Development of chitosan/agar-silver nanoparticles-coated paper for antibacterial application

Abstract The radical proliferation of pathogenic bacteria and their infections causes significant issues for human health and the environment. Today, biopolymers are used to produce different nanoparticles. In the present investigation, the fabricated chitosan/agar-silver nanoparticles (Cht/Ar-AgNPs)-coated papers were tested for antibacterial applications. Agar was used as a reducing agent for the synthesis of AgNPs. Synthesized Ar-AgNPs were examined through optical, phase crystallinity and topological analysis. Cht and Ar-AgNPs solution was mixed with various ratios of 9:1, 8:2, 7:3, 6:4, and 5:5 by weight. In addition to that, the conformity of Cht/Ar-AgNPs-coated papers was characterized by structural, spectral, and morphological analysis. However, Cht/Ar-AgNPs-coated papers were subjected to antibacterial properties. The ratio of (6:4) Cht/Ar-AgNPs-coated paper showed excellent antibacterial agent, and it can be used as extending the food product shelf life.


Introduction
Biopolymers are considered an ideal candidate for nanotechnology's recent advent due to the biocompatibility, reducing/capping ability, and different physicochemical properties, especially nontoxicity to an environment. Depending on the applications, biopolymers are varied. Moreover, they are highly edible as well as biodegradable films in food packaging. The high recyclable natural biomoleculesbased packaging materials like fatty acids, amino acids, and polysaccharides are convenient environmental agents than petrochemical-based polymers. Biopolymer films and coatings are used for many applications: it serves as the best food packaging material, minimizes food deterioration, extends food life by serving as solute and gas barriers, and acts as various additives (antioxidants, antimicrobials, coloring agents, and nutrients) [1][2][3][4][5].
The biopolymers and papers-associated materials are providing an eco-friendly approach. Paper has unique characters as well as used for multiple applications: less weight, low cost, superior mechanical properties, low environmental impact, paper sensors, filters, indicators, writing/printing prepossess, packaging, household products, resistance from insufficient grease and oil, low barrier properties [6][7][8][9][10], and due to its lipophilic nature, it is prone to react with fatty molecules and leads to damage in printed papers, and enables usage as synthetic polymers, which leads to recycling issues and causes environmental problems [6][7][8][9][10][11]. Moreover, many researchers pushed towards the advanced and new approaches for developing antimicrobial papers by administering eco-friendly synthesis processes [12][13][14].
Many researchers are likely attracted by agar (Ar) because it is highly renewable, biodegradable, and of low cost. Ar is a mixture of agarose and agaropection polysaccharides: agarose 70% of agar (linear polymer, along with agarobiose repeating unit, a disaccharide making up of D-galactose and 3,6-anhydrous-L-galactopyranose) and agaropection 30% of agar (smaller molecule of a heterogeneous mixture, made up of alternating units of D-galactose and L-galactose highly modified with sulfate and pyruvate, an acidic side-group) [15].
Chitosan (Cht) is a biodegradable polycationic polymer obtained from chitin, a natural polymer that exhibits antibacterial features. It consists of (1,4)-linked 2-amino 2-deoxy-b-D-glucopyranose [16]. Moreover, it has antibacterial and nontoxic characteristics and therefore, been used as individual health properties and metal nanoparticle chitosan material [17]. Cht is an excellent chelating agent consisting of functional NH 2 and OH groups [18]. Silver (Ag) ion revealed a strong bacterial inhibiting effect, even a low quantity of Ag nanoparticles (NPs) providing maximum antibactericidal effect [19]. The composite mixture of Cht and AgNPs provided a significant bacterial inhibition effect [20].
AgNPs were used as an effective antimicrobial agent against bacteria, viruses, and fungi [21,22]. Synthesis of AgNPs has been carried out by various methods such as microwave irradiation/sonochemical process [23][24][25], and along with bio-based reduction and capping agent has been proving to develop chemical-free AgNPs [26][27][28][29][30][31]. Previously, researchers have made various attempts to carry out the agar-medicated synthesis of AgNPs and reported that the synthesized Ar/AgNPs composites significantly improve the mechanical and antimicrobial properties. Similarly, the amino acids of tyrosine and tryptophan-mediated synthesized AgNPs had been incorporated into the agar polymer matrix. These Ar/AgNPs nanocomposite films revealed intense antimicrobial activity against Escherichia coli than Listeria monocytogenes [32]. The red seaweed of Gracilaria dura-derived agar extract-mediated synthesized AgNPs were formulated with agar polymer help by the microwave heating process. Synthesized Ar/AgNPs composite films showed a more significant bactericidal effect (99.9%) on Bacillus pumilus -HQ318731 [33]. Rhim et al. [34] reported that the chemically synthesized AgNPs were introduced to the agar polymer by various weight contents. Linearly enhancing the water vapor barrier properties and surface hydrophobicity increases the content of silver without affecting the mechanical strength. However, 1 wt% silver containing the composite film exhibited intense antimicrobial activity against L. monocytogenes and E. coli O157:H7. The same research group has investigated the preparation of AgNPs by a laser ablation method and AgNPs/agar composite films were prepared by the solvent casting method with varying the content of AgNPs. The inclusion of metallic AgNPs somewhat increased water vapor permeability and water contact angle. At the same time, it exhibited potent antimicrobial activity with a higher content at 40 and 80 mg of AgNPs [35].
The present investigation intended at the fabrication of active antibacterial paper using AgNPs asserted biobased coating material. The AgNPs were synthesized by agar (Ar). The chitosan and Ar-AgNPs solutions were prepared at different ratios, which coated over cellulose paper, and it was assessed by antibacterial activity against human pathogens.

