In Vitro Antimicrobial Activity of Lavender, Mint, and Rosemary Essential Oils and the Effect of Their Vapours on Growth of Penicillium spp. in a Bread Model System

The chemical composition, antioxidant activity, and antimicrobial properties of three commercially available essential oils: rosemary (REO), lavender (LEO), and mint (MEO), were determined in the current study. Our data revealed that the major components of REO, MEO, and LEO were 1,8-cineole (40.4%), menthol (40.1%), and linalool acetate (35.0%), respectively. The highest DPPH radical-scavenging activity was identified in MEO (36.85 ± 0.49%) among the investigated EOs. Regarding antimicrobial activities, we found that LEO had the strongest inhibitory efficiencies against the growth of Pseudomonas aeruginosa and Candida (C.) tropicalis, MEO against Salmonella (S.) enterica, and REO against Staphylococcus (S.) aureus. The strongest antifungal activity was displayed by mint EO, which totally inhibited the growth of Penicillium (P.) expansum and P. crustosum in all concentrations; the growth of P. citrinum was completely suppressed only by the lowest MEO concentration. The lowest minimal inhibitory concentrations (MICs) against S. enterica, S. aureus, and C. krusei were assessed for MEO. In situ analysis on the bread model showed that 125 µL/L of REO exhibited the lowest mycelial growth inhibition (MGI) of P. citrinum, and 500 µL/L of MEO caused the highest MGI of P. crustosum. Our results allow us to make conclusion that the analysed EOs have promising potential for use as innovative agents in the storage of bakery products in order to extend their shelf-life.


Introduction
Bread is an important staple food worldwide. However, its fungal spoilage during storage is a serious problem that can result not only in economic losses, but also in human health hazards because of the presence of mycotoxins [1]. In general, bread rot is caused by microscopic fungi, such as Penicillium and Aspergillus, as well as Mucor, Cladosporium, Fusarium, and Rhizopus [2]. One of the potential alternatives to prevent the spoilage of bakery goods appears to be the application of essential oils (EOs) as natural preservatives [3].
EOs are volatile secondary metabolites derived from plants responsible for their typical smell and taste. They can be obtained from about 17,500 angiosperm plants (e.g., Rutaceae, Lamiaceae, Zingiberaceae, Myrtaceae, Asteraceae), and among them, only approximately 300 species of EOs are commercially available [4]. These highly concentrated

Results
The vapour-phase antifungal activities of three selected EOs obtained from rosemary, lavender, and mint against Penicillium spp. inoculated on bread samples were evaluated in the current study. The data expand our findings related to bread preservation using natural alternatives, such as EOs [20,21].
The strongest antimicrobial activity of REO was shown to be against S. aureus, with an inhibition zone of 10.33 ± 0.58 mm, which significantly differed from those of MEO and LEO. Additionally, the REO activities against S. enterica and C. tropicalis were considerably (p < 0.05) higher as compared to LEO and MEO, respectively.  Data from the inhibitory effects of the analysed EOs against three tested Penicillium (P.) spp. fungi (P. crustosum, P. citrinum, P. expansum) are shown in Table 4. Our results revealed that the growth inhibition of fungi strains depends on the type and concentration of the EO used. Remarkable antifungal activity was observed for the MEO among all investigated EOs, which completely inhibited the growth of P. crustosum and P. expansum in all used concentrations (125, 250, and 500 µL/L). The growth of P. citrinum was also totally inhibited by MEO in a concentration of 125 µL/L, whereas it showed significantly (p < 0.05) different zones of inhibition (6.67 ± 0.58 mm; 9.00 ± 1.00 mm, respectively) in the 250 and 500 µL/L concentrations. On the other hand, LEO (125 and 250 µL/L) and REO (in all concentrations) displayed no inhibitory impact on the growth of P. crustosum, and P. citrinum and P. expansum were significantly (p < 0.05) inhibited by the EOs in the highest concentrations.

Minimum Inhibitory Concentrations of EOs against Gram-Negative and Gram-Positive Bacteria, and Yeasts
The MIC values of tested EOs against Gram-negative and Gram-positive bacteria and yeasts are represented in Tables 5 and 6. The EOs displayed a variable degree of inhibition activity against the different tested strains, with significant differences (p < 0.05) among the analysed EOs, as well as the microorganisms that were used. LEO had the lowest MICs against C. albicans regarding the effectiveness of selected EOs, whilst EO was the most effective against S. enterica. MEO exhibited the weakest action against the growth of P. aeruginosa, and also against C. glabrata and C. albicans. On the other hand, MEO was the most effective against S. enterica and C. krusei. Finally, REO had weak antimicrobial activity against C. albicans and a stronger effect against S. enterica.

