**3. Discussion**

The studied EO in the current study was hydrodistilled from the new cultivar "Kristinka" of *M. piperita*. The parameters of EO differ from the standard ones for *M. piperita.* The cultivar Kristinka was bred and certificated to obtain a higher amount of the main component, menthol [24]. The newly bred cultivar of *M.* × *piperita* is characterised by a higher amount of menthol than found in other commercial cultivars [20,25]. The amount of EO depends on external factors influencing the vegetation season (environmental and climatic conditions) and may vary [25,26]. Plant biodiversity is also represented by different amounts of the main chemical components. This variation present great opportunity for the research to study the different effects of antibacterial activity.

Generally, the explanation of components of EO analysed by the GCMS is by peak area and it is explained in %. In particular, the major components of the EO of *M.* × *piperita* in different publications are menthol, menthone, menthofuran and menthyl acetate in the amounts of about 40, 30, 7 and 10%, respectively, of the whole amount of EO content [2,7,27–29]. Another study conducted by Kamatou et al. [18] on *Mentha canadensi* EO reported that the main components were identified as isomenthone (27.4%), menthol (24.3%), menthone (9.2%), limonene (5.8%), 1,8-cineole (5.6%), menthofuran (4.4%) and isomenthol (3.2%).

Many studies have highlighted the promising antibacterial and antifungal activity of peppermint EO against some human- and phytopathogens such as *Botrytis cinerea*, *Cladosporium cladosporioides*, *Penicillium aurantiogriseum*, *Staphylococcus aureus*, *Streptococcus pyogenes*, *Escherchia coli* and *Klebsiella pneumonia* [2,7,27]. On the other hand, the antimicrobial activity of peppermint EO might be correlated to its chemical composition due to the hydrophobic nature of those above-mentioned compounds, which allows them to interact with microbial membranes causing cell lysis, interrupting the proton's motor force, electron flow and transport activity, and inhibiting protein synthesis [30,31]. Particularly, the obtained results of the current study of the bioactivity of menthol and menthone have confirmed their role in antimicrobial activity, as previously hypothesized.

Regarding the antifungal activity of peppermint EO, Tsao and Zhou [32] concluded that menthol was able to inhibit the postharvest fungi *Botrytis cinerea* and *Monilinia fructicola* [32]. Furthermore, different stereoisomers of menthol were active against *Fusarium verticillioides*, commonly reported as fungal species infecting maize (*Zea mays*) [33]. Tyagi et al. [34] have reported the efficacy of peppermint oil and its vapours against yeasts causing food spoilage in fruit juice such as *Saccharomyces cerevisiae*, *Zycosaccharomyces bailii*, *Aureobasidium pullulans*, *Candida diversa*, *Pichia fermentans*, *Pichia kluyveri*, *Pichia anomala* and *Hansenula polymorpha.*

A recent study conducted by Hsouna et al. [5] underlined the antibacterial activity of peppermint EO against *Agrobacterium tumefaciens*, the causal agent of crown gall disease in over 140 species of eudicots, where the tested concentration 200 mg/mL was able to completely inhibit the formation of tumours on tomato plants when inoculated with *A. tumefaciens* ATCC 23308<sup>T</sup> [5].

In the current research, peppermint EO showed promising antifungal activity against the postharvest tested pathogenic fungi by measuring the growth of mycelium in plates. In addition, the studied EO explicated moderate to acceptable antibacterial activity, especially against *P. syringae* pv. *phaseolicola* and *C. michiganensis*, by measuring the diameter of the inhibition zones compared to the respective positive controls.

The results of the inhibitory effect against some phytopathogens are in agreement with some other important studies, especially those conducted by Afridi et al. [35]. The latter authors have attributed the biological activity of peppermint EO to their ability to penetrate the plasma membranes and cell walls of fungal cells, increasing their permeability, causing a significant decomposition of the walls, and later leading to the death of the fungal cells [32]. The last interpretation is what we tried to clarify through this research by conducting the CMP assays of cell membranes and their rate of electrical conductivity in the broth culture media of the tested fungi [36,37]. This, in turn, gave a clear indication of a change in the normal rate of permeability of the cell wall compared to the control cells due to the influence of biological oil as well as mono active compounds.

Consistently with this interpretive context, Ultee et al. [38] have also attributed the promising biological activity of peppermint EO to its rich content of menthol and its related compounds. These compounds can destabilize the cytoplasmic membrane and act as a proton exchanger to reduce pH gradient. Therefore, this action can destroy the proton motive force cause the depletion of ATP and hence increasing the possibility of cell death.
