Antimicrobial activity of Titanium dioxide and Zinc oxide nanoparticles supported in 4A zeolite and evaluation the morphological characteristic

In this study, the antimicrobial activity of titanium dioxide (TiO2), zinc oxide (ZnO), and TiO2/ZnO nanoparticles supported into 4A zeolite (4A z) was assessed. Based on antimicrobial experiments, minimum inhibitory concentration (MIC90), minimum bactericidal concentration (MBC), fractional inhibitory concentration (FIC) and disc diffusion test were determined after 24 h of contact with the prepared nanocomposites. These results are in agreements with the results of disc diffusion test. During the experiments, the numbers of viable bacterial cells of Staphylococcus aureus, Pseudomonas fluorescens, Listeria monocytogenes and Escherichia coli O157:H7 decreased significantly. The crystallinity and morphology of nanoparticles were investigated by X-ray diffraction patterns (XRD), elemental mapping at the microstructural level by scanning electron microscopy (SEM) with energy dispersive X-ray spectrometry (EDS), and transmission electron microscopy (TEM). As a result, it was demonstrated that TiO2/ZnO nanoparticles supported in 4A zeolite could lead to an optimum activity as antimicrobial agents.

were tested with an excellent antimicrobial activity in the efficient removal of pathogens. MONP may not show a considerable antimicrobial activity in form of metal oxide and metal salt alone, because of their tendency of aggregation 15 . However, the stability and slow release of metal ions from MONP are effective properties that can be tuned by appropriate synthesis. For this reason, new methods to immobilize MONP into zeolite membranes have been suggested 16 . One of the most important factors in antimicrobial activity is light. MONP, particularly ZnO and TiO 2 , become activated against pathogenic bacteria when exposed to ultraviolet (UV) light 17,18 . The mechanism of this procedure involves substrate adsorption in the composite surface, depending on pH, temperature, composite stability, area and substrate concentration [19][20][21][22] . Some of the disadvantages are low adsorption ability (small active area) and photo corrosion 23,24 . Therefore, with precise preparation of ZnO and TiO 2 , especially regarding morphologies and sizes, most of these caveats can be overcome [25][26][27] .
MONP also show significant antimicrobial activity through connection to microbial DNA and proteins, and caused to preventing bacterial duplication, avoiding metabolic enzymes of the bacterial electron transport chain, leading to their inactivation 2 .
The semiconductor properties of zinc oxide (ZnO) and titanium dioxide (TiO 2 ) can be tuned in nano scale [28][29][30][31] . Manufacturing nanoparticles involves protocols of different complexities with aggregation of ZnO and TiO 2 nanoparticles as an important limitation in the process 32,33 . These compounds are useful in pharmaceutical utilization 34 , pigmentation 35 , antimicrobial activities, anti-fungal and anti-viruses properties 36 . In addition, ZnO and TiO 2 are important components in the novel and active packaging industry.
Tiny ZnO and TiO 2 nanoparticles with a wide surface can maintain their photoactivity; however, their recovery at the end of the procedure is very difficult. Accordingly, a useful approach is to support ZnO and TiO 2 in a zeolite structure which can enhance adsorption and subsequently, increase the composite efficiency and ease of recovery by ordinary deposition 37,38 . The use of effective composites made up with ZnO and TiO 2 impregnated in zeolites is a new strategy to produces nanoparticles with supporter. Recent research reported some properties of ZnO supported in zeolites including ZSM-5, Y, X and A 39,40 . In this research, we have focused on the description and development of ZnO and TiO 2 nanoparticles supported in the channels of a porous matrix (4A zeolite) for antimicrobial application. First of all, we described a simple synthetic route for preparation of ZnO and TiO 2 impregnated into 4A zeolite (4A z). Secondly, the structural properties and antimicrobial effects of the materials were determined. In fact, zinc and titanium in high quantities may have toxic effects [41][42][43][44][45] ; hence, in this work we have tried to use 4A z as a support of metal nanoparticles. These nanoparticles are encapsulated inside zeolite cavities or external channels to decrease the possibility of leaching as low as possible. This process reduces the toxicity of nanoparticles and their release from the matrix and therefore, this complex (nanoparticles and zeolite) can be used in polymer matrix for food packaging. In this work, we aimed to select the lowest effective amount of nanoparticles into 4A z with antimicrobial activity.