Ar-AgNPs synthesis
Initially, Ar-AgNPs solution synthesis added 2.5 g of agar and 100 mL of 10 mM AgNO 3 solution, and it was mixed well using a magnetic stirrer at 90°C for 1 h under dark condition [33,34]. Then, the mixture was cooled down at 37°C for back-up.

Preparation of Cht/Ar-AgNPs coating solution
At first, 2.0 v/v% acetic acid was prepared from 10% acetic acid used to prepare the chitosan solution. The 2.0 v/v% acetic acid was encountered through stirring for 8 h at 90°C to obtain 1.5 wt% of Chitosan solution, which was cooled at 25 ± 1°C and 2.5 pH. Finally, through 10 min vigorous mechanical stirring, the chitosan solution and Ar-AgNPs solutions were mixed at various ratios of 9:1, 8:2, 7:3, 6:4, and 5:5 by weight, respectively.

Fabrication of Cht/Ar-AgNPs-coated papers
The Cht/Ar-AgNPs cellulose-coated paper was prepared by the bar-coating method. Initially, the uncoated paper was firmed by a stable glass plate, then Cht/Ar-AgNPs coating solution was smeared using a #20 Mayer rod. Cht/Ar-AgNPs cellulose-coated paper allowed the oven for the drying process (90°C for 10 min). Air-tight polyethylene covers are used to ensure coated paper protection against light. Table 1 summarizes the detailed confirmation of uncoated and coated papers at various ratios of chitosan and Ar-AgNPs used in the study.

Characterization of Ar-AgNPs analysis
Various spectral and microscopic methods analyzed the synthesized Ar-AgNPs solution: UV-Vis spectrometer (Shimadzu, UV-1800 spectrophotometer operating absorbance measurements with a resolution of 1 nm, with a 200-800 nm wavelength range). Shape and size of the synthesized Ar-AgNPs solution were characterized using transmission electron microscopy (Tecnai F20 model) at accelerating voltage of 200 kV. The crystalline phase of Ar-AgNPs solution, plain paper, and coated papers was analyzed by XRD spectrometer (PANalyticalX'Pert Pro, wavelength: 1.5418 Å, diffraction patterns were collected at 25°C, over an angular range of 10-80°w ith a step size of 0.05°and a step time of 10.16 s per increment). The functional groups of plain paper and coated papers were analyzed by FT-IR spectrometer Thermo Nicolet 380, from 500 to 4,000 cm −1 , and the surface topology analyzed through FE-SEM microscopy (Hitachi S-4500).