Moisture Content and Water Activity of Bread Samples
The results from the moisture content (MC) and water activity (a w ) measurements showed that the parameters of bread analysed in our study had values of 41.65 ± 0.55% and 0.944 ± 0.001, respectively.

Discussion
It is generally known that the antibacterial effects of EOs depend on their chemical composition [22], which can be influenced by various factors, such as the plant developmental state, the plant part used for extraction, plant geographical location, and physical and chemical characteristics of the soil and climate in question [23].
Mentha EOs consist mainly of oxygenated monoterpenes as a major fraction [29]. Soković et al. [30] determined in the EO from Mentha piperita, that the most abundant substances were menthol (37.4%), menthyl acetate (17.4%), and menthone (12.7%). The chemical composition of the EO from M. piperita during two seasons (summer and winter) was analysed in the study by Hussain et al. [29]. The authors found that the main components

Discussion
It is generally known that the antibacterial effects of EOs depend on their chemical composition [22], which can be influenced by various factors, such as the plant developmental state, the plant part used for extraction, plant geographical location, and physical and chemical characteristics of the soil and climate in question [23].
Mentha EOs consist mainly of oxygenated monoterpenes as a major fraction [29]. Soković et al. [30] determined in the EO from Mentha piperita, that the most abundant substances were menthol (37.4%), menthyl acetate (17.4%), and menthone (12.7%). The chemical composition of the EO from M. piperita during two seasons (summer and winter) was analysed in the study by Hussain et al. [29]. The authors found that the main components of the EOs collected during summer and winter were menthone (28.13% and 25.54%), menthyl acetate (9.51% and 9.68%), limonene (7.58% and 7.73%), and isomenthone (4.04% and 7.63%), respectively. However, the content of the compounds (menthone 16.8%, menthyl acetate 9.1%, menthol 40.1%, limonene 1.8%, and isomenthone 2.8%) was different in our study, indicating that the aforementioned factors might contribute to the discrepancies in the chemical composition of the MEO analysed in the three studies. We assume that a lower content of methyl acetate in our MEO as compared to that by Soković et al. [30] may be connected with its higher antifungal activity since it was found that this compound causes a decrease in the antifungal properties of the EOs [28].
As mentioned above, EOs are especially known for their variable range of biological functions, including an antioxidant purpose [36]. The antioxidant ability is dependent on compounds that protect the biological system against the deleterious influences of processes causing excessive oxidation exponentiation of reactive oxygen forms [37]. The DPPH, i.e., stable free radical, is a compound often used in methods for determining antioxidants' free radical scavenging activities [38].
The antioxidant activity of EOs may vary depending on their chemical composition [39]. Our results revealed that the effect of the MEO was stronger as compared to other analysed EOs. This fact may be related to the presence of individual volatile compounds, especially menthol and menthone, containing the hydroxyl radical (-OH), which improves the antioxidant activity strength [40]. We assume based on the data obtained from recent studies that other minor components in MEO, including 1,8-cineole, carvone, and γ-terpinene, could also increase the variable [41,42].
The antimicrobial activity of L. officinalis EO (10 µg/disk) against P. aeruginosa was also evaluated in the study by Gavanji et al. [48]. However, the authors found that the zone of inhibition of their EO was 7.83 ± 0.03 mm, and P. aeruginosa proved to be more resistant toward a broad range of L. officinalis EO concentrations (0.08-100 µg/disk) as compared to S. aureus, which is inconsistent with our findings. Indeed, the LEO used in our study possessed a better inhibitory effect on the growth of P. aeruginosa, whilst the antibacterial activity against S. aureus was weak. This discrepancy between the two studies could be associated with the different chemical compositions of both lavender EOs employed. A particularity of P. aeruginosa is its high intrinsic resistance to antiseptics and antibiotics, which is partly caused by its low permeability of the outer membrane [49]. However, the study by Trombetta et al. [50] suggests that the antimicrobial effect of EO components, such as linalool acetate, may (at least partially) result from a perturbation of the lipid fraction of bacterial plasma membranes, thereby leading to alterations of membrane permeability and leakage of intracellular materials. In effect, the amount of linalool acetate was, in our LEO, quantified as a high content, whilst in the EO from L. officinalis used in the research by Gavanji et al. [48], it was completely absent. The hypothesis is also supported by the research of Hanamanthagouda et al. [51], in which EOs from dried leaves of L. bipinnata containing 3.37% of linalyl acetate exhibited low activity against P. aeruginosa (inhibition zone: 7 mm).