Materials and Methods
Materials. Here, 4A z (Si/Al ≅ 2) was synthesized according to the formula below. Zinc acetate dehydrate, orthotitanate and ethanol were purchased from Merck. All the applied reagents were of analytical grade. It should be noted that in order to test antimicrobial activity, bacterial strains were obtained from Biological and Genetic Resource Center, Tehran, Iran. Nutrient broth and Nutrient agar were all purchased from Micromedia, Canada.
The degree of crystallinity is measured by peak height method 46 based on Eq. (1): where CrI is crystallinity degree (%), I, is peak height at the angle of diffraction (2θ) related to crystalline section and I 0 is peak height at the angle of diffraction (2θ) related to non-crystalline section. Scanning electron microscope (SEM) (Philips XL30) was evaluated to catch SEM images and to carry out elemental analysis. The SEM sample was gold coated prior to examination and SEM was operated at 5 kV. MAP was obtained by wavelength-dispersive X-Ray spectroscopy (WDS) system to show the spatial distribution of elements in a sample and be extremely useful for displaying element distributions in polymer matrix, particularly for showing compositional zonation. Transmission electron microscopy (TEM) was determined by electrons microscope systems and was also used to indicate the interactions between the electrons and the atoms in order to observe crystal structure and features in the structure like dislocations and grain boundaries. The minimum inhibitory concentration (MIC 90 ), minimum bactericidal concentration (MBC) and fractional inhibitory concentration (FIC) of MONP dropped into 4A z were also determined.

Methods
Preparation of 4A zeolite. 4A z (alumina silicates) with a silica to alumina ratio (Si/Al ≅ 2) was synthesized by hydrothermal method. In this process, clinoptilolite and alumina were used as sources of silicon and aluminum, respectively, and then mixed with NaOH. In the final stage, the solution was conveyed to teflon reactor at 90 °C, in order to produce 4A z nanocomposite 37 . Preparation of ZnO doped into 4A zeolite. In this research, ZnO clusters were synthesized by the following stage response: in the first step, a solution of 0.17 g Zn(CH 3 CHOO) 2 ·2H 2 O was prepared, mixed with a ratio of 5:95% w/w (nanoparticles:4A z) and was shaken for 30 min. In the second step, integration of zinc acetate to 4A z using an ion exchange process was done (24 h at 60 °C). Next, by the use of distilled water, the solution was washed and dried at 80 °C. The final powder was calcined at 500 °C for 2 h to produce ZnO/4A z nanocomposite.
www.nature.com/scientificreports www.nature.com/scientificreports/ preparation of tio 2 doped into the 4A zeolite. In this research TiO 2 salt was created by the following stage response: in the first step, 0.2 g ortho titanate was dissolved in ethanol. Then, this solution was mixed with a ratio of 5:95% w/w (nanoparticles:4A z) and was shaken for 30 min. In the second step, integration of ortho titanate to 4A z using an ion exchange process was done (6 h at 90 °C). Next, by the use of distilled water, the solution was washed and dried at 80 °C. The final powder was calcined at 500 °C for 2 h in order to produce TiO 2 /4A z nanocomposite.
