Optimized Synthesis of Xanthan gum/ZnO/ TiO 2 Nanocomposite with High Antifungal Activity against Pathogenic Candida albicans

Increased resistance of fungal pathogens to common antimicrobial agents is known as one of the most important human problems. Due to the limited variety of antifungal drugs available, the identi ﬁ cation and use of new antifungal drugs are essential. This study aimed to determine the optimal conditions for synthesizing a novel nanocomposite of xanthan gum/ZnO/ TiO 2 with the highest antifungal activity against Candida albicans ( C. albicans ). For this purpose, nine experiments were designed using the Taguchi method. In the designed experiments, three factors of xanthan gum, ZnO, and TiO 2 nanoparticles have been investigated at three di ﬀ erent levels, and the best ratio with the highest antifungal activity was determined. The results showed that in the presence of the synthesized nanocomposite in experiment 3 (xanthan gum 0.01M, ZnO 0.09M, and TiO 2 0.09M), the inhibition of fungal growth reached 92.51%. The properties of the synthesized nanocomposite and its components were investigated using di ﬀ erent characterization methods, which con ﬁ rmed the formation of nanocomposites with desirable properties. The antifungal activity results showed that the synthesized nanocomposite as an antifungal agent has an e ﬀ ective performance and can be used well in various ﬁ elds.


Introduction
The dramatic increase in microbial resistance to antimicrobials has become a pervasive challenge and a serious threat to public health worldwide. C. albicans is one of the most common fungal pathogens in humans. The widespread use of a limited number of antifungal agents has led to drug resistance in treating C. albicans infections, a problem that is becoming increasingly important [1]. The range of human infections caused by the C. albicans yeast and several related species is significant. They cause various infection levels, from relatively trivial conditions such as oral candidiasis and genital infections to fatal and systemic infections in patients who have already had serious illnesses. There has been great interest in fighting Candida infections, especially C. albicans, as fatal infections have become more common and new disorders have been identified with Candida [2].
Antifungal drugs often belong to two groups of polyenes and azoles. These compounds act on fungal cells by disrupting the metabolism of RNA or DNA and causing intracellular accumulation of peroxide. The currently available antifungal agents used topically and systemically to treat C. Albicans have shown serious side effects that have greatly limited their use. Toxicity and drug resistance are the main reasons for extensive research on new antifungal compounds [3]. Since discovering and using a new class of antimicrobial compounds is very time-consuming, expensive, and usually unsuccessful, an alternative is to use modern science and design and synthesize compounds with antimicrobial properties. Nanotechnology has solved many current problems by using structures with at least one dimension of 1 to 100 nanometers [4][5][6]. In recent years, significant studies have been conducted in applications of nanomaterials, and their effectiveness has been observed in many fields. Metal oxides with antimicrobial properties are among the most widely used compounds in various fields [6,7]. The use of nanomaterials can be effective in combating fungal infections. Due to their special properties, such as proper penetration into target cells and tissues, these particles have been able to provide more inhibitory power with lower concentrations compared to conventional compounds [8].
Extensive sources of zinc metal, reasonable price, antimicrobial, antioxidant, and anticancer properties make zinc a valuable metal as a raw material in the production of various compounds such as mouthwashes and toothpaste that reduce plaque and bad breath [9,10]. ZnO nanoparticles have successfully inhibited pathogenic microbes such as Streptococcus mutans and Staphylococcus aureus [11].
Titanium dioxide (TiO 2 ) has unique properties such as high photocatalytic activity, anticancer, antimicrobial properties, biocompatibility, reactivity, cheap and safe production, nontoxicity, temperature stability, and chemical stability [12,13]. These properties of TiO 2 are used in the production of food, pharmaceutical, biomedicine, environment, cosmetics, air purification, water disinfection, solar cell production, and antimicrobial compounds [14]. It is an optical catalyst whose antimicrobial applications have been investigated more than any other material with this property [15]. TiO 2 NPs can inactivate microorganisms and exhibit antimicrobial properties by producing hydroxyl free radicals and superoxide [16,17].
Similar properties are found in natural materials. Xanthan gum is a type of polysaccharide that is naturally produced by the Xanthomonas bacterium [18]. Its main chain consists of β-D-glucose units interconnected in positions 1 and 4; in addition, two mannose and glucuronic acid are also involved in its structure [19]. Among the desirable properties of xanthan are nontoxic, inexpensive, with high biocompatibility, thermal stability, resistance to acid and base, high stability and solubility in water, and antimicrobial properties [20,21].
Due to the tendency of nanoparticles to agglomerate, which leads to the loss of their desirable properties, it is necessary to use strategies to prevent this. The use of nanoparticles in the form of nanocomposites can largely prevent them from agglomerating. For this purpose, this study was designed and conducted for the first time to determine the optimal conditions for the synthesis of xanthan gum/ZnO/ TiO 2 with the highest antifungal properties.

