Extracellular biosynthesis and characterization of zinc oxide and nisin-loaded zinc oxide nanoparticles using Bacillus subtilis ZBP4

In recent years, the biosynthesis of zinc oxide nanoparticles (ZnO NPs) is gaining considerable interest as an alternative to chemical and physical routes, due to its cheap, environmentally friendly, and large-scale manufacturing potential. This study was conducted in the laboratories of the Food Engineering Department at Sakarya University from 1 January 2021 to 1 June 2022. Firstly, the biosynthesis of ZnO NPs was screened using 45 different Bacillus strains previously isolated from soil and food samples. The biosynthesis reactions were performed extracellularly after centrifugation of bacterial culture grown in nutrient broth for 24 h. The best isolate (Bacillus subtilis ZBP4) was selected and the reaction conditions affecting the biosynthesis of ZnO NPs were optimized, including reaction pH (5-9), temperature (30-40°C), ZnSO4.7H2O concentration (2- 10 mM) and incubation time (0-72 hours) and the optimum conditions were determined at 8 mM ZnSO4.7H2O concentration, pH 7.5, 33 oC and 24 h. The color change from white to yellow was used to preliminary examine the production of ZnO NPs, and the sharpe peak was identified to be at 341 nm.The crystalline nature of synthesized ZnO NPs are characterized with UV–VIS spectroscopy. Also, the size and shape of the synthesized NPs were determined by Field Emission Scanning Electron Microscopy (FESEM) and Transmission electron microscopy (TEM), and the NPs displayed a quasi-spherical form with nanoscale of 26 nm. Fourier Transform Infrared Spectroscopy (FTIR) and Energy-dispersive X-ray spectroscopy (EDS) were used to confirm nanoparticle synthesis. The formation of nisin loaded Zinc Oxide nanoparticles (N-ZnO NPs) were verified by using UV-VIS spectroscopy, the sharp peak and high absorbance of the reaction mixture was observed at 341 nm created by the surface plasmon resonance (SPR) of nanoparticles. TEM was used to analyze the size and shape of the N-ZnO NPs and NPs shapes were quasi-spherical with diameters ranging from 14-40 nm. The nanoparticles synthesis were validated by Energy-dispersive X-ray spectroscopy analysis. X-ray diffraction used to identify the natural of NPs, which showed amorphous and alone broad peaks at 2θ angles (2θ = 26.99°). The active groups attributed to presence of protein were detected by FTIR that acted as reducing and stabilizing agents. The zeta potential measurements of NPs exhibited negative surface charges were ˗19.0 and ˗17.7 mV for ZnO and N-ZnO NPs respectively, indicating that the particles are moderately stable. Results showed that N-ZnO NPs synthesized by Bacillus remained stable for 120 days without color change. Kirby-Bauer Disk Diffusion Susceptibility Test was used to assess the antimicrobial activity of ZnO and N-ZnO NPs against Gram-positive (Bacillus cereus, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes ATCC 7644) and Gram-negative (Escherichia coli Type 1, Escherichia coli O157:H7 NCTC 12900, Pseudomonas aeruginosa, Salmonella Enteritidis ATCC 13076 and Salmonella Typhimurium). The ZnO NPs perform well against a variety of food pathogens, including Gram-positive and Gram-negative. Additionally, N-ZnO NPs showed high efficacy against pathogenic bacteria compared to the action of ZnO NPs and free nisin alone; the inhibition zones formed by N-ZnO NPs at 10 mg/mL against Gram-negative bacteria were 16 mm and 15.8 mm for S. Enteritidis ATCC 13076 and P. aeruginosa respectively. In addition, results revealed that the N-ZnO NP has strong bactericidal activity in liquid media against pathogenic bacteria compared with ZnO NPs and free nisin, which killed 94.98 and 96.79 % of the L. monocytogenes ATCC 7644 and S. aureus ATCC 25923 respectively at 150 µg/ mL within 6 hours of treatment. As a result of this study, it was determined that Bacillus subtilis ZBP4, a local isolate, is a microorganism that can be used for biosynthesis ZnO NPs, and synthsized ZnO NPs can be a good antimicrobial agent. In addition, it has been demonstrated that when nisin is loaded to the biosynthesis medium, both the antibacterial and antioxidant properties of the synthesized nanoparticles can be significantly improved.