preparation of Zno and tio 2 doped into 4A zeolite. In order to prepare ZnO/TiO 2 doped into 4A z, ZnO nanoparticle was prepared and mixed with a ratio of 2.5:95% w/w (nanoparticles:4A z). Then, 2.5% w/w titanium dioxide ethanol dried powder was added to the mixture of 4A z and ZnO and dried at 80 °C. The final powder was calcined at 500 °C for 2 h in order to produce TiO 2 and ZnO/4A z nanocomposite. The MBC is the lowest concentration of an antibacterial agent required to kill a particular bacterium 47,48 . It can be determined from broth dilution MIC tests by sub culturing to agar plates that do not contain the test agent. The minimum bactericidal concentration is identified by determining the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by ≥99.9%. The MBC is complementary to MIC; whereas, MIC test demonstrates the lowest level of antimicrobial agent that inhibits growth, MBC test demonstrates the lowest level of antimicrobial agent that results in microbial death 49,50 . The fractional inhibitory concentration was used for combination of materials based on the following equation:

Determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration(MBC) and fractional inhibitory concentration (FIC
where A refers to TiO 2 nanoparticles and B refers to ZnO nanoparticles. Determination of antibacterial activity of nanoparticles doped in 4Azeolite. The disc diffusion test was performed to determine the antibacterial activity of nanoparticles doped in 4A z 51 . Pseudomonas fluorescens, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O 157 :H 7 suspensions were collected from 18 h nutrient broth cultures then adjusted to 0.5 McFarland standard turbidity (1.5 × 10 −8 CFU/mL) and diluted (1:10) to the desired bacterial density (1.5 × 10 −6 CFU/mL). The Mueller-Hinton agar medium was then inoculated with 0.1 mL of the bacterial suspensions (1.5 × 10 −6 CFU/mL). Next, the suspensions of nanoparticles doped in 4A z and also 4A z individually, were prepared under sterile conditions and then situated on the surface of Mueller-Hinton agar plates (100 λ). The plates were incubated at 37 °C (for L. monocytogenes, E. coli O 157 :H 7 , and S. aureus) or 25 °C (for P. fluorescens) for 24 h, after which the inhibition zone around the discs were measured by a digital micrometer. Indeed, inhibition zone have shown the antibacterial effect of nanoparticles. The greater the diameter of the inhibitory zone, the greater antimicrobial activities of nanoparticles.

Inductively coupled plasma mass spectroscopy (ICP-MS).
Migration tests were performed according to EU regulation for plastic materials and articles intended to be in contact with food (Regulation 10/2011/ EU) 52 . Migration of TiO 2 and ZnO nanoparticles was assessed using a simulant (deionized water with pH = 6-7) over 12 days at 25-30 °C. Prior to the migration tests, the concentration of ZnO and TiO 2 nanoparticles in 4A z was determined. In ICP-MASS assay, we first produced a nano bio-composite film with selected polymer and nanoparticles zeolite. Then, this nano bio-composite film was converted to food packaging by thermal processing. Next, to measure the migration of nanoparticles zeolite from food packaging to food product, we have used food simulant. Food simulant was filled in food packaging, packed and stored for 12 days. Next, the simulants were filtered through a 220 nm Millipore filter and then migrated nanoparticles were analyzed using ICP-MS (Agilent 7800 Quadrupole ICP-MS, Perkin Elmer, California, United States). In the final stage, the migration results were normalized to nanoparticles migrated/cm 2 after 12 days. It should be noted that, on the first day, the measurement of nanoparticles zeolite in food packaging was conducted regardless of food simulant. This was to discover the exact amount of nanoparticles embedded in 4A z and polymer matrix.
Ethical approval. This article does not contain any studies with human participants or animals performed by any of the authors.

