Antifungal Efficacy of Chitosan-Modified Zinc Oxide Nanoparticles on Tube Sedge Products

INTRODUCTION
Zinc oxide (ZnO), an n-type direct band gap semiconductor, is renowned for its applications in industry. Currently, more eco-friendly routes are developed to synthesize ZnO in forms of nanostructures [1-4]. Nanostructured ZnO exhibits multifunctionality based on its electric, optical, photocatalytic as well as antimicrobial properties. Direct contacts of ZnO on bacteria cells release Zn2+ and reactive oxidation species (ROS) which are detriment to bacterial growth. For fungi, the exposure to ZnO nanoparticles deforms the surface of fungal hyphae with increased nucleic acids and carbohydrates through the stress response in fungal hyphae [5]. Hydrogen peroxide generated from ZnO is also a major contributor of antifungal activity [6]. The rupture and the damage of the cell membrane reduce the fungal enzymatic activity and lead to cell deaths [7-10]. A high surface-to-volume ratio of ZnO nanoparticles then enhances the antimicrobial activity compared to the bulk form. Lalithambika demonstrated that antibacterial efficiency of ZnO nanopowder was higher than that of NiO [2]. The antimicrobial activity was successfully enhanced by doping ZnO with Ag [11], Co [12], Ni [13]. The doping of ZnO by either Co or Ni also led to dilute magnetic semiconductors. To increase antibacterial activity with the surface-to-volume ratio, the size of ZnO was regulated by capping agents such as polyethylene glycol [14] and chitosan [15]. Furthermore, a large variety of nanocomposites with antimicrobial activity were obtained by incorporating ZnO into other functional materials, e.g. polyaniline [16], CuO [3], rare-earth elements [17]. By virtue of these doping, surface modification and composites, the antimicrobial properties were enhanced by synergistic effects.
The antimicrobial agents from ZnO, doped-ZnO and ZnO nanocomposites are majorly exploited in nanomedicine and medical uses. Besides, these materials can be useful for food and handicraft products which are susceptible to microbial growths. Jamdagni and co-workers used ZnO in conjunction with common agricultural fungicide [8]. Such applications greatly benefit the community aiming to reduce the yield loss and health risk as well as to add the value of local products. This work emphasizes on the fungi inhibition on tube sedge (Lepironia articurata (Retz.) Domin) basketry which are used and sold by locals in several countries including the southern region of Thailand. The values of local handicrafts can be added by the ZnO coating for sun-screen and fungi protections.

MATERIALS AND METHODS
ZnO nanosuspensions were synthesized from zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O; Ajax Finechem, 99.5%) and sodium hydroxide (NaOH; Merck, 99%) with chitosan  as a capping agent. Zn(CH₃COO)₂·2H₂O of 1.0975 g, 2.195 g, and 4.390 g was separately dissolved in 100 mL of distilled water to obtain the concentration of 0.05 M, 0.1 M, and 0.2 M whereas the chitosan (low MW-4000) of 1.25 g and 2.5 g was dissolved in 2% acetic acid. With varying concentrations shown by Table 1, both solutions were then mixed with 0.2 M NaOH solutions, giving rise to 6 samples referred to as C1-C6 in Table 1. All nanosuspensions were continuously stirred for 15 h. The pH was adjusted to 7 by NaOH solutions and the stirring was continued for another 4 h. The nanosuspensions were then sonicated for 4 h to enhance ZnO dispersion in nanosuspensions. 
To characterize ZnO nanoparticles, nanosuspensions were centrifuged and the precipitates were filtered. The nanoparticles were repeatedly washed by ethanol and then dried at 60 °C for 15 h. Morphology of each sample were obtained from the scanning electron microscope (SEM, FEI Quanta 450 PEG) and transmission electron microscope (TEM, Tecnai G2 20) whereas optical properties were characterized by the UV-Vis spectrophotometer (Shimadzu UV-2450). Surface area and porosity were analyzed by the static volumetric nitrogen gas adsorption method (Micromerit ASAP 2460) according to Brunauer-Emmett-Teller (BET) theory.
The structure was characterized by the simultaneous thermal analyzer (Perkin Elmer, STA8000), Fourier transform infrared spectrophotometer (FTIR, Agilent Cary 630) and X-ray diffractometer (XRD, Philips X′ Pert MPD). The average crystallite sizes of the particles (D) were calculated by using Debye-Scherrer equation:

where λ is the X-ray wavelength (1.54 Å). θ is the diffraction angle and β is the full width at half maximum of the major XRD peak.
To test antifungal efficacy using the zone of inhibition method, the suspension of the Aspergillus sp. was adjusted to approximately 106 conidia spore/mL by 0.85% normal saline and spread on potato dextrose agar (PDA) (Himedi, India). The nanosuspension was also sprayed on 1 inch² pieces of tube sedge basketry from local market. Before the fungi application, the tube sedge samples were heated at 60 °C for 4 h and coated with ZnO nanosuspension. Two other samples, one lacquer coating and a control without any coating, were also prepared for the comparison. The Preti dishes containing PDA and samples were incubated at room temperature. The growth of pathogenic fungi was monitored by taking photographs of the tube sedge pieces after 1, 10, 20 days of inoculation.

RESULTS AND DISCUSSION
TGA and DTG
As the temperature is raised up to 1000 °C in Fig. 1, the thermogravimetric analysis (TGA) curve exhibits the reductions in weight with changing slopes. Corresponding to the derivative thermogravimetry (DTG) curve, the different slopes indicate the occurrence of different processes. The drastic weight losses below 100 °C [18] and over 200 °C [3] are respectively due to the dehydration and the thermal decomposition of chitosan. The next weight loss is gradual in an extended range from 350 to 750 °C, covering both combustion of organic residuals and crystallization of nanostructured ZnO [19]. Around 760 °C, the remaining weight is only 35%.

FTIR
The characteristic vibration of each bonding can be detected by FTIR spectroscopy and the spectra of chitosan-modified ZnO are compared with the ZnO and chitosan standards in Fig. 2. For every spectrum, a broad peak centered around 3295 cm^-1 corresponds to the O−H vibration of adsorbed water molecules [1-3, 12, 13, 17]. This range also covers the vibration of amino group of chitosan [15]. The much smaller peaks at 2867 cm^-1 are attributed to the −CH symmetric stretching [3, 13, 15]. Whereas the peak at 2350 cm^-1 due to the absorption of atmospheric CO₂ on the metallic radicals is not clearly observed, chitosan and chitosan-rich samples have small but broad peaks at slightly lower wavenumbers [1, 13]. The peak at 1560 cm^-1 is attributed to chitosan. Between 1000 cm^-1 and 1500 cm^-1, there are a few sharp peaks corresponding to −NH, −CH, −NH,C=O and other residual free charges from impurities in the products [1, 2, 17]. The absorption peak around 470-530 cm^-1 from the Zn-O stretching mode of vibration in ZnO structure is exclusively observed in ZnO sample [1-3, 12, 15-17]. Overall, the addition of chitosan in the ZnO synthesis largely affects the intensity in IR absorption. Whereas all characteristic ZnO peaks appear at identical positions, the peak intensity is decreased with increasing chitosan.

XRD and microscope images
The phase identification by XRD is consistent with the characteristic vibration in FTIR spectra. XRD patterns in Fig. 3 exhibit peaks at 31.69°, 34.34°, 36.19°, 47.38°, 56.47°, 62.75°, 66.27°, 67.84°, 68.97°, and 72.37° by the diffraction respectively from (100), (002), (101), (102), (110), (103), (200), (112), (201), and (004) planes of the single phase ZnO with the hexagonal wurtzite structure (JCPDS card No.36-1451) [2, 3, 14, 17]. In addition to the characteristic ZnO peaks, broad peaks below 30° are assigned to the chitosan. With increasing Zn(CH3COO)2·2H2O in the synthesis, the peaks of samples C5 and C6 are shifted to a higher angle and grow at the expense of ZnO peaks. This evolution of XRD pattern signifies the encapsulation ZnO by chitosan. On the other hand, the lattice parameters of ZnO remains rather constant as diffraction peaks from ZnO are at the same positions. The crystallite sizes evaluated from the major ZnO peak width are shown in Table 1. The values as regulated by the chitosan are comparable to the literature ranging from 14 to 54 nm [2, 3, 8, 14].
The crystallite sizes are consistent with the TEM images and the samples can then be divided into 2 groups. Samples C1-C3 in the first group have the crystallite size of 38.8-56.5 nm and overlapping ZnO nanoparticles are observed in Figs. 4(a)-(c). In the other group, the crystallite size is reduced to 21.1-28.2 nm in Figs. 4(d)-(f). The smaller ZnO crystallites without overlapping are obtained in the case of samples C5 and C6. However, agglomerations into microscale aggregates are observed in every sample by SEM images. Interestingly, samples C1-C3 with larger crystallize sizes tend to agglomerate into bulky aggregates with smaller number of pores as shown in Figs. 5(a)-(c).

