Development of a feed-compatible thymol–carvacrol nanoemulsion with enhanced antibacterial activity against avian pathogenic Escherichia coli

The emergence of antimicrobial resistance in poultry production has intensified the search for alternative antimicrobial strategies.Phytogenic compounds such as thymol and carvacrol exhibit promising antibacterial properties; however, their practical application in animal production systems is limited by poor aqueous solubility and instability.This study aimed to develop a feed-compatible thymol-carvacrol nanoemulsion and evaluate its physicochemical properties, antibacterial efficacy, antibiofilm activity, cytocompatibility, and storage stability.The nanoemulsion was prepared using high-speed homogenization followed by ultrasonication with Tween 80 as surfactant.Dynamic light scattering analysis showed that the optimized formulation exhibited a mean particle size of 102 ± 3 nm, polydispersity index (PDI) of 0.18 ± 0.01, zeta potential of -28.6 ± 1.7 mV, and encapsulation efficiency of 93.4 ± 1.9%, indicating a stable nanoscale dispersion.Antibacterial activity against avian pathogenic Escherichia coli (APEC) and Salmonella enterica demonstrated improved efficacy of the nanoemulsion compared with the free compound blend, with MIC values reduced from 125 to 31.25 µg/mL for APEC and from 250 to 62.5 µg/mL for S. enterica.The nanoemulsion also showed strong antibiofilm activity, inhibiting biofilm formation by 77 ± 3%, compared with 42 ± 3% for the free compound mixture.Cytocompatibility analysis revealed cell viability above 90% at antimicrobial concentrations near MIC values (≤50 µg/mL), indicating acceptable biocompatibility.Stability studies demonstrated a gradual increase in particle size during 60 days of storage, ranging from 102 to 120 nm at 25°C and 102 to 112 nm at 4°C, with no evidence of phase separation.These findings indicate that thymol-carvacrol nanoemulsion enhances the antimicrobial performance of phytogenic compounds and may serve as a promising feed-compatible antimicrobial delivery system for poultry production.