**1. Introduction**

Many microorganisms cause different plant diseases in field and/or postharvest. Without proper treatments, they can cause losses or decrease the shelf life of fruits and vegetables [1,2]. Although synthetic pesticides efficiently control diseases, their application is restricted, particularly postharvest, because of consumer concern for human health conditions, the harmful effects on the environment and the development of new resistant strains [3–5].

There are strict regulations worldwide regarding the minimum pesticide residue levels in the edible portion of the fresh vegetable and fruits for protecting human health and the environment [1,5]. On the other hand, in Europe, synthetic fungicides are prohibited in postharvest applications. For that reason, the discovery of new natural substances, such as plant essential oils (EOs), for controlling phytopathogens, especially in postharvest conditions, has attracted great interest recently. Several research projects reported the antifungal efficacy of plant EOs against postharvest fruit pathogens, being considered natural, safe and biodegradable alternatives [6–9].

*Mentha* × *piperita* L. (peppermint) is a perennial plant that is widespread throughout the Mediterranean region [10]. Peppermint, a plant in the Family *Lamiaceae*, has long been considered an economically important [11]. It was already known in Egyptian, Greek and Roman medicine for its wide benefits for human health, especially for digestive and diuretic problems and as a remedy for coughs and colds [12]. Peppermint has several medicinal uses such as treating stomach-aches, chest pains and for treating irritable bowel

**Citation:** Camele, I.; Grul'ová, D.; Elshafie, H.S. Chemical Composition and Antimicrobial Properties of *Mentha* × *piperita* cv. 'Kristinka' Essential Oil. *Plants* **2021**, *10*, 1567. https://doi.org/10.3390/ plants10081567

Academic Editor: Ki Hyun Kim

Received: 8 July 2021 Accepted: 27 July 2021 Published: 30 July 2021

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syndrome [13]. Peppermint EO can be extracted from the aerial parts of the flowering plant, from dried leaves or from fresh flowers [11,14]. Many studies reported the chemical composition of peppermint EO, which is composed mainly of menthol, menthone, menthofuran, 1,8-cineole, and menthyl acetate [15]. Previous studies revealed that most peppermint EO is rich in pulegone, menthon, menthol, carvone, 1,8-cineole, limonene and β-caryophyllene [16]. Regarding the chemical composition of peppermint EO, some previous studies have analysed its chemical composition and principal single constituents and found that the respective percentage of different peppermint species varied depending upon the origins of the plant, species as well as the possible variation within the same species [16]. In addition, there are some factors, such as physiological and environmental conditions, genetic and evolution that can also determine the chemical variability of peppermint EO [17].

Bibliographic research revealed that the plant EOs from different species of peppermint possess potential antimicrobial activity against different plant pathogens [18] as well as insecticidal activity against stored product [19]. Several researchers have also reported the promising biological activities of peppermint EO against different phyto- and food pathogens, especially against Gram-positive bacteria such as *Staphylococcus aureus* and *Enterococcus faecalis*, as reported by Jirovetz et al. [11]. Researchers at the University of Prešov bred a variety of peppermint with a very high content of the main constituents menthol and menthone [20].

The current study aims to evaluate the in vitro antimicrobial activity of peppermint EO against some common postharvest fungal pathogens and some pathogenic bacteria. This research aims also to study the mode of antimicrobial actions and the minimum fungicidal concentrations, both for the EO and for its main components.

In particular, the main objectives of the current study were to: (i) identify the main components of the *Mentha* × *piperita* cv. 'Kristinka' in the harvest season 2020 cultivated in Prešov, Slovakia; (ii) screen the antifungal effect of the extracted EO against *Monilinia fructicola*, *Aspergillus niger*, *Penicillium expansum* and *Botrytis cinerea*; (iii) evaluate the antibacterial affect against *Clavibacter michiganensis*, *Xanthomonas campestris*, *Pseudomonas savastanoi* and *P. syringae* pv. *phaseolicola*; (iv) study the effect of EO and its two main constituents (menthol an menthone) on the fungal cell membrane permeability (CMP); (v) determine the minimum fungicidal concentration (MFC) of the studied EO and its two main constituents.

#### **2. Results**

#### *2.1. Identification of M. piperita EO Components*

Essential oil of *M.* × *piperita* cv. 'Kristinka' was hydrodistilled and qualitatively analysed using GC-MS for determining the main components as mentioned below. Average amount of EO was 0.4 ± 0.02% from plant materials. Qualitative parameters are summarised in Table 1. The most principal component identified was menthol (70.08 ± 0.05%), followed by menthone (14.49 ± 0.01%). This is the typical characteristic of the new cultivar Kristinka, where menthol is the dominant component with higher quantity. Other dominant components were limonene (4.32 ± 0.03%), menthyl acetate (3.76 ± 0.01%) and *β*-caryophyllene (2.96 ± 0.04%). Oxygenated monoterpenes presented 89.13% of the identified chemical group. Sesquiterpenes hydrocarbons (5.46%) and monoterpenes hydrocarbons (5.26%) followed with the almost the same quantity. Among the different chemical groups, oxygenated sesquiterpenes, such as spathulenol compound, were also present in very low quantities (0.03%).


**Table 1.** Identification of *Mentha* × *piperita* cv. 'Kristinka' EO components.

Percentage was calculated as an average from three replication ± SD; Ki–Kovats index calculated by researchers; Ki lit. Kovats index from literature (MS Finder) for comparison.