Antibacterial activity
The antibacterial activity examined Gram-positive bacteria of B. subtilis and three Gram-negative bacteria of E. coli, S. dysenteriae, and P. vulgaris strains. The aforementioned bacterial strains were grown in nutrient broth at overnight culture. 20 mL of autoclaved nutrient agar culture media was poured into agar plates. The respective bacteria were evenly spread over the respective nutrient agar media with a cotton swab. The 1 × 1 cm of plain and coated papers were placed on the medium and plain paper considered as control. All the plates were maintained at 38°C for 24 h. After completing the incubation period, the inhibition zone was calculated by each coated paper and photographed. Further, this experiment was repeated three times [36].

Statistical analysis
Data from antibacterial experiments were expressed as mean ± SE and we employed ANOVA followed by Tukey's HSD test (P ≤ 0.05) using the SPSS software package 16.0 version.

Optical, structural, and morphological analysis of Ar-AgNPs
During AgNPs synthesis, in the reduction process, the colorless agar with AgNO 3 mixture changed to brown color, and it is the necessary conformity of AgNPs synthesis. The Table 1: Different ratios of chitosan and agar-AgNPs-coated cellulose paper and their respective coating weight (g)

Sample code
Chitosan ( UV-visible spectrum showed an absorption hump at 403 nm by the synthesized agar-mediated AgNPs (Figure 1). This absorbance peak indicates a reduction of Ag + to Ag 0 . It confirms the formation of AgNPs owing to the surface plasmon resonance and similar observation reported by previous biopolymer reports [37]. The XRD patterns of Ar-AgNPs appeared 2θ value at 38.13°, 44.27°, 64.47°, and 77.30°, which indexed to the planes (111), (200), (220), and (311), respectively (Figure 2), and it revealed the crystalline phase of face-centered cubic structure. Further, Figure 3a and b prove the TEM images of Ar-AgNPs reveal the spherical shape, size of particles ranging from 5 to ∼20 nm, and the mean value of 10 nm; Figure 3c depicted the selected area diffraction patterns well-correlated with the XRD indexed planes.

XRD analysis of coated papers
The plain chitosan and chitosan combined agar-mediated silver nanoparticles-coated paper were considered by XRD analysis (Figure 4a). Two sharp characteristic intense peaks were observed at 2θ = 16.36°and 22.56°in the plain cellulose paper. Further, chitosan-coated cellulose paper revealed peaks at 2θ = 15.90°, 22.71°, 29.86°, and 39.80°. However, the first peak position slightly shifted towards front side. The second peak showed high crystalline nature as compared to the pure cellulose paper peak. Besides, a newly raised peak at 2θ = 29.86°confirmed chitosan on the cellulose paper surfaces. The even ratio of Cht: Ar-AgNPs-coated cellulose paper peaks was located at 2θ = 15.62°, 22.71°, 29.51°, 39.57°, and 43.21°, respectively. Whereas, the peak position at 15.62°and 22.71°crystalline decreased due to the composition of Cht: Ar-AgNPs coated on the cellulose paper surface as compared to the chitosancoated paper. Interestingly, the newly appeared two peaks located at 39.57°and 43.21°correspond to the AgNPs' presence on the cellulose paper surface (Figure 4b).

