Harnessing Levilactobacillus brevis as a Microbial Nanofactory for the Sustainable Production of Multifunctional...

Green synthesis of silver nanoparticles (AgNPs) using probiotic microorganisms has emerged as a sustainable strategy for producing multifunctional nanomaterials with enhanced biomedical potential. In the present study, the probiotic bacterium Levilactobacillus brevis, isolated from raw milk and identified by 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing under GenBank accession number PZ476340, was employed as a biological reducing and stabilizing agent for AgNP biosynthesis.

Nanoparticle formation was initially confirmed by a characteristic color change and subsequently characterized using ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and zeta potential analysis. The biological activities of the biosynthesized AgNPs were evaluated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging antioxidant assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, in vitro scratch wound-healing assay, and anticoagulant assays based on prothrombin time (PT) and activated partial thromboplastin time (APTT).

UV-Vis spectroscopy revealed a distinct surface plasmon resonance peak at approximately 410 nm, confirming the formation of AgNPs. FTIR analysis demonstrated the involvement of extracellular proteins and polysaccharides in nanoparticle reduction and surface capping, while TEM and SAED analyses showed predominantly spherical, crystalline nanoparticles with sizes ranging from 7 to 55 nm and an average diameter of 27.18 ± 12.7 nm. Zeta potential analysis yielded a value of -2.57 mV, indicating weak electrostatic stabilization; however, FTIR data suggested that colloidal stability was primarily maintained through steric hindrance provided by a biomolecular capping layer derived from L. brevis metabolites. The nanoparticles exhibited strong concentration-dependent antioxidant activity, achieving 79.7% DPPH radical scavenging at 1000 μg/mL, with a half-maximal inhibitory concentration (IC₅₀) of 61.4 μg/mL. MTT assays demonstrated significant dose-dependent cytotoxicity against Vero normal kidney epithelial, MCF-7 human breast adenocarcinoma, and HepG2 human hepatocellular carcinoma cell lines, with IC₅₀ values of 110.29, 81.28, and 102.64 μg/mL, respectively, accompanied by marked morphological alterations. In vitro scratch assays revealed moderate wound-healing activity, resulting in approximately 34.5% wound closure after 48 h. Furthermore, the AgNPs exhibited anticoagulant activity by prolonging PT and APTT, suggesting interactions with coagulation pathways.

Collectively, these findings demonstrate that L. brevis can serve as an efficient microbial nanofactory for the eco-friendly synthesis of multifunctional AgNPs. The combination of nanoscale dimensions, crystalline structure, and biomolecular surface functionalization contributed to significant antioxidant, cytotoxic, wound-healing, and anticoagulant activities, highlighting the potential of probiotic-mediated nanotechnology for biomedical and therapeutic applications.