Antifungal Properties and BET Analysis
The BET surface area listed in Table 1 is clearly related to the amount of chitosan used in the synthesis. Samples C2, C4 and C6 respectively exhibit surface areas of 4.69, 6.67, 3.64 m2/g whereas those from similar reagents with a lower amount of chitosan (1.25 g) are only 1.24-1.87 m2/g. This finding confirms the encapsulation of ZnO by chitosan. In Fig. 6, photographs of pieces of tube sedge basketry without coating (the control) and conventional lacquer coating indicate the onset of fungi growth after 10 days. For 20 days, fungi have grown and substantially covered the surface. By coating with chitosan-modified ZnO, Aspergillus sp. still increasingly grows from 1 to 20 days with an exception of sample C5. It is unusual that the fungi inhibition is effective in the case of lowest BET surface area. However, the antifungal activity in this sample is related to the lowest crystallite size of ZnO encapsulated by chitosan.
The fungi inhibition by this chitosan-modified ZnO is attributed to the stress response in fungal hyphae and generation of hydrogen peroxide [5-10]. The reduction in crystallite size promotes the fungicide. The discrepancy regarding the BET surface area is probably explained by the agglomeration of smaller crystallites in close contacts. Only one fungal strain is tested in this work but antifungal activities of ZnO have been previously demonstrated on others. In addition to Aspergillus sp., there are reports on the inhibitions of Botrytis cinerea [5], Penicillium expansum [5], Trichoderma harzianum [9], Fusarium sp.[10], Rhizopus stolonifer [9, 20], Candida albicans [20], A. flavus [9], A. nidulans [9], A. brasiliensis [21], A. niger [22] and T. reesei [22].

UV-Vis absorption
Fig. 7 compares UV-Vis absorption spectra of ZnO with varying chitosan in the synthesis. Again, absorption characteristics are divided into 2 groups. For samples C1-C3, the absorption is pronounced in the entire UV range of 200-400 nm without any distinct peak. The absorbance then drops to minimum in the visible regime. The spectra, resembling those of ZnO with a different morphology in the previous reports [23], indicate the potential use as sun-screen coating. By contrast, samples C4-C6 with low amounts of chitosan do not have a flat response in the UV range. The UV absorbance is lower and exhibit a peak around 270 nm. The peak is related to the size of ZnO nanoparticles and the capping agents adsorbed at the surface of ZnO inhibit the growth of ZnO nanostructures in certain directions [14]. The different UV-Vis absorption characteristics in samples C1-C3 is due to the crystallite size regulated by chitosan [3, 8, 12, 13] and the agglomeration into microscale aggregates which could also affect photocatalytic properties [24].

CONCLUSIONS
ZnO nanoparticles were synthesized from the reaction between Zn(CH₃COO)₂·2H₂O and NaOH. Chitosan of varying amounts was added to regulate the crystallite size of ZnO. The BET surface area of ZnO is increased with increasing chitosan used in the synthesis. Large UV absorbance in an extended wavelength range was obtained when large ZnO crystallites agglomerate into bulky aggregates due to an excessive amount of chitosan. On the other hand, the inhibition of Aspergillus sp. on tube sedge basketry was more effective in the case of smaller ZnO crystallite size and BET surface area. It follows that chitosan-modified ZnO can be implemented in sun-screen and antifungal coating on local handicrafts.

ACKNOWLEDGEMENTS
This work is funded by SciUS-TSU and NECTEC-NSTR. The authors would like to thank Scientific Equipment Center of Prince of Songkla University and Thaksin University for the access to characterization facilities.

CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest regarding this article.