Nano zinc oxide – zinc supplement to replace zinc salts and zinc chelate for livestock and poultry

Objectives:  This study aims to investigate the antibacterial and cytotoxic effects of nano zinc oxide (ZnO-NPs) in vitro for supplemental application in animal feed.

Materials and methods:  Nano ZnO-NPs were synthesized by wet chemical precipitation method using zinc acetate as the precursor and sodium hydroxide was used to desalinate the precursors. The properties of the synthesized powders were characterized using ultraviolet (UV) visible spectroscopy, Fourier transform infrared (FTIR), scanning electron microscopy (SEM) and ray diffraction. X (XRD), respectively. In vitro antibacterial activities  were analyzed against avian pathogenic bacteria Escherichia coli , Staphylococcus aureus , Klebsiella pneumoniae and Streptococcus aeruginosa. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to analyze the cytotoxic effects of nano zinc oxide ZnO-NPs.

Result:  SEM showed a spherical ZnO-NPs in the range of 70-100 nm. Particle size and sample purity were confirmed by XRD. The nano-sized ZnO-NPs exhibited a maximum UV absorption at 335 nm. In FTIR spectroscopy, the pure ZnO-NPs nanoparticles show stretching oscillations at 4000-5000 cm -1 . ZnO-NPs nano zinc oxide exhibited significant antibacterial activity against bacterial strains E. coli , S. aureus , K. pneumoniae and S. aeruginosa . Cell viability was significantly dose-dependently reduced in cytotoxicity studies.

Conclusion:  From the broad-spectrum antibacterial activity and lower cytotoxicity observed at the prescribed dose, it is concluded that ZnO-NPs nanopowder is a potential alternative zinc supplement for livestock.

(Copyright by NanoCMM Technology)

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introduction

Zinc is the second essential trace element in all living systems from animals to humans, playing an essential role in many metabolic processes of the body [ 1 ]. Daily dietary zinc intake is required to regulate cell division by regulating protein and DNA synthesis [ 2 ]. The two main sources of Zn used in the animal feed industry are ZnO-NPs and ZnSO 4 .H 2 O [ 3 ]. Zinc deficiency in cattle leads to inappropriate growth, reduced feed intake, reduced milk yield, and reduced cycle and conception rates [ 4 , 5 ]. Milk yield was increased when Zn was added in the form of zinc methionine or zinc lysine to cattle [ 6]. The National Research Council recommends 30 ppm (mg/kg) as the dietary requirement of zinc on a dry matter basis for cattle. nano zinc oxide supplementation significantly reduced the number of somatic cells in milk of cows with subclinical mastitis and improved milk production compared with cows supplemented with macroscopic zinc oxide [ 7 , 8 ]. Zinc deficiency in sheep leads to loss of wool, reduced growth rate and improper testicle development [ 9 ], weight loss during lactation, development of skin lesions and excessive salivation [ 10] ]. Increased phytate due to poor intestinal zinc absorption due to improper zinc supplementation leads to prolonged enteritis and dermatological disease [ 11]. Continuous zinc supplementation in the form of zinc sulfate (10 mg/kg/day) or zinc methionate (1.7 mg/kg/day) is usually required for maintenance [ 12 ]. Zinc has effects on disease resistance, cellular immunity, spleen development, and changes in high-density lipoprotein cholesterol in poultry [ 13 , 14 ]. Zinc supplements used for poultry are zinc sulfate or zinc chloride [ 15 ]. Zinc in the form of Zn methionine shows higher bioavailability than zinc from inorganic sources [ 16 ]. The recommended level of zinc in various poultry diets is between 40 and 75 ppm [ 17 ].

Zinc oxide is the most commonly used zinc supplement with high antibacterial, antifungal and growth promoting activity [ 18 ]. Zinc oxide produces hydrogen peroxide that can pass through cell walls, disrupting metabolism and thereby inhibiting microbial growth. The affinity of zinc oxide for bacterial cells is the most important factor for antibacterial activity [ 19 ]. It alleviates zinc deficiency and its results are reduced growth retardation and reduced infertility rates [ 20]. However, the bioavailability of ZnO-NPs could be enhanced by altering the size effect. The reduced size of ZnO-NPs at the nanoscale would enhance bioavailability by increasing zinc ionization. Generally, an organic source of zinc yields good results due to its higher bioavailability in all animal growth and production activities. However, production costs and additional dosages are not sufficient to induce artificial farming in a cost-effective manner. Nano ZnO-NPs can make a positive effect to overcome the zinc deficiency problem with cost-effectiveness and lower dosage ratio.

We hypothesized that the higher bioavailability of nano zinc oxide ZnO-NPs could be readily absorbed from the gut and interfere with subcellular mechanisms. Moreover, the highest antibacterial effect against different bacterial species has been reported in recent works. Nano ZnO-NPs in the feed mix will provide the dual function of a Zn supplement and as an antibacterial agent during feed preservation. To test this hypothesis, we synthesized nano ZnO-NPs by wet chemical precipitation at 80 °C using zinc acetate. Confirmation of the presence, concentration, morphology and size of ZnO-NPs nanoparticles was characterized using ultraviolet (UV), Fourier transform infrared (FTIR) visible spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. The antibacterial effect against  Escherichia coli ,Staphylococcus aureus , Klebsiella pneumoniae , and Streptococcus aeruginosa at an in vitro level was assessed by the antibacterial disc diffusion test. MTT cytotoxicity assay was performed to determine the biocompatibility and cytotoxic effects of nano ZnO-NPs.

