Green Synthesis of Zinc Oxide Nanoparticles Mediated By Lacticaseibacillus Rhamnosus: Evaluation of Their Antioxidant and Hemolytic Activities

Using probiotics to biosynthesize metal oxide nanoparticles is a new, more biocompatible, and environmentally harmless approach compared to traditional physical and chemical synthesis methods. In the current study, we isolated the probiotic Lacticaseibacillus rhamnosus from local dairy products. Instead of directly using the living cells of this bacterium, we extracted its cell-free extracellular supernatant, the clear liquid remaining in the upper layer of the culture medium after the bacterium’s metabolism, and successfully synthesized zinc oxide nanoparticles (ZnO-NPs). The first sign that confirmed the successful synthesis was a distinct color change visible to the naked eye. After preliminary confirmation that the synthesis was complete, we conducted a comprehensive and rigorous set of tests on the physical and chemical properties of these nanoparticles. After observation using Field Emission Scanning Electron Microscopy (FE-SEM), it was found that the synthesized nanoparticles were nearly spherical, with an average diameter of 41.5 nanometers, and the size of all particles fell within the range of 27 to 56 nanometers. The XRD analysis confirmed the crystalline nature of the biosynthesized ZnO nanoparticles, with an average crystallite size of 22.13 nm calculated by Scherrer’s equation. FTIR spectrum showed a characteristic Zn–O stretching vibration at 563 cm⁻¹ and the bands observed in the range of 1448–1580 cm⁻¹ were attributed to carboxylate and amide II groups, indicating the possible involvement of bacterial biomolecules in the capping of nanoparticles. AFM images showed the vertical height values from −0.726 nm to 0.920 nm and the peak to valley height difference of 1.646 nm. The nanoparticles showed strong DPPH radical scavenging activity, and it was found to be concentration-dependent, with 91.89% inhibition at 1000 µg/mL. Moreover, in vitro hemolysis assays with human erythrocytes showed 0% hemolysis at concentrations of 125–500 µg/mL and 1.60% at 1000 µg/mL, indicating low hemolytic activity under the tested in vitro conditions. The present results suggest that the synthesized ZnO nanoparticles derived from L. rhamnosus could be potential potent and non-toxic candidates for biomedical and pharmaceutical applications.