Nanotechnology is becoming increasingly popular in pharmaceuticals and cosmetics and is important as an approach to killing or reducing the activity of many microorganisms. Natural antibacterial materials, such as zinc and silver, are said to have good antibacterial properties. Nano-sized particles of zinc oxide have been confirmed to have more pronounced antibacterial activity than large particles. Antibacterial/antifungal activity of ZnO nano zinc oxide against five pathogens (Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160, Bacillus subtilis MTCC 441, Aspergillus niger MTCC 281, Candida albicans MTCC 227) and the effect of size The granules of this inorganic powder on its antibacterial/antifungal efficacy were examined in this study.
Nanotechnology is being envisioned as a burgeoning field that has the potential to revolutionize pharmaceuticals and cosmetics. Nanotechnology, or the use of materials with compositional dimensions at the atomic or molecular scale, is increasingly being applied to pharmaceuticals and cosmetics and is of great interest as an approach to kill or reduce the activity of many microorganisms. Some natural antibacterial materials, such as zinc and silver, are said to have good antibacterial properties. Microbial spoilage in cosmetic formulations has always been of particular concern to the cosmetic industry. The use of preservatives permitted by the new regulation 1123/209 is required to prevent product spoilage caused by microorganisms and to protect the product from unwanted contamination by consumers. throughout the shelf life. However, since several years now, the cosmetic industry is facing a number of restrictions regarding the use of certain preservatives. Therefore, there has been considerable interest in the development of new preservatives. Among the raw materials that exhibit antibacterial/antifungal properties, inorganic powders such as zinc oxide (ZnO) are a promising alternative to these chemical preservatives [1, 2]. Zinc oxide is a non-toxic II-VI semiconductor with a wide band gap (3.37eV) and a natural n-type conductivity [3, 4]. Zinc oxide because of its interesting properties, such as optical transparency, electrical conductivity, piezoelectricity, near ultraviolet emission [5, 6, 7, 8, 9, 10] and morphologies various, has become one of the most attractive nanomaterials for research purposes. Its important properties have made it applicable in UV emitters, variants, transparent high power electronics, surface acoustic wave devices, voltage transducers, gas sensors, etc .
The use of zinc oxide in cosmetic creams and gels renders them sun-protective and antibacterial . The effectiveness of their activity largely depends not only on the concentration of the active substance, zinc oxide, but also on the size of its particles, their variability and the degree of polychromatic dispersion. Furthermore, zinc oxide (ZnO) is listed by the US Food and Drug Administration as “generally recognized as safe” (GRAS) (21CFR182.8991). Nanoscale ZnO particles have been confirmed to have more pronounced antibacterial activity than large particles, considering the fact that the small size (below 100 nm) and high surface volume ratio of the nanoparticles can may allow better interactions with bacteria. Recent studies have shown that these nanoparticles are selectively toxic to bacteria but exhibit minimal effects on human cells [1, 13, 14, 15, 16, 17, 18, 19 ]. Even if ZnO has been used for a long time in cosmetics or pharmaceutical ointments, its antibacterial properties have not been fully studied in the context of cosmetic preservation. A systematic and detailed study was designed, taking into account the above claims, to investigate the enhanced antibacterial and antifungal properties of nano zinc oxide compared with conventional research. Therefore, the aim of this paper was first and foremost to demonstrate the antibacterial/antifungal potential of ZnO on 5 pathogens (Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160, Bacillus subtilis MTCC 441, Aspergillus niger MTCC 281, Candida albicans) MTCC 227) which is used for challenge tests, and second to determine the effect of the particle size of this inorganic powder on its antibacterial/antifungal efficacy.
Zinc oxide nanoparticles with a diameter of ~65 nm were synthesized and used in this study. Representative TEM images of ~65 nm zinc oxide nanoparticles are shown in Fig. 1. Ordinary zinc oxide nanoparticles with a diameter of ~1000 nm were used for comparison.
Selection of experimental pathogens
The pathogenic microorganisms selected for the study included three bacteria, viz., Escherichia coli (MTCC 443), Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 441) and two fungi, viz., Aspergillus niger (MTCC 281) and Candida albicans (MTCC 227).
Prepare dilutions of synthetic compounds
10 mg of each particle (nano zinc oxide and conventional zinc oxide) was accurately weighed and dissolved in 10 ml of DMSO to give a solution of 1mg/ml concentration. 1 ml of the above solution was again diluted to 10 ml with DMSO to give a solution with a concentration of 100 µg/ml.
Preparation of Agar nutrient medium (for bacteria)
5.6g Agar was dissolved in 150ml distilled water and heated. The medium was sterilized by autoclaving at 115oC for 30 min.
Prepare Sabouraud Dextrose Agar (for mushrooms)
9.20g Sabouraud Dextrose Agar is dissolved in distilled water and heated. The medium was sterilized by autoclaving at 115oC for 30 min.
Prepare the nutrient broth
0.75 g of medium (Bacteria/fungi) was dissolved in 30 ml of distilled water and heated. The medium was then sterilized by autoclaving at 115oC for 30 min.
Preparation of bacterial and fungal skewers
Five Nessler bottles were sterilized by hot sterilization in an oven at 160oC for 30 minutes. Laminar Blower Cabinets are wiped with cotton dipped in ethanol and UV ON for 15 min. The dried bacterial and fungal media were poured into 5 sterilized Nessler cylinders (3 bacteria, 2 fungi) and left in an inclined position until the medium in the cylinders solidified. Rigid ring wire is used to transfer bacterial and fungal strains to smaller cylinders. Then, labeled nessler cylinders and cotton buttons were attached to their mouths and incubated at 37oC minus aspergillus niger (incubated at 25oC) for 24 h. From each strain, a small portion was transferred to 3 ml of separate nutrient broth and incubated at 37 °C for 24 h. 0.1 ml of the five intermediates was transferred into five different conical flasks with lids containing 0.9% NaCl solution.
Antibacterial activity: Determination of minimum inhibitory concentration and minimum bactericidal/fungicide concentration
The minimum inhibitory concentration (MIC) determined for conventional and nano-sized zinc oxide nanoparticles showed antibacterial and antifungal activity against the tested pathogens by serial dilution method. The broth dilution method was performed to determine the MIC value. 1ml of medium was taken into a test tube, to which, 1ml of test solution (100µg/ml) was added. Then, 0.1ml of the microbial strain (bacteria/fungi) prepared in 0.9% NaCl was put into a test tube containing the medium and test solution. Serial dilutions were performed five times for concentrations of 50, 25, 12.5, 6.25, 3.75, 1.5 µg/ml. Cover the test tube with cotton and incubate at 37°C. The incubation time is different for different strains (bacteria/fungi), i.e. 24 hours for bacteria and one week for fungi.
The MIC values were taken as the lowest concentration of particles in the tube that were not cloudy after incubation. Turbidity of the components in a test tube is understood as the visible growth of microorganisms. Minimum bacterial/fungicides (MBC/MFC) concentrations determined by subculture of 50 µl from each test tube showed no obvious growth. The least reagent concentration that showed no visible growth when the subculture was obtained was MBC/MFC.
RESULTS AND DISCUSSION
Nano zinc oxide have been fully characterized. Representative TEM images of ZnO nanoparticles (~65 nm) are shown in figure 1. Second, the antibacterial properties of common ZnO particles and ZnO nanoparticles were investigated. Both the common and nanoparticle particles showed antibacterial activity against Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160 and Bacillus subtilis MTCC 441 with size-dependent effects. Figures 2 & 3 depict the behavior of bacterial populations after incubation with conventional ZnO particles and ZnO nanoparticles for 24 h. The minimum inhibitory concentration of ZnO nanoparticles (as shown in table 1) against
three types of bacteria, viz., Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160 and Bacillus subtilis MTCC 441 were found to be 6.25µg/ml, 6.25µg/ml, 12.5µg/ml, respectively, very low concentrations. than that of common zinc oxide particles (25 g/ml, 12.5 µg/ml, 12.5 µg/ml, respectively). Similarly, the minimum number of bacteria for ZnO nanoparticles in each case was less than for conventional ZnO particles (tables 1 & 2). Figures 4 & 5 depict strong bacterial growth on the plate in the presence of common zinc oxide particles and less bacterial growth on the plate in the presence of ZnO nanoparticles. The difference in sensitivity to the same test substance between these three strains may be due to structural and chemical differences.
their bacterial cell walls . According to a study by Yamamoto et al., 2000 , the presence of reactive oxygen species (ROS) generated by ZnO nanoparticles is responsible for their bactericidal activity. Zhang et al., 2010 , further suggested that the antibacterial behavior of ZnO nanoparticles could be due to chemical interactions between hydrogen peroxide and membrane proteins, or between other chemicals produced when presence of ZnO nanoparticles and the bacterial outer lipid bilayer. The generated hydrogen peroxide penetrates the cell membranes of bacteria and kills them. The study also showed that nano-sized ZnO particles are responsible for inhibiting the growth of bacteria . Furthermore, Padmavathy and Vijayaraghavan, 2008 , showed the bactericidal activity of ZnO nanoparticles. According to their findings, once hydrogen peroxide was generated by ZnO nanoparticles, the nanoparticles remained in contact with the dead bacteria to prevent the bacteria from continuing to function and continuing to generate and release hydrogen peroxide into the environment. . The results of the present study correspond with the results of the above authors, showing that ZnO nanoparticles have excellent antibacterial activity. Zinc oxide also exhibits antifungal activity but to a milder extent than antibacterial as no fungicidal activity has been reported. ZnO nanoparticles showed activity against Aspergilllus Niger and Cadida ablicans at concentrations of 12.5 µg/ml and 6.25 µg/ml, respectively. Again this concentration is higher for conventional ZnO particles, i.e. 25 and 12.5, respectively (table 1). The minimum fungal counts for zinc oxide nanoparticles were found to be the same as the Minimum Inhibitory Concentrations, i.e. 12.5µg/ml and 6.25µg/ml, respectively. Similar is the case for the common ZnO particles in the case of Aspergillus niger, where the MFC (25 µg/ml) is the same as the MIC (25 µg/ml). But the antifungal activity against candida albicans showed a different pattern given that the MFC (25 g/ml) was more than the MIC (12.5 µg/ml). Figures 6 & 7 represent strong fungal growth on the plate in the presence of common zinc oxide particles and less fungal growth on the plate in the presence of ZnO nanoparticles.
The results in this study indicate that nano zinc oxide have strong antibacterial and antifungal activity against selected bacterial and fungal strains compared with conventional zinc oxide particles. In conclusion, the present study suggests that zinc oxide nanoparticles can be an antibacterial and antifungal agent for the treatment of bacterial and fungal infections. In the future, these nanoparticles can replace conventional preservatives in cosmetics. However, the antibacterial/antifungal effects, safety, and detailed mechanisms of zinc oxide nanoparticles need to be further investigated in vitro and in vivo.