wf Wi Wi
where Wi is the initial weight of dried sample and Wf is the weight of sample in swollen state.

Scanning Electron Microscopy (SEM)
Surface morphology of BC-Chitosan nanocomposites was analyzed by SEM (JEOL JSM 7100 F, Tokyo, Japan) using a LED filter. Samples were graphite coated using a Vacuum Evaporator EMITECH K950X, France. where I002 is the maximum intensity of the lattice diffraction and IAM is the minimum in intensity at 2 between 18º and 19º, which corresponds to the amorphous part of cellulose.

Fourier transform infrared spectroscopy (FTIR)
The infrared spectra of samples were obtained using FTIR spectroscopy (Fourier Transform IR spectrometer Perkin Elmer Frontier, Waltham, Massachusetts, U.S.). Spectra were obtained at wave numbers ranging 4000 to 400 cm -1 recorded at 4 cm -1 resolution.

Antimicrobial activity
The antimicrobial activity of BC-Ch composites was tested against the Gram-positive bacteria Staphylococcus aureus, the Gram-negative bacteria Pseudomonas aeruginosa and the yeast Candida albicans. To obtain the inoculum for the antimicrobial tests, the strains were grown overnight in Luria Bertani (LB) broth at 37 ºC in shaking conditions. Then, these cultures were centrifuged for 5 minutes at 18407 RCF and the pellet resuspended in 0,3 mM KH2PO4 to remove the culture medium. BC-Chitosan nanocomposites were cut in squares of 1 cm 2 and sterilized prior to the assay. Two antimicrobial tests were performed, the Drop over paper test and the Dynamic contact condition test.

Drop over paper test
Three l of the corresponding microbial suspension (about 10 5 microorganisms per mL) were inoculated over the 1 cm 2 BC-Chitosan composite placed on the surface of Tryptic Soy Agar (TSA) medium plates. The growth over a sample of BC paper was used as positive control.
After overnight incubation at 37 ºC, the samples of composites and BC-paper were submerged in 1mL of 0.3 mM KH2PO4 and the microorganisms were detached by intense shaking. The metabolic activity of the resuspension was measured by the resazurin assay. In a medium with viable cells resazurin is reduced to resorufin and this reduction can be quantified by a fluorometer. 22 For the assay, 30 L of resazurin (7-Hydroxy-3H-phenoxazin-3-one-10-oxide) were added to 100 L of each microbial resuspension in a 96-well plate. where t0 is the time 0 h and tx is the time at which the percentage of reduction is calculated.

Antibiofilm activity
Antibiofilm properties of BC-Ch nanocomposites were assayed with Pseudomonas aeruginosa, a well-known biofilm producer. 1mL aliquots of a 1:100 dilution of an overnight culture of P. aeruginosa (about 10 6 bacteria) were pipetted into a 24-well plate where samples of BC-Ch and BC paper were previously placed. After overnight incubation at 37°C, the medium was removed, and the samples were rinsed three times with Phospate-buffered saline (PBS).
Resazurin assay and SEM analysis were carried out on BC-Ch nanocomposites and BC paper.

Antioxidant activity
The antioxidant activity was assessed by a procedure that allows to determine the antioxidant Where Ai is the absorbance value of the blank and Af the absorbance value of the sample.