REO exhibited the strongest antibacterial activity against S. aureus in our research. Gomes Neto et al. [52], in line with this finding, reported significant inhibition of S. aureus viability and growth in meat broth induced by the effects of R. officinalis EO and by its majority compound 1,8-cineole itself, which was also quantified in our REO in the highest amount. The inhibitory and bactericidal efficiencies of R. officinalis EO, with the main component being 1,8-cineole (23.56%), against S. aureus were also reported by Jardak et al. [53].
Generally, MIC is a parameter that is often used for the measurement of Eos' antimicrobial activity, expressing the lowest concentration of the compound able to inhibit the growth of the analysed microorganisms [54].
Our results indicate that even antimicrobial highly resistant isolates, including S. aureus [55], C. albicans [56], and E. faecium [57], showed sensitivity to lavender, mint, and rosemary EOs, predicting their potential usage as promising detergents with the ability to inhibit the growth of a wide range of microorganisms. The differences in the susceptibility of the analysed bacteria and yeasts to the test EOs can be linked to variation in the rate of samples' penetration through the cell wall and cell membrane structures [58]. In addition to the EOs used, their concentration, and the type of microorganism tested, the differences in MIC values may be influenced by the cell size, cell damage, and EO oxidation [59].
EOs are known for their hydrophobicity through which they are capable of interacting with the fungal plasma membrane, leading to disruption of the membrane structures (leakage of some cellular components) or to alterations of the membrane permeability, reflecting their antifungal effects [60].
Many studies have shown strong antifungal activity of diverse plant EOs with a wide inhibition spectrum, pointing out their high potential as innovative preservative agents to replace synthetic fungicides [61]. Among other factors, the variations in the fungicidal activity of these aromatic compounds can be related to their active compounds, such as phenols, aldehydes, and ketones [62], which is consistent with the GC-MS analysis carried out in our study.
The results from MC and measurements showed that the parameters of bread analysed in our study had values of 41.65 ± 0.55% and 0.944 ± 0.001, respectively. Bread is considered as an intermediate moisture food, with MC typically ranging from 35-42%, and a w above 0.95, which is consistent with our findings. Therefore, baked goods, including bread, are susceptible to microbial spoilage with high growth of various fungi strains [63], thereby offering its application as a suitable type of substrate in such experiments.
Generally, antimicrobial agents are applied in food products for two main reasons: (i) to control natural spoilage processes (preservation of food), and (ii) to prevent or control the growth of microorganisms (food safety) [43]. Our study was focused primarily on an evaluation of the antifungal effects of EOs on bread as a model substrate for fungal growth (in situ conditions) since fungal spoilage occurs more often than bacterial spoilage [64]. Vapour diffusion exposure was applied based on the fact that most of the antimicrobial activity of EOs is attributed to volatile compounds [65].
The EOs' antifungal activity upon solution contact (broth dilution and agar dilution methods) has been studied by many researchers. However, the activity by vapourphase contact has been reported more rarely [66,67]. Different types of fungi, including Penicillium spp., are responsible for bread spoilage [68]. Although P. expansum is mainly associated with the degradation of apples, it was used in our study for its higher resistance than other species of the Penicillium strains [69]. Despite the high resistance, the antifungal effectiveness of all EOs tested against P. expansum, ranging from 36.05 ± 1.73% (250 µL/L of REO) to 86.48 ± 3.55% (125 µL/L of REO), was reported in the current research. Therefore, we assume that the EOs used can also be effective against other resistant species of microorganisms. Interestingly, P. citrinum was the most sensitive to the lowest concentration (125 µL/L) of MEO. We propose that the finding can be associated with the lower concentration of methyl acetate as compared to the MEO higher concentrations used as the chemical compound decreases the antifungal activity of MEO [28]. The results are in accordance with our previous studies, in which the antifungal effects of other EOs, such as coriander EO [20] or Citrus aurantium EO [21], against the same fungi species analysed (P. citrinum, P. expansum, P. crustosum) were confirmed.

