# Olive Oil with Ozone-Modified Properties and Its Application
## 2 Results and Discussion
### Preservative Efficacy of Cosmetic Emulsions  
The cosmetic emulsions formulated on the basis of the refined and ozonated olive oils were subjected to the challenge tests [22]. To check the preservative efficacy of the ozonated olive in the formulation, no synthetic preservatives were added. The survival of _E. coli_ and _P. aeruginosa_ was not suppressed during 28 days of incubation in any of the formulations irrespective of the olive oil treatment (Figure 6a,b). No statistically significant differences  
Figure 4: Antimicrobial activity of olive oil measured by the growth inhibition zones in the agar disc diffusion method: refined olive oil (dotted), ozonated olive oil 0.04 mole O\({}_{3}\)/100 g oil (diagonally); ozonated olive oil 0.10 mole O\({}_{3}\)/100 g oil (striped); the same letters indicate no statistically significant differences (\(p<0.05\)); different letters indicate statistically significant differences (\(p<0.05\)). Figure 5: Homogeneity of emulsions based on different types of olive oil: (**a**) Refined olive oil; (**b**) Ozonated olive oil 0.04 mole O\({}_{3}\)/100 g oil; (**c**) 0.10 mole O\({}_{3}\)/100 g oil; Scanning-Electron Microscope JEOL JCM-6000. were observed among the formulations with olive oils. In the emulsions with the refined oil and the one treated by 0.04 mole O\({}_{3}\)/100 g olive, the number of _S. aureus_ viable cells was gradually declining, reaching the maximum drop by about 3 log units at the end of incubation period (Figure 6c). The preservative activity of the emulsion with olive oil ozonated 0.04 mole O\({}_{3}\)/100 g was the strongest within two weeks of incubation, reducing the staphylococci level by 2 logarithmic units CFU. All the tested emulsions promoted the growth of _C. albicans_ by 0.4-1.06 logarithmic units CFU up to 14 days (Figure 6d). After the next two weeks, the number of viable yeasts decreased by 1.9 logarithmic units in the emulsion with the olive oil treated by 0.10 mole O\({}_{3}\)/100 g. All the emulsion formulations enabled the growth of moulds _A. brasiliensis_ (increase by 1.5-1.9 logarithmic units CFU) after 21 days of incubation. Extending the incubation time for the next 7 days resulted in the inhibition of the moulds growth by 1.9 log units in the emulsion with olive ozonated with 0.10 mole O\({}_{3}\)/100 g only (Figure 6e). Figure 6: Inhibition of microorganisms growth in cosmetic emulsions with refined olive oil (continued line), ozonated olive oil 0.04 mole O\({}_{3}\)/100 g oil (discontinued line), ozonated olive oil 0.10 mole O\({}_{3}\)/100 g oil (dotted line): (**a**) _E. coli_; (**b**) _P. aeruginosa_; (**c**) _S. aureus_; (**d**) _C. albicans_; (**e**) _A. brasiliensis_; * marks statistically significant differences (\(p<0.05\)). The literature data of preservative efficacy of cosmetics containing olive oil are scarce and primarily focussing on the formulation's biophysical and microbiological stability [23]. A variety of beneficial actions of olive oil topically applied on a skin were shown in in vivo experiments, and the researchers are rather focusing on the cosmetics' cutaneous effects [24; 25; 26]. However, the olea ointment with the olive oil as one of the main compounds was proved to prevent burns infections [27], indicating its antibacterial activity. Although the emulsions tested by us did not fulfil either A or B criteria for cosmetics' preservative efficacy, the emulsion with olive ozonated by 0.04 mole O\({}_{3}\)/100 g expressed vivid anti-staphylococcal activity. This finding is in agreement with the antimicrobial activity tests of olive oils, where the 0.04 mole O\({}_{3}\)/100 g ozonated olive oil showed the statistically significant (\(p<0.05\)) increase in anti-staphylococcal activity compared to the other olive specimens. The weak inhibitory effect against C. _albicans_ and _A. brasiliensis_ was also demonstrated for the emulsion with the addition of olive oil with the highest dose of ozone.