Abstract: Mentha spicata essential oil was subjected to gas chromatography-mass spectrometer (GC-MS) analysis and its
major components were determined as piperitenone (39.74%) and limonene (32.56%). A Mentha spicata oil-organic salt
microemulsion system was then formulated. It was found that sodium ascorbate and sodium sorbate showed no significant
effect on increasing the microemulsion area as indicated by the pseudo-ternary phase diagram. Minimum inhibitory
concentration (MIC), minimum bactericidal/fungicidal concentration (MBC/MFC) and bactericidal kinetics were investigated
to assess the antimicrobial activities of Mentha spicata oil microemulsion (ME) against Escherichia coli, Staphylococcus
aureus, Saccharomyces cerevisiae and Saccharomyces baillii. The antimicrobial mechanism was explored in terms of cell
morphological change and membrane integrity by scanning electron microscope (SEM) and measurement of optical density
at 260 nm (OD260 nm), respectively. ME showed antimicrobial activities against all tested strains, with MIC values ranging
from 8 to 32 μL/mL, and MBC/MFC values ranging from 32 to 128 μL/mL. Time-killing curves revealed that a complete
loss of viability was achieved in E. coli and S. aureus after treated with minimal bactericidal concentrations of ME for 60
and 90 min, respectively. Furthermore, leakage of nucleic acid and cell damage were observed in ME-treated E. coli. These
results suggest that ME inhibits E. coli by acting on their cell membrane to cause damage to the cell membrane, release of
intracellular materials, and changes in the cell membrane structure and function.

Key words: Mentha spicata oil, microemulsion, antimicrobial, mechanism of action