Results and Discussion
Evaluation of XRD patterns. XRD showed the impact of homogenization on crystalline structure of the combination 53,54 . XRD patterns of ZnO, TiO 2 , and ZnO/TiO 2 supported into 4A z are shown in Fig. 1 41°. According to Scherrer equation (τ = κ λ/β cosθ), the average crystallite size of nanoparticle was approximated to be 50 nm. In this equation, τ is the average crystalline size, κ is the shape factor (about 0.9), λ is the wavelength of X-ray radiation, β is the full wide at half the maximum intensity and θ is the Bragg diffraction angle 55 . As shown in Fig. 1, the peaks of 4A z are visible at certain 2θ with high intensity. The diffraction peaks for ZnO and TiO 2 were in good agreement with those given in the standard data (PCPDF, 79-0207 and JCPDS, 21-1272, respectively) with acceptable crystallinity. According to XRD patterns, it was found that ZnO and TiO 2 was crystallized with hexagonal wortzite and anatase phase, respectively. On the other hand, it is clear to see that the width of the reflections is considerably broadened, which indicates a small crystalline domain size. In the spectra of oxide nanoparticles doped into 4A z, the presence of nanoparticles can be confirmed based on the observed peaks, in particular 2θ = 4-70. Regarding the significant peaks with miller indices of TiO 2 (642), (664) and ZnO (842), (664), they have considerable overlap with the peaks of 4A z (Fig. 1), and therefore, the oxide nanoparticle peaks are not clearly visible.
But according to MAP pattern, the presence of elements (titanium and zinc) has been confirmed which affects the intensity of 4A z peaks (Fig. 2). Also, it should be noted that in ZnO/TiO 2 spectrum, the intensity of crystal-  Fig. 2A, cube-shaped particles are completely apart from one another and the deformed structures are not visible. The image ( Fig. 2A) showed that the matrix of ZnO and TiO 2 nanoparticles had homogeneous and smooth surfaces without any roughness and cracks, which indicated proper synthesis of the materials. The size of 4A z particles, based on Fig. 2A,B, is 400 to 600 nm; besides the oxide nanoparticles are very thin and smaller than 50 nm. According to the obtained images (Fig. 2B,C), the dispersion of ZnO and TiO 2 nanoparticles into 4A z structure is uniform and no accumulation has been observed. Also, cubic particles with square layers are present. Figure 2B shows a typical TEM micrograph of ZnO/TiO 2 nanoparticles powder. This powder is formed by 10-50 nm size particles having equiaxed morphology. The particles are well separated from each other. Dark field imaging displayed that each particle was a single crystal. The nanoparticle sizes from TEM examination were in good agreement with both XRD and MAP.  (Table 1). TiO 2 /4A z, ZnO/4A z, and TiO 2 /ZnO/4A z nanocomposite were activated in the concentrations of 1-4 mg/mL against E. coliO157:H7, S. aureus, P. fluorescens, and L. Monocytogenes, separately. These results were in agreement with the outcome of prior studies 57,58 . The best concentrations of MIC were selected for TiO 2 /4A z, ZnO/4A z, and TiO 2 , ZnO/4A z nanocomposite as 2, 1 and 1 mg/mL, respectively; against microorganisms (Table 1). This concentration in MBC corresponded to 2, 2 and 2 mg/mL, respectively; and 0.25 mg/ ml concentration of TiO 2 , ZnO/4A z nanocomposite against E. coli O 157 :H 7 , had an excellent FIC value (Table 3). Based on the results, gram negative bacteria were more sensitive compared to antimicrobial agents. Gram positive bacteria have a thick layer of peptidoglycan in their cell walls which have caused more resistance compared to gram negative bacteria against antimicrobial agent. Gram negative bacteria contains a thin peptidoglycan layer which facilitates the mobility of metal ion nanoparticles to the cell, and it also assists the interaction between nanoparticle and bacterial cell walls due to lack of thick peptidoglycan layer. Another aspect of gram negative www.nature.com/scientificreports www.nature.com/scientificreports/ bacteria is the negative charge of lipopolysaccharide layer. This charge has a role as a significant incorporation factor for positive ions, which consequently leads to nanoparticle emission, intracellular damages and destruction of DNA and proteins (Fig. 4).      www.nature.com/scientificreports www.nature.com/scientificreports/ Antibacterial activity of nanoparticles doped in 4A zeolite. The disc diffusion procedure was conducted to assess the antimicrobial activity of nanoparticles doped in 4A z (Table 4). Inhibition zone diameter indicated that TiO 2 , ZnO/4A z had a greater antimicrobial effect against bacteria compared to the combination of individual nanoparticles with 4A z (Fig. 3). Also it should be noted that, 4A z individually do not have an antimicrobial activity against bacteria 56 . Based on the results, Gram-negative bacteria (particularly E. coli O 157 H 7 ) were more sensitive to the antimicrobial agents. The main purpose of using 4A z, as a carrier with nanoparticles, is to control the release of them and increase the antimicrobial activity. The highest zone of inhibition was seen against E. coli O 157 H 7 including ; 6.86 ± 0.03 mm, 9.13 ± 0.03 mm, and 10.73 ± 0.04 mm for ZnO/4A z, TiO 2 /4A z and ZnO and TiO 2 /4A z, respectively; while S. aureus had the lowest inhibitory zone in all treatments (P < 0.05). Indeed, nanoparticles disturb the permeability of both the cell wall and cell membrane, thereby affect biomolecules such as DNA and protein, and prevent processes such as DNA replication and protein synthesis 59 .