Determination of the concentration of chitosan
The method to measure chitosan was adapted from Badawy. 26  indicating that the ratio BC:Ch on the composites was about 10:1, and suggesting that chitosan was well incorporated into the nanofibers of cellulose network structure. In the mass impregnation process, the BC fibers were suspended in water, which would facilitate the access of the chitosan and favor its interaction with the cellulose molecules. In contrast, in the impregnation by immersion procedure, the molecules of cellulose could be less accessible to chitosan since BC fibers were compacted and dried during the process of papermaking.
However, not substantial differences were found in the amount of chitosan loaded throughout the two approaches, suggesting that chitosan in BC-ChI composites not only coated the paper surface but penetrated the porous matrix of fibers of BC. increase in the smoothness of the paper surface, more obvious in the case of BC-ChI probably due to the differences on the methodology to produce the nanocomposites. In the case of BC-ChI, the chitosan was coated on the paper surface, being placed in the surface pores of the composite. Paper-like supports made of bacterial cellulose are characterized by excellent barrier properties to air, water and oil 18,37 which is important for applications that need impermeability, as for packaging material. 11 Results showed that the blend of chitosan and BC increased the impermeability to the air, indicated by the lower value in air permeance of the BC-Ch nanocomposites. Hydrophylicity is a characteristic inherent to most matrices of polysaccharides. Bordenave et al., 11 reported that the association of paper produced from vegetal cellulose with chitosan was water sensitive and they improved the hydrophobic character of chitosan-coated vegetal papers after chemical modification of chitosan and bounding with fatty acids. However, BC paper features water and vapor barriers properties without the need of the addition of hydrophobic components or the chemical modification of the molecule. 18,37 The results obtained in this work indicated that the presence of chitosan did not increase the wettability of the resulting BC-Ch nanocomposites estimated by WDT ( Table 1), suggesting that the BC paper maintained its hydrophobic character.
Water absorption capacity (WAC) of BC-Ch nanocomposites was evaluated and compared with that of BC paper. The WAC of BC paper was about 6.5 times of its dry weight, while the   * Results expressed as the percentage of reduction of the microbial activity with respect the activity over BC paper The yeast C. albicans showed less sensitivity to chitosan than the bacteria strains. Moreover, chitosan was more active against the Gram-positive (S. aureus) that against the Gram-negative (P. aeruginosa) bacteria. The differences in the effectiveness of chitosan can be explained by its varied mechanisms of action as well as by the differences in the structure of the cell envelopes of the three microorganisms. 7 However, the exact mechanism of chitosan antimicrobial action it is not totally understood. Factors as MW and degree of acetylation of chitosan, and pH of the medium may influence its antimicrobial action. Results suggested that the external lipidic membrane in Gram-negative could confer some protection hindering the access of chitosan to the cell. Nevertheless, in the literature there is not a general agreement regarding the degree of susceptibility of Gram-positive, Gram-negative and fungi to the chitosan. 40 While we could expect more chitosan to accumulate on the surface of the compounds obtained by the immersion procedure than in those obtained by mass impregnation, the results indicated that BC-ChM were more effective in preventing microbial growth on their surface. The SEM images of the BC-Ch nanocomposites ( Figure 4) revealed that BC-ChM presented a less compact surface that would allow better contact of the bacteria with the nanofibers facilitating the action of chitosan.
One aspect to consider was that results stated above indicated that BC-Ch nanocomposites had less water absorption capacity than BC paper ( Figure 5). Moreover, SEM analysis showed that chitosan covered the nanofibers and could be filling the matrix pores. These circumstances could be limiting the diffusion of water and nutrients dissolved in water during the drop over paper test analysis and artificially increase results of antimicrobial activity. To preclude this possibility, the biocidal ability of the BC-Chitosan composites was assayed under dynamic liquid condition. Suspensions of the microorganisms on 0,3 mM KH2PO4 solution were incubated in contact with the BC-Ch composites and BC paper, at room temperature and lightly agitation.
Viable cell counts were determined at different times, and the percentage of cell viability reduction was calculated ( Table 3). Suspensions of the microorganisms in contact with samples of BC paper did not experiment a decrease of viability over 24 h incubation time (results not shown). However, the microorganisms in contact with BC-Ch nanocomposites showed a remarkable diminution of viability after one hour of incubation and, after 24 h hours, the reduction of viability was 100% (Table 3). These results indicated that BC-Ch composites, not only inhibited the microbial growth, but exhibited, also, strong biocidal activity against the tested strains. Moreover, the antimicrobial effectiveness of the two types of nanocomposites was similar, suggesting that this property did not depend on the procedure followed for the production of the nanocomposites. Table 3. Viable cell counts (CFU/mL) and cell viability reduction (%) of microorganisms in dynamic contact with BC-ChI and BC-ChM composites. Paper of BC is a matrix with great mechanical resistance and does not disintegrate in water, which allows its reusability. An interesting characteristic of the composites produced was to know if after being in an aqueous environment for a period and then dried retained their antimicrobial activity for further applications. To do this, samples of BC-Ch and BC paper were incubated in water at room temperature for 24 hours. Then, the papers were dried and the drop over paper test was performed with a suspension of S.aureus. Both nanocomposites still showed antimicrobial activity after being in contact with water for 24 h and then dried (Table 4). BC-ChM and BC-ChI nanocomposites maintained 63% and 51% of its antimicrobial capacity compared to its initial antimicrobial activity (t0, Table 4), respectively. BC paper did not show reduction of activity (results not shown). Differences in the results of the two types of composites were consistent with the fact that BC-ChM showed less migration of chitosan from the BC matrix than BC-ChI.

CONCLUSIONS
In this work, the combination of BC and chitosan rendered composites of paper with improved physical, chemical and biological characteristics. The two impregnation methods tested allowed a stable binding of chitosan to the BC matrix without the requirement of crosslinking molecules, and produced BC-Ch nanocomposites with similar characteristics. BC-Ch nanocomposites had the consistency and stiffness of paper and showed great durability, retaining their properties for a long time without the need of special storage in terms of temperature or humidity. They had good resistance to the passage of air and water. They exhibited antimicrobial activity against bacteria and yeasts, and prevented the formation of biofilm in their surfaces. Moreover, BC-Ch paper showed scavenging capacity of oxidizing radicals. The BC-Ch composites developed in this work generated paper-like supports, which can expand the previously described biomedical applications for chitosan embedded in never-dried BC membrane and BC film. Their physical and biological properties, and the organic nature of its components, make them suitable to be part of the design of environmentally friendly materials in the area of bioactive-paper.