Functional group analysis of coated papers
The FT-IR spectral analysis of plain chitosan and chitosan combined agar-mediated silver nanoparticlescoated papers exhibited several characteristic peaks at 3,321-3,342, 2,899-2,920, 1,631-1,652, 1,547-1,552, 1,403-1,423, 1,140-1,161, and 1,014-1,025 cm −1 . It shows the functional groups OH-stretching, C-H bending, C═O stretching, N-H bending, CH 3 wagging, anti-symmetric stretching of (C-O-C) bridge, and C-O stretching ( Table  2 and Figure 5). Cellulose, chitosan, and agar biopolymers approximately had similar functional groups structure. Notably, the chitosan-coated paper showed characteristic peaks at 3,321, 1,403, and 1,014 cm −1 somewhat shifted to lower frequencies than the plain cellulose paper. Moreover, the Cht/Ar-AgNPs-coated paper showed characteristic peaks at 3,342, 2,909, and 1,161 cm −1 shifted to higher frequencies than cellulose and chitosancoated paper. Also, the newly raised peak at 549 cm −1 corresponded to metal (Ag) peak. This metal peak confirmed the presence of AgNPs on the cellulose paper surface.

Scanning electron microscope
The SEM analysis evaluated the AgNPs dispersion on the paper. Due to the interwoven fibers, plain cellulose paper showed a rough and uneven surface, and the chitosancoated paper appeared evenly spread and of smooth surface. Besides, Cht/Ar-AgNPs' well-distributed coating surface exhibited spherical shape AgNPs (Figure 6a-c).

Antibacterial properties
Safety and maintaining food quality have a vital role in antibacterial food packaging applications [38]. The antibacterial activity of coated paper was measured by a disk diffusion method, as shown in Figure 7. Obtained results, 6:4 (Cht/Ar-AgNPs) ratio coated paper, showed excellent antibacterial activity in Gram-positive (B. subtilis) and Gram-negative (E. coli, S. dysenteriae, and P. vulgaris) bacterial strains (Figure 8). This ratio promotes better   electromotive interaction with the negatively charged bacterial cells to positively charged AgNPs. As per the Swiss norm 1,95,920-ASTM E2149-0, the antibacterial activity showed the result in over than one mm, as considered for the superior bactericidal agent [23,39]. The inhibition zones' appearance around the Cht/Ar-AgNPscoated papers was showed on the antibacterial mechanism of AgNPs. Generally, AgNPs enhanced the reactive oxygen species (ROS) generation. Hitherto, the antibacterial activity of AgNPs' different mechanisms has been reported [40,41]. The best antibacterial activity mechanism was achieved by the electrostatic interactions of negatively charged bacterial cell walls and positively charged AgNPs [41]. At first, the bacterial cell lose the cell wall integrity. Consequently, bacterial electrolytes (Proteinaceous constituents and K + ions) leakage retards the cell division. It causes osmatic imbalances and inhibits bacterial growth. Besides, it enhanced the ROS generations, superoxide (˙O 2 − ), hydroxyl ion (˙OH − ), hydroxyl radical (˙OH), and hydrogen peroxide (H 2 O 2 ) [42,43], which leads to oxidative cellular damage. On the other hand, when nanoparticles were in contact with the cell surface, they migrate into the intercellular matrix and bind with sulfur-containing amino acids like cysteine and methionine. These S-H bondings with the AgNPs act as a sulfa drug related to folic acid synthesis metabolism. Besides, it had damaged the DNA replication, protein synthesis, intracellular signal communication, intracellular organelle of mesosome, adenosine triphosphate synthesis, and the regularity of electron transport chain. Finally, bacteria lose survivability. However, nanoparticles' antibacterial activity depended upon the dose of the treatment and species specificity [44][45][46][47][48].

Conclusion
This study was performed to develop the functional coated papers and to apply for antibacterial application. AgNPs are successfully synthesized by a single-step method. Synthesized Ar-AgNPs revealed a face-centered cubic crystalline structure and the spherical morphology with   a mean value of 10 nm. Different ratios of chitosan and Ar-AgNPs solution-coated cellulose papers were investigated against the antibacterial activity. Overall obtained results, the optimum composition level of Cht:Ar-AgNPs 6:4 (weight ratio), showed excellent antibacterial activity. Appreciably, the facile-synthesized AgNPs can promote a potential candidate for food packaging application in future market.