Materials and Methods
2.1. TiO 2 Nanoparticle Synthesis. To prepare TiO 2 NPs using the sol-gel method, 10 ml of isopropanol was combined with 10 ml of deionized water, and 20 ml of titanium isopropoxide solution was added dropwise with continuous stirring. The resulting solution was placed at 60°C for 60 min, the yellow gel formed in the oven was dried at 80°C, and the gel powder was calcined in air for 3 h at 650°C [22].
2.2. ZnO Nanoparticle Synthesis. Zinc acetate (Zn(CH 3 CO 2 ) 2 ·2H 2 O) and sodium hydroxide (NaOH) were used to prepare ZnO nanoparticles. First, 0.1 M zinc acetate and 0.2 M sodium hydroxide solutions were prepared by dissolving them in deionized water.
Then two solutions were poured into a beaker and stir at 750 rpm for 2 h at 60°C. After 2 h, a clear milky white solution was formed. The next step was centrifugation at 5000 rpm for 5 min to precipitate a white product. The precipitate was washed first with deionized water and then with acetone. ZnO NPs were obtained in powder form by drying the product using an oven at 75°C for 6 h [23].

Xanthan Gum/ZnO/TiO 2 Nanocomposite Synthesis.
To determine the optimal conditions for the synthesis of xanthan gum/ZnO/TiO 2 nanocomposite with the highest antimicrobial activity, using Qulitek-4 software according to the Taguchi method, 9 experiments containing different ratios of xanthan gum, ZnO, and titanium NPs were designed. To evaluate the antimicrobial activity of synthesized nanocomposites, using in situ method and 0.01, 0.02, and 0.03 M levels of xanthan gum and 0.03, 0.06, and 0.09 M levels of ZnO and TiO 2 NPs, nine samples of nanocomposites were tested. To synthesize the studied nanocomposite, xanthan gum was prepared commercially; ZnO and TiO 2 NPs were prepared by coprecipitation and sol-gel methods, respectively. In the synthesis of nanocomposites, first, the solutions of the components were stirred separately by a magnetic stirrer for one h. All three solutions were then dispersed at room temperature for 15 min using an ultrasonic homogenizer. Finally, solutions of ZnO and TiO 2 NPs were added simultaneously and dropwise to the solution containing xanthan gum. The final solutions were stirred for one h and then dispersed for 15 min by an ultrasonic homogenizer. Finally, the prepared solution was placed in an oven at 60°C for 24 h to form nanocomposite sediments. The resulting precipitate was separated from the container with a spatula and ground in a mortar to prepare the final nanocomposite powder [24].

Antifungal Activity.
The antifungal activity of synthesized xanthan gum/ZnO/TiO 2 nanocomposites on C. albicans (ATCC 10231) was studied using the colony forming unit (CFU) method. For this purpose, C. albicans was incubated on a Sabouraud dextrose agar (SDA) medium for 48 h at 37°C to prepare an isolated colony. After that, a colony was taken and dissolved in twice distilled water to obtain an approximate 10 6 CFU/ml concentration. To evaluate the antifungal activity of the synthesized nanocomposites, solutions containing SDA and 9 synthesized nanocomposites (1 mg/ml) were prepared. The resulting solutions were poured into the Petri dish, and after freezing the culture medium, 100 μL of the fungal suspension prepared on the 2 Journal of Nanomaterials culture medium was completely cultured using a swap. In the control group, 100 μL of fungal suspension was cultured on a pure SDA medium. The number of colonies was counted for 9 experimental and control groups after 96 h of incubation at 28°C. The inhibition of fungal growth by each of the synthesized nanocomposites is calculated using the following equation: where Cg is the average growth of colonies in the control group and Eg is the average growth in the experimental group. Thus, the optimal conditions for synthesizing nanocomposites with the highest antifungal activity were determined from 9 experiments. All experiments were repeated three times.