Titanium dioxide (TiO 2 ), is also called titania, is one of the most widely used semiconductor nanoparticles with specific hydrophilic and photocatalytic properties, which caused to antimicrobial and ultraviolet (UV) protecting characteristics 51,60 . These nanoparticles are extensively used in producing polymer nanocomposites in food packaging. TiO 2 nanoparticle works in two phases: first, the ability to degrade bio polymeric compound (such as polysaccharides and proteins) 61 on its surface and second, to alter the surface properties of the objects to hydrophilic state when TiO 2 is placed on that surface. In Europe, TiO 2 (E171) is confirmed and categorized as a color additive in confectionaries, dairy products, and soft drinks under Directive of 1994/36/EC 62 . TiO 2 is more prone to oxidization when exposed to ultraviolet (UV) in a wavelength lower than 385 nm. As a result, TiO 2 can produce active oxygen species when exposed to sunlight 63 . Furthermore, the antimicrobial activity of TiO 2 is related to its crystal structure, the kind of artificial light, UVA light intensity, shape and size 64 as well as production of ROS, active radical species, hydrogen peroxide, superoxide radical, and hydroxyl radical [64][65][66] . These active species destroy the outer membrane of the bacteria, namely phospholipids, proteins and lipopolysaccharides and finally damage the bacteria.
ZnO is recognized as a colorless, wide band gap semiconductor with an optical band gap in UV region which makes it applicable as an impressive absorbent of UV radiation. Currently, ZnO is listed as generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA) and is consumed as a food additive, considering the fact that zinc is an important trace element in nutrition 67 . The antimicrobial activities and potential applications of ZnO nanoparticles in food preservation have been approved 68 . In this regard, ZnO nanoparticles were combined in polymeric matrix in order to supply the packaging material with antimicrobial activity and to improve some packaging properties 69,70 . Furthermore, the antimicrobial properties of ZnO is related to photocatalytic activity of H 2 O 2 . Both Zn +2 and ZnO particles hold antibacterial activities. The antimicrobial activities of ZnO at nanoscale would yield affordable and safe innovative strategies 69,71 . Moreover, ZnO has been blended into the linings of food cans used for meat, fish, corn, and peas to preserve colors and hinder spoilage because of its antimicrobial action.
