Thesis title: Enhancement of Ozonated oil for Veterinary Applications
This doctoral research focuses on the production, characterization, and biological effects of extra virgin olive oil (EOO), with a specific emphasis on its potential applications in veterinary medicine, particularly in the management of bovine mastitis. The therapeutic potential of ozonated EOO has garnered increasing interest due to its broad-spectrum antimicrobial, antifungal, antioxidant, and regenerative properties.
EOO was synthesized through a controlled ozonation process, optimizing reaction parameters to ensure stable ozonide formation while preserving the bioactive constituents of the oil. Analytical assessments, including titration and gas chromatography-mass spectrometry (GC-MS), confirmed a direct correlation between ozonation time and ozonides concentration, highlighting the structural modifications induced by ozone exposure.
Considering the rising prevalence of antibiotic-resistant pathogens in bovine mastitis, this study explored the antimicrobial and antibiofilm properties of EOO against key mastitis-associated bacteria, including Gram-positive (Staphylococcus aureus, Streptococcus agalactiae) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) strains. The results revealed a dose- and time-dependent bactericidal effect, with S. aureus and S. agalactiae exhibiting significant viability reduction at lower concentrations, whereas E. coli and P. aeruginosa required higher doses due to their outer membrane resistance. Notably, EOO effectively inhibited biofilm formation, particularly in S.aureus, by reducing biofilm biomass up to 70% following 24-hour treatment. These findings support the potential of EOO as an alternative therapeutic strategy for mastitis control, potentially reducing the reliance on antibiotics in the dairy industry.
Beyond its antibacterial activity, EOO exhibited potent antifungal effects against Candida albicans and Candida glabrata. The study demonstrated a dose-dependent inhibition of fungal viability and biofilm formation, with over 90% cell mortality in C. albicans at a concentration of 3% (v/v) ozonated oil. Transmission electron microscopy (TEM) revealed substantial structural damage to fungal cell walls, while reactive oxygen species (ROS) levels increased by 60% within 10 minutes of exposure. Upregulation of autophagy-related genes (atg-7 and atg-13) further indicated a stress-induced cell death response, suggesting EOO as a promising antifungal agent, particularly in biofilm-associated infections.
Additionally, EOO demonstrated antioxidant and regenerative properties in eukaryotic cells, reinforcing its therapeutic and cosmetic potential. In 3T3 fibroblasts and HaCaT keratinocytes, EOO enhanced cell viability, mitochondrial function, and resistance to oxidative stress. JC-1 and MitoSOX assays indicated improved mitochondrial membrane potential and reduced superoxide production, while catalase activity assays confirmed its role in mitigating intracellular oxidative stress. Notably, protein carbonylation levels were significantly reduced, further supporting its cytoprotective effects.
These findings establish EOO as a multifunctional bioactive compound with antimicrobial, antifungal, and antioxidant properties, positioning it as a viable candidate for veterinary medical applications. Future research should focus on elucidating its molecular mechanisms and expanding its potential for sustainable therapeutic solutions beyond bovine mastitis.