Results and Discussion
3.1. Antifungal Activity. Optimal conditions for the production of xanthan gum/ZnO/TiO 2 nanocomposites with the highest antifungal activity were determined based on 9 experiments designed by the Taguchi method. Table 1 shows the effect of nanocomposites synthesized under different conditions on the growth inhibition of C. albicans. The results showed that the nanocomposites produced in       Table 2 shows the effect of xanthan gum, ZnO, and TiO 2 NPs factors on the growth inhibition of C. albicans.
The results indicate that the xanthan gum factor at level 2 and ZnO and TiO 2 NPs at level 3 had the greatest effect in inhibiting the growth of C. albicans.
The interaction of factors on the growth inhibition of C. albicans is presented in Table 3. Xanthan gum in the first level and TiO 2 in the third level had the most interaction and the growth inhibition of C. albicans at 45.33%. Xanthan gum in the first level and ZnO in the third level showed a significant interaction on the growth inhibition of C. albicans by 15.75%. The lowest percentage of interaction intensity index belonged to ZnO and TiO 2 in the third level at 1.23%.
The analysis of variance of the parameters affecting the growth inhibition of C. albicans is presented in Table 4. The highest effect on inhibiting the growth of C. albicans was shown by ZnO with an effect of 56.09%, TiO 2 by 40.47%, and xanthan gum by 2.71%, respectively.
After reviewing the data and analyzing the effect of each factor and their interaction, the optimal conditions for the synthesis of xanthan gum/ZnO/TiO 2 nanocomposite with the highest antifungal activity were estimated (Table 5). Based on these results, ZnO had the highest contribution, and xanthan gum had the lowest contribution in inhibiting the growth of C. albicans, and TiO 2 showed an effect between these two factors and close to ZnO. The second level was the most appropriate level for the xanthan gum factor and the third level for ZnO and TiO 2 NPs factors. Based on the results, it was estimated that the synthesized nanocomposite in optimal conditions inhibited about 95% of fungal growth, which was most consistent with the results of experiment 3.
Previous similar studies have reported the optimal antimicrobial properties of nanomaterials and their components. Nanoparticles currently used in the clinical field are limited by agglomeration. Many studies have used the modification of impurities and the synthesis of nanocomposites to prevent nanoparticles from agglomerating and disperse nanoparticles in different environments. In other words, the modification in this way is one of the most effective ways to regulate and control the interaction of nanoparticles and germs [25][26][27][28].
The larger area and high density of nanoparticles allow them to show antimicrobial activity more favorably on the surface of fungi with more interaction. In addition, nanoparticles combined with polymers or coated on the surfaces of biocompatible materials have been used to improve their antimicrobial properties. The study of the mechanism of antimicrobial activity of nanocomposites containing metal oxides indicates that these nanostructures can cause the destruction of the fungal cell wall and the penetration of nanoparticles into the cell. Nanoparticles that pass through the cell wall are not fully understood. However, their transport, diffusion and endocytosis, and entry into the cell can lead to the production of reactive oxygen species (ROS) and thus interfere with the function of many intracellular organs [29,30]. Reactive oxygen species seem to play a major role in antifungal activity. By inducing oxidative stress, they can destroy all biomolecules in the cell (including proteins and DNA) and disrupt the function of intracellular organs [31]. Nanostructures can also cause fungal death by damaging cellular enzymes and disrupting the electron transfer chain (Figure 1). In the FTIR spectrum of xanthan gum (diagram a), two peaks at positions 3420 and 2922 cm -1 were observed for the tensile bonds of -OH and -CH, respectively. Asymmetric and symmetric tensile vibrations related to carboxylate group -COO-bonds were shown at 1620 and 1420 cm -1 , respectively. A wide absorption peak was observed at 1069 cm -1 due to the tensile vibration of the glycosidic bond [32].
In the FTIR spectrum of ZnO NPs (diagram b), a wide adsorption peak in the range of 3100-3700 cm -   5 Journal of Nanomaterials 424 cm -1 due to the tensile vibrations related to oxygen and zinc. Zn-O tensile bands were observed at 424 and 569 cm -1 for pure samples of ZnO NPs [33].
In the FTIR spectrum of TiO 2 nanoparticles (diagram c), the peaks observed at 3414 cm -1 and 1635 cm -1 refer to the adsorbed moisture and the O-H surface groups. The peak observed in the 1618 cm -1 region corresponds to the bending vibrations of the water molecule, and the peak index of the 1385 cm -1 region is due to the carbonate group, which is created by the adsorption of ambient carbon dioxide by nanoparticles. Sharp peaks observed in the range below 850 cm -1 showed the binding of oxygen to titanium [34,35]. The peaks  25,38,48,54,55,63,69,70. and 75 degrees, respectively [37].
X-ray diffraction pattern obtained from synthesized nanocomposite (diagram c) showed the presence of component peaks and their intensity reduction, flattening or removal, and displacement of some peaks relative to the X-ray diffraction pattern of components in the synthesized nanocomposite X-ray diffraction spectrum. In addition, the average crystallite size was calculated for the highest peak of 28 nm.

SEM and Elemental
Mapping Analysis. The appearance and elemental mapping of the xanthan gum/ZnO/TiO 2 nanocomposite were examined by field emission scanning electron microscopy ( Figure 4). Figures 4(a) and 4(b) show the placement of metal oxide nanoparticles inside the xanthan gum, resulting in the formation of the final nanocomposite. Elemental mapping was performed by energydispersive X-ray spectroscopy (EDS) to evaluate the  3.5. TEM Analysis. The morphology of the xanthan gum/ ZnO/TiO 2 nanocomposite was investigated by TEM micrograph preparation of the synthesized nanocomposite. TEM micrograph analysis showed the formation of this nanocomposite ( Figure 5). The morphology and distribution of oxide nanoparticles were also examined in more detail by TEM. The results showed that the metal oxide nanoparticles were well in contact with the xanthan gum and the final nanocomposite was prepared by distributing different sizes of particles.
3.6. UV-Vis Analysis. The optical properties of xanthan gum/ZnO/TiO 2 nanocomposite and its components were investigated using ultraviolet-visible spectroscopy in the range of 200 to 800 nm ( Figure 6). No specific absorption band was observed in the xanthan gum spectrum (diagram a). In the spectrum of ZnO NPs (diagram b), a sharp and specific peak showed the production of adsorption in the range of 370 nm for this sample. In the spectrum of TiO 2 NPs (diagram c), a wide absorption band with the highest intensity in the range of 274 nm was observed. The absorption spectra of the final synthesized nanocomposite (diagram d) showed the presence of a wide low-intensity absorption peak in the range of 270 nm and a specific low-intensity peak in the range of 371 nm. This confirmed the interaction of the components of the nanocomposite [24].
3.7. Thermal analysis. The thermal properties of xanthan gum/ZnO/TiO 2 nanocomposite composition and its thermal stability were evaluated by TGA and DTA analysis (Figure 7). The behavior of thermogravimetric analysis and differential thermal analysis of the final nanocomposite showed the three main stages of weight loss by 44.15%.
The first stage, consisting of 11% weight loss, occurred in the range of 25-250°C due to the moisture loss and dehydration of the prepared nanocomposite. The second stage, with a weight loss of 28%, in the temperature range of 250-550°C, is related to the thermal decomposition of the elements of impurity components and carbon bonds in the nanocomposite. In the last stage of weight loss, with a weight loss of 5.15%, in the temperature range of 550-800°C, the structural water, which is in the form of bonded hydroxyl groups, begins to decompose and release. In addition, at this stage, impurities with higher thermal stability were decomposed. In the diagram obtained from the differential thermal analysis of the final nanocomposite, exothermic peaks were observed upwards, and endothermic peaks were observed downwards.

Conclusions
By analyzing the results of experiments designed by the Taguchi method, the antifungal activity of xanthan gum/ ZnO/TiO 2 nanocomposite synthesized by in situ method against C. albicans fungal strains was evaluated. The optimal antifungal properties of synthesized nanocomposite were confirmed. Based on the antifungal activity results, it was estimated that the synthesized nanocomposite under the optimal conditions prevents fungal growth up to 95%. Due to the antifungal properties predicted for this nanocomposite, its use as an antimicrobial agent in medicine and dentistry can be effectively useful, and it dramatically reduces pathogenic microbes. Identifying and synthesizing novel and effective antimicrobial compounds can reduce the prevalence of many microbial diseases and prevent increased treatment costs. It is recommended that clinical studies be performed locally and systemically to evaluate the effectiveness of this nanocomposite in the treatment of oral and vaginal candidiasis.  Journal of Nanomaterials

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.