Indeed nanoparticles have the ability to decline or remove the microorganism resistance by two mechanisms: (a) free metal ion toxicity arising from dissolution of metals from the surface of nanoparticles and (b) oxidative stress via generation of reactive oxygen species (ROS) using hydrogen peroxide (H 2 O 2 ) and organic hydro peroxides (OHP) on the surface of nanoparticles 72 . Actually, nanoparticle can effect on the survival of microorganisms by agglomeration on the surface of bacteria and alter the structure of lipids, peptidoglycan, proteins and their DNA 73 . But there may be differences in the impact of nanoparticles on types of specific microorganisms; for instance, Sierra et al. 74 showed a higher antimicrobial effect against Streptococcus mutans of Cu nanoparticles at lower concentrations than zinc. In another study, Ruparelia et al. 75 showed that copper nanoparticle has great impact as an antimicrobial agent against E. coli, Bacillus subtilis, and Staphylococcus aureus in comparison with TiO 2 . Our results are in agreement with those of Lou et al. 59 and Yael N et al. 76 .
Inductively coupled plasma mass spectroscopy. Migration is a mass transfer process where low molecular mass particles are released into the surroundings. To assess migration, the system must be simplified and particles must be analyzed separately. The package is assumed to be homogeneous, the food is substituted by food simulants and the substance is introduced at the recognized concentration. In this research, nanoparticles ion releasing capacity in food simulants of the composites was quantified by ICPMASS.
Zinc and Titania ions are doped into 4A z through ionic exchange, preferably with Na + ions 77 . Next, ions are released from composites through ionic exchange with cations/protons present in food simulants, and their concentration determines the amount of released nanoparticles. Therefore, for applications where a long-term antibacterial activity is expected, nanoparticles zeolites should be placed in solutions with low ionic strength 78 . The immersion of matrix (nanoparticles doped in 4A z) in distilled water (as a food simulant) demonstrated that nanoparticles were sensitive to water. When the matrix was immersed in water for a long period of time, the water molecules interacted with polar groups in nanoparticles were doped in 4A z, which led to the swelling and deformation of the matrix. According to ICP-MS results (  www.nature.com/scientificreports www.nature.com/scientificreports/ ppb, and decreased to 943.3 ppb (ZnO/4A z) after 12 days of immersion in food simulant. These values were 655 and 3.1 ppb for TiO 2 /4A z, respectively; while they were recorded to be 1259 and 344.1 ppb for ZnO and TiO 2 /4A z. Based on the results, the migration values for ZnO, TiO 2 , and ZnO and TiO 2 were approximately 47%, 0.47% and 21%, respectively. It is worth mentioning that ZnO and TiO 2 have been approved by the US Food and Drug Administration (FDA) as safe compounds to be used in food and food contact materials at restricted quantities below 2 and 1% of the food weight for ZnO and TiO 2 nanoparticles, respectively 79,80 . Moreover, low amounts of ZnO and TiO 2 nanoparticles are beneficial for the human body because of their antimicrobial and anticancer properties. ICP-MS was used to detect the metal ions with high sensitivity, and it could detect very low signals of ZnO and TiO 2 nanoparticles from the digested solutions. These results revealed that the slight migration of nanoparticles occurs in food simulant; however,this amount is in accordance with the standard rate by FDA 81 . Our results regarding TiO 2 nanoparticles are in accordance with those of Lian et al. 82 .
conclusions TiO 2 , ZnO and TiO 2 /ZnO nanoparticles supported into 4A z were successfully synthesized through hydrothermal method, and were evaluated for antimicrobial activity for the first time. The size of 4A z particles were around 400 to 600 nm and the average crystallite size of nanoparticles was approximately 50 nm. The XRD, SEM, MAP and TEM analysis demonstrated that crystallographic plate, size, shape and morphology of nanocomposite (nanoparticles with 4A z) are very close to 4A z itself. Moreover, the results of antimicrobial test confirmed a considerable antimicrobial activity against gram positive and negative bacteria of the nanoparticles supported in 4A z. Indeed, the antimicrobial activity of nanoparticles embedded into 4A z was dependent on the type of nanoparticles and on the species of microorganism (gram positive or gram negative). The most sensitive bacteria were P. fluorescens and then E. coli O157:H7. Embedding of nanoparticles in 4A z caused to control the release of them, and enhance their antimicrobial properties. Hence, using these composites can be a promising approach to create new active packaging in food industry.