Antifungal Effects of Essential Oils of Zataria multiflora, Mentha pulegium, and Mentha piperita

Background: Among important fungi associated with foods are Aspergillus spp., Penicillium spp., and Geotrichum spp. In this study, we evaluated antifungal effects of Essential Oils (EOs) of Zataria multiflora, Mentha pulegium, and Mentha piperita. Methods: Antifungal properties of EOs of M. piperita, M. pulegium, and Z. multiflora against Aspergillus spp., Penicillium spp., and Geotrichum candidum were determined by agar well diffusion and broth macrodilution method. Data were analyzed by SPSS 20. Results: Among three studied plant EOs, Z. multiflora EO had the strongest antifungal activity (p<0.05) on tested fungi; so that the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) were 0.01 and 0.3% for G. candidum, 0.005 and 0.3% for Penicillium spp., and 0.1 and 0.3% for Aspergillus spp. Conclusion: All three studied plant EOs showed antifungal activities. However, as Z. multiflora EO showed the most antifungal effect, it could be specially suggested as natural powerful antifungal preservatives in the food industry. © 2021, Shahid Sadoughi University of Medical Sciences. This is an open access article under the Creative Commons Attribution 4.0 International License.

Some fungi associated with foods, such as Aspergillus spp., Penicillium spp., and Geotrichum spp., may cause food spoilage and diseases (Derek and Gary, 2014). Control of fungi in foods is usually done using synthetic chemical additives, but some synthetic preservatives may have mutagenic, carcinogenic, and allergic effects. Thus, in recent years, researches on using natural compounds, especially Essential Oils (EOs) of the plants have been focused to prevent fungal growth and spoilage in foods in vitro study, we evaluated antifungal effects of EOs of Z. multiflora, M. pulegium, and M. piperita.

EOs
EOs of M. piperita, M. pulegium, and Z. multiflora used in this study were obtained from Barij ® Essence Pharmaceutical Company (Kashan, Iran). The dilutions of EOs and itraconazole were prepared using dimethyl sulfoxide (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Organisms
The tested fungi included Aspergillus spp., Penicillium spp., Geotrichum candidum that were isolated from cheese culture. Ten g of each cheese sample was homogenized using a stomacher 400 with 90 ml of sterile physiological saline for at least 2 min. Decimal dilutions of the homogenized samples were prepared in sterile physiological saline and were plated in duplicates onto Potato Dextrose Agar (PDA; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) plates and were incubated at 30 ˚C for 3-7 days. After that, the plates were observed for the presence of mold colonies. The fungal isolates were identified and purified using a combination of macroscopic observation and microscopic Tease mount test (Reiss et al., 2012). Isolated strains were cultured on plates and incubated at 30 ˚C for 7 days. The 7 day old culture slants were flooded with 5 ml of 0.01% tween 80 ® (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with a sterile Pasteur pipette and shaken vigorously to give inoculums; then, the conidia was counted with a haemocytometer and diluted in sterilized distilled water to correspond to a final inoculums concentration of 2.4×10 6 spores/ml.

Determination of antifungal effects -Agar well diffusion method
After mixing a solution of fungi, 0.1 ml of fungal suspension at a concentration of 2.4×10 4 Colony Forming Unit (CFU)/ml was transferred to PDA plates and was spread using a sterile bent glass rod. At the center of plates, 6 mm wells by sterile cork borer were made and 50 μl of different concentrations of EOs were poured into each well. The plates were incubated at 30 ˚C for 3-7 days. The inhibition zone diameter (mm) was measured and as the control experiments were used itraconazole and dimethyl sulfoxide 5% into wells instead of EOs. The experiment was repeated in triplicate and an average value was reported (Fontenelle et al., 2007).

-Broth macrodilution method
A completely synthetic medium of RPMI-1640 ® containing glutamine and pH indicator (Sigma, Germany) was provided and buffered with MOPS ® (Sigma, Germany). The various concentrations of each EO were induced in broth medium, and then fungal inoculums (2.4×10 6 CFU/ml) were added to all tubes. After shaking, for determination of Minimum Inhibitory Concentration (MIC), all tubes were incubated at 30 ˚C for 3-7 days and readings were taken when the turbidity of the growth control tube was evident. For determining Minimum Fungicidal Concentration (MFC), a bent glass rod was used to spread 0.1 ml of each MIC tube showed complete inhibition on the plates. The plates were incubated at 30 ˚C for 3-7 days and were observed daily for the presence of fungal colonies.

Statistical analyses
The data were analyzed by SPSS 20 software using descriptively for MIC and MFC and, one way ANOVA for comparing the inhibition diameter zones. P<0.05 was considered as a significant differences among groups.

Results and discussion
Among three studied plant EOs, Z. multiflora EO showed the strongest antifungal activity (p<0.05) on tested fungi by macrodilution method (Table 1); so that the MIC and MFC were 0.01 and 0.3% for G. candidum, 0.005 and 0.3% for Penicillium spp., and 0.1 and 0.3% for Aspergillus spp.
By increasing the concentration of EOs, the diameter of the inhibitory zone was also increased in agar well diffusion method (Tables 2-4). Z. multiflora EOs had the most antifungal activity (p<0.05) against tested fungi; and the inhibition zone for Aspergillus spp., Penicillium spp., and G. candidum were 11.6, 5.7, and 7.1 mm, respectively.
Fungi are of the important agents of food spoilage due to the production of different enzymes and break down the nutrients (Ledenbach and Marshall, 2009). In this study, the antifungal effect of M. piperita, M. pulegium, and Z. multiflora on the growth of Aspergillus spp., Penicillium spp., and G. candidum were determined. The results of the present study showed that all the tested plants EOs had the acceptable antifungal activities on tested fungi. However, the most antifungal activity was recorded for Z. multiflora EOs.
The findings of this study are close to that of Gandomi et al. (2009) Saharkhiz et al. 2012;Sokovic et al. 2009;Verma et al. 2011). Hammer et al. (1999 reported that the antifungal activity of thyme (Thymus vulgaris) EO was stronger than the peppermint oil which is in agreement with the results of the present study. Similar findings were also reported by Ameziane et al. (2007) who showed perfect antifungal activity of M. pulegium against three fungal species, including Penicillium digitatum, Penicillium italicum, and G. candidum.

Conclusion
All EOs of Z. multiflora, M. pulegium, and M. piperita showed antifungal activities. However, as Z. multiflora EO showed the most antifungal effect, it could be specially suggested as natural powerful antifungal preservatives in food industry.

Author contributions
M.B. and H.M. designed the study; Z.Z.C. and A.E. conducted the experimental work; M.B. and A.E. analyzed the data; Z.Z.C. and M.B. wrote the manuscript. All authors revised and approved the final manuscript.

Conflicts of interest
There is no conflict of interest in the study.