Materials and methods

Moral approval

Ethics Committee approval was not required for this study because we conducted the in vitro  trial.

Precipitation method

Zinc oxide nanoparticles were synthesized using zinc acetate as the precursor and sodium hydroxide as the reducing agent. The 0.1 M zinc acetate homogeneous mixture was dissolved in twice distilled water at pH 11 for 2 h with the aid of a magnetic stirrer. Add 0.1 M NaOH solution slowly to zinc acetate solution, stirring continuously. The final solution was stirred for 4 h at pH 7. The final precipitate was filtered with Whatman filter paper No.1 and then the zinc oxide colloid was lyophilized. The powdered zinc oxide nanoparticles were then collected and stored for further processing.

Zn(CH3 COO) 2 + 2NaOH → Zn(OH) 2 + 2CH 3 COONa

Zn(OH) 2 → ZnO-NPs + H 2 O

Characterization

The obtained samples were characterized by powder XRD with CuKα X-ray irradiation (λ = 0.15496 nm). The surface morphology of the sample was detected by SEM (TESCAN, VEGA3 LMU). The composition of the elements was analyzed using FTIR spectroscopy (PerkinElmer Spectrum RX I) and optical absorption spectroscopy of ZnO-NPs nanopowders derived from UV-visible spectroscopy (UV 1800, SHIMADZU).

Antibacterial activity

Bacterial strains such as  E. coli ,  S. aureus ,  K. pneumoniae , and  S. aeruginosa  were purchased from the Microbial Culture Collection, Chandigarh, India. Actively growing test bacterial strains were propagated on four wells made in nutrient agar plates. Zinc oxide solutions with different concentrations such as 50, 100 and 150 µg/ml were loaded into each well, while one well was filled only with broth medium as a control. Then, the plates were incubated at 37 °C for 24-48 h. The antibacterial activity was expressed as the diameter (mm) of the inhibition zone.

Cytotoxicity test

The cytotoxicity assay of the prepared ZnO-NPs nano zinc oxide was measured by the MTT assay. Mouse fibroblasts (L-929) at a density of 1 × 10 6 cellll were pipetted into tissue culture with 12 wells, incubated for 24 h, and treated with different concentrations (50-500 l/ml ) ZnO-NPs nanoparticles. After ZnO-NPs nanoparticle treatment, the medium was changed and the cells were washed twice with (Dulbecco’s Modified Eagle’s Medium/Ham’s 12-nutrient blend) without fetal calf serum for removal. dead cells. Cells were incubated with 200 µl (5 mg/ml) of MTT reagent for 6-7 h at 37 °C in a 5% CO 2 cytotoxic incubator. The MTT tetrazolium salt is converted to colored formazan by mitochondrial dehydrogenases indicating the presence of surviving cells. Color development was measured at 595 nm using a spectrophotometer. In this test, cells without nanoparticles were used as controls. The cell viability is calculated as follows:

Results and Discussion

SEM analysis

The SEM image of the sample is shown in Figure 1.  The SEM image of the ZnO-NPs samples obtained by the precipitation method shows the presence of spherical nanoparticles with minimal agglomeration. A similar structure was observed in ZnO-NPs nanoparticles by Kim and Park [ 21 ], Ong et al . [ 22 ]. A capping agent can be used to reduce the particle size during precipitation. The grain size varies from 70 to 100 nm as observed from the SEM images shown below. Reducing the rate of sodium hydroxide addition with zinc acetate can reduce particle size formation during precipitation.

Figure 1 Scanning electron microscope image of ZnO nanoparticles.

XRD . Research

Figure 2 shows the XRD patterns of the ZnO-NPs samples. Bragg reflections with 2θA 32.18°, 36.78° and 47.54° observed at the (100), (101) and (102) planes confirm the presence of ZnO-NPs nanoparticles. Furthermore, the less intense peaks at the values ​​of 48°, 54°, 57°, 64° and 77° (2θ) indicate the high crystallinity of the ZnO-NPs samples and the high purity of the nanopowder. ZnO-NPs. The crystal sizes of the ZnO-NPs samples were calculated using Scherrer’s formula. The average grain size of the sample obtained by this precipitation method was calculated using the full width at half the maximum of the higher intensity peak corresponding to 101 planes located at 32.18° by Scherrer’s formula. The average crystal size was found to be 74.67 nm. Similarly, the XRD model was reported by Kim and Park [ 21 ], Mohana and Renjanadevi [ 23], and Jenkins and Snyder [ 24 ].

Figure 2 Spectra of ZnO zinc oxide nanoparticles obtained by X-ray diffraction spectroscopy.

FTIR . Spectrum

The FTIR spectra of the synthesized ZnO-NPs nano zinc oxide show (Figure-3) the fundamental vibrational mode at 3410.69 corresponding to OH stretching vibration, 2924.78 corresponding to CH stretching vibration. , and 1377.13 corresponds to the asymmetric stretching oscillation C = O. The peaks 1647.58 and 619.27 correspond to the stretching and strain oscillations of ZnO-NPs. The absorption at 857 cm −1  is due to the formation of a tetrahedral coordination of Zn. The frequencies observed for zinc oxides are in agreement with the documented values [ 25 – 27 ] that reported similar FTIR spectra of zinc oxide nanoparticles in their investigation.

Figure-3 Fourier transform infrared spectrum of ZnO – NPs nanoparticles.

The visible absorption spectrum of UV

UV visible absorption spectroscopy is a commonly used technique to examine the optical properties of nanoparticles. It is evident from Figure 4 that the zinc oxide nanopowder exhibits a strong absorption band at about 335 nm, which is below the 388 nm band wavelength of bulk ZnO-NPs. The excited absorption of powdered ZnO-NPs and bulk ZnO-NPs materials appearing at ~327 nm and ~373 nm have been reported. The excitation peak at 335  Figure 4 is similar to that reported previously [ 28 , 29 ].

Figure 4 Visible ultraviolet absorption of ZnO – NPs nanoparticles.

Antibacterial activity

The antibacterial activity of the control together with ZnO-NPs zinc oxide nanoparticles was studied against pathogenic bacteria such as E. coli , S. aureus , K. pneumoniae and S. aeruginosa .Table 1 states that ZnO -NPs exhibited significant antibacterial activity against tested bacterial strains. It has been demonstrated that nano-sized suspensions of ZnO-NPs are active in inhibiting bacterial growth. In this study, ZnO-NPs nanoparticles were found to have broad-spectrum antibacterial activity. A significant rate of inhibitory effect was observed against the selected bacteria  E. coli , S. aureus , K. pneumoniae  and  S. aeruginosa . It seems that the reactive oxygen species generated by the ZnO-NPs nanoparticles may be responsible for the antimicrobial activity. The antibacterial activity of nano ZnO-NPs against  E. coli has been reported, and the reactive oxygen species generated by nano ZnO-NPs are responsible for inhibiting the growth of bacteria [ 20 , 30 ].

Table 1 Antimicrobial activity of ZnO-NPs nanoparticles.

Cytotoxicity test

The cytotoxic effect of ZnO-NPs nanoparticles was determined using L-929 mouse epithelial cells by MTT assay. The cytotoxicity rate was increased with increasing concentration of ZnO-NPs nanoparticle (Figure-5). Significant cytotoxic effects start at a concentration of 180 µg/ml, while up to 180 µg/ml, the minimum acceptable toxicity level is 30%. Therefore, ZnO-NPs nanoparticles can be used as animal feed supplements at dosage rates up to 180 µg/ml.

Cytotoxicity test

The cytotoxic effect of ZnO-NPs nanoparticles was determined using L-929 mouse epithelial cells by MTT assay. The cytotoxicity rate was increased with increasing concentration of ZnO-NPs nanoparticle (Figure-5). Significant cytotoxic effects start at a concentration of 180 µg/ml, while up to 180 µg/ml, the minimum acceptable toxicity level is 30%. Therefore, ZnO-NPs nanoparticles can be used as animal feed supplements at dosage rates up to 180 µg/ml.

Figure-5 Cytotoxicity of ZnO-NPs zinc oxide nanoparticles on the cell line L-929 (mouse fibroblasts).

conclusion

Larger granular ZnO is not commonly used in animal feed supplements due to its low bioavailability. However, nanomolecular ZnO-NPs may provide a better surface-to-volume ratio for the physiological digestibility of zinc. Therefore, the bioavailability of zinc could be enhanced with the addition of nano ZnO-NPs compared with the larger granular ZnO-NPs or zinc methionine supplements. In this context, ZnO-NPs nanoparticles were synthesized by precipitation method using zinc acetate. SEM analyzes showed that the synthesized ZnO-NPs were spherical in shape with a diameter of 70-100 nm. XRD of the same size and purity of the sample. The nano-sized ZnO-NPs exhibited a maximum UV absorption at 335 nm. In FTIR spectroscopy, the pure ZnO-NPs nanoparticles show stretching vibrations at 4000-5000 cm −1. The antibacterial test demonstrated that the prepared ZnO-NPs could resist the growth of the tested bacteria. The cytotoxicity study showed a lethal dose just above 180 µg/ml. Therefore, the antibacterial activity (150 µg/ml) and the dose level of cell viability (up to 180 µg/ml) were unique in the experiment conducted. Therefore, it is proposed to conduct animal feeds with ZnO-NPs to evaluate their feed value.

Reference source: Nano zinc oxide – An alternate zinc supplement for livestock

K. Geetha,1 M. Chellapandian,2 N. Arulnathan,2 and A. Ramanathan3