Essential Oils
The following EOs were applied in this study: Lavender (LEO; Lavandula angustifolia x latifolia), mint (MEO; Mentha x piperita L.), and rosemary (REO; Rosmarinus officinalis). All essential oils were purchased from Hanus s.r.o. (Nitra, Slovakia) to complement our previous results [20,21] from such experiments, thus creating a comprehensive view of the biological actions of various commercially available EOs obtained from the same company.

Fungal Strains
Three Penicillium (P.) strains (P. crustosum, P. citrinum, P. expansum) were isolated from berry samples of Vitis vinifera and consequently classified using a reference-based MALDI-TOF MS Biotyper. The obtained results were also validated by comparison with the taxonomic identification obtained by 16S rDNA sequences analysis.

Evaluation of Antioxidant Activity of the EOs
The antioxidant activity of the three analysed EOs was assessed on the basis of the scavenging activity of the stable radicals 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the methodology used in the studies [20,21].

Chemical Characterization of EO Samples by Gas Chromatography/Mass Spectrometry (GC/MS) and Gas Chromatography (GC-FID)
Gas chromatography/mass spectrometry analyses of the selected EO samples were performed using an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to a quadrupole mass spectrometer 5975B (Agilent Technologies, Santa Clara, CA, USA). A HP-5MS capillary column (30 m × 0.25 mm × 0.25 m) was used. The temperature program was as follows: 60 • C to 150 • C (increasing rate 3 • C/min) and 150 • C to 280 • C (increasing rate 5 • C/min). The total run time was 60 min. Helium 5.0 was used as the carrier gas with a flow rate of 1 mL/min. The injection volume was 1 L (EO samples were diluted in pentane), while the split/splitless injector temperature was set at 280 • C. The investigated samples were injected in the split mode with a split ratio at 40.8:1. Electron-impact mass spectrometric data (EI-MS; 70 eV) were acquired in scan mode over the m/z range 35-550. The mass spectrometry ion source and MS quadrupole temperatures were 230 • C and 150 • C, respectively. Acquisition of data started after a solvent delay time of 3 min. Gas chromatography (GC-FID) analyses were performed on an Agilent 6890N gas chromatograph coupled to an FID detector. Column (HP-5MS) and chromatographic conditions were the same as for GC-MS. The FID detector temperature was set at 300 • C.
The individual volatile constituents of injected EO samples were identified based on their retention indices [69], and a comparison with reference spectra (Wiley and NIST databases). The retention indices were experimentally determined using the standard method [70], which included retention times of n-alkanes (C6-C34), injected under the same chromatographic conditions. The percentages of the identified compounds (amounts higher than 0.1%) were derived from their GC peak areas.

Evaluation of Antimicrobial Activity of the EOs
The evaluation of the antimicrobial activity of the EOs was performed using the agar disc diffusion method. For this purpose, there was an aliquot of 0.1 mL of fungal and bacterial suspension in Mueller Hinton Broth (MHB; Merck, Gernsheim, Germany) inoculated to Mueller Hinton Agar (MHA; Merck, Germany; 60 mm). Subsequently, the discs of filter paper (6 mm) were impregnated with 10 µL of the analysed EO samples and then applied on the MHA surface. Inoculated MHA plates were kept at 4 • C for 2 h and incubated aerobically at 37 • C for 24 h (bacteria). In the case of fungi, 10 µL of the analysed oils were applied at three concentrations (125, 250, and 500 µL/L, diluted in 0.1% dimethyl sulfoxide (DMSO)), and incubated at 25 • C for 5 days. Two antibiotics (Cefoxitin, Gentamicin) and one antifungal (Fluconazole) were used as positive controls for Gram-negative and Gram-positive bacteria and yeasts, respectively. Disks impregnated with ethanol served as negative controls.
The diameters of the inhibition zones were measured in mm after incubation. Each test was repeated three times (one repeat reflecting one separate plate).

Determination of Minimum Inhibitory Concentration
The minimum inhibitory concentration (MIC) was detected according to the National Committee for Clinical Laboratory Standards (NCCLS) as it was recently described by Kačániová et al. [20,21]. Chloramphenicol and nystatin, and DMSO served as positive and negative controls, respectively. MIC was detected at 570 nm with a spectrophotometer (Promega Inc., Madison, WI, USA).

Bread Preparation
Wheat bread used for analyses was baked in the Laboratory of Cereal technologies (AgroBioTech Research Centre) according to the methodology described by Kačaniova et al. [20,