Curcuma longa nano-emulsions against bovine mastitis inducing bacteria: Machine-learning based optimization and characterization

Abstract This study examined the physicochemical characteristics, antibacterial activity, and anti-biofilm efficacy of Curcuma longa nano-emulsions formulated using a lactic acid–menthol–based hydrophobic deep eutectic solvent as the oil phase and Tween 80–propylene glycol or Tween 20–propylene glycol (1:1, v/v) as surfactant systems, designated C. longa -T-80-8 and C. longa -T-20-8, was evaluated. Droplet size, PDI, and zeta potential were determined for both formulations, and their morphological characteristics were analysed using SEM. The C. longa -T-80-8 formulation demonstrated superior physicochemical performance, exhibiting a smaller droplet size (235.2 ± 6.4 nm), lower PDI (0.219 ± 0.008), and greater electrostatic stability (− 36.6 ± 3.7 mV). Antibacterial assays revealed strong inhibitory activity against Staphylococcus aureus (0.1–0.3 / 0.1–0.6% v/v), Staphylococcus haemolyticus (0.1–0.6 / 0.3–1.25% v/v), and Staphylococcus chromogenes (0.1–1.25 / 0.3–2.5% v/v) isolated from bovine mastitis, with C. longa -T-80-8 exhibiting consistently lower MIC and MBC values. Both nano-emulsions significantly inhibited biofilm formation and effectively eradicated established biofilms. At 32× MIC, C. longa-T-80-8 achieved up to 92.2%, 90.9%, and 90.5% reduction in mature biofilms of S. aureus , S. haemolyticus , and S. chromogenes , respectively. A significant negative correlation between MIC values and biofilm inhibition/eradication indicated a close association between antibacterial potency and antibiofilm efficacy. Furthermore, Random Forest–based machine-learning models provided data-driven validation of the experimental findings, demonstrating predictive performance for growth inhibition, biofilm formation, and bacterial viability, and identifying dose and formulation type as the primary determinants of antimicrobial efficacy. Overall, the Tween 80-based nano-emulsion exhibited superior stability and biological activity, highlighting its potential as a natural alternative to conventional antibiotics for bovine mastitis management. However, the underlying mechanisms of antimicrobial action were not directly investigated in this study and require further validation.