Different ratios of CH:TQ (1:0.25, 1:0.5, 1:0.75, and 1:1) were evaluated in order to perform the optimal formulation. For antimicrobial analysis, the NPCH:TQ ratio 1:1 was chosen to perform the assay because this ratio has better loading efficiency values than the other ratios (50.7%) plus similar sizes and PDIs.
The antimicrobial activity and minimum inhibitory concentration (MIC) of terpenes and nanoparticles were tested against the most common pathogenic microorganisms in cosmetics and those that the UNI EN ISO 11930:2012 recommend for preservative efficacy evaluation. Antimicrobial activity of nanoparticles against P. aeruginosa, E. coli, S. aureus, A. brasiliensis, and C. albicans was tested using the broth microdilution method [17 (link),40 (link)]. Stock cultures were prepared from Culti-Loops ™ (Sigma-Aldrich, Madrid, Spain) in Nutrient Broth (NB) and Potato Dextrose Broth (PDB) at 37 °C. Standardized inoculum was then created by dilution in Müller–Hinton medium to a final density of 0.5 McFarland units by densitometer McFarland type DEN-1B (Biosan, Riga, Latvia). TQ, NPCH-TQ, and NPCH were tested in concentrations of 1000 µg/mL to 15.6 µg/mL. Gentamicin (for bacteria) and Tebuconazole (for mold and yeast) were used as standards. After treatment, plates were incubated 24 h at 37 °C for bacteria and 48 h at 30 °C for yeast and fungi.
The antimicrobial activity and minimum inhibitory concentration (MIC) of terpenes and nanoparticles were tested against the most common pathogenic microorganisms in cosmetics and those that the UNI EN ISO 11930:2012 recommend for preservative efficacy evaluation. Antimicrobial activity of nanoparticles against P. aeruginosa, E. coli, S. aureus, A. brasiliensis, and C. albicans was tested using the broth microdilution method [17 (link),40 (link)]. Stock cultures were prepared from Culti-Loops ™ (Sigma-Aldrich, Madrid, Spain) in Nutrient Broth (NB) and Potato Dextrose Broth (PDB) at 37 °C. Standardized inoculum was then created by dilution in Müller–Hinton medium to a final density of 0.5 McFarland units by densitometer McFarland type DEN-1B (Biosan, Riga, Latvia). TQ, NPCH-TQ, and NPCH were tested in concentrations of 1000 µg/mL to 15.6 µg/mL. Gentamicin (for bacteria) and Tebuconazole (for mold and yeast) were used as standards. After treatment, plates were incubated 24 h at 37 °C for bacteria and 48 h at 30 °C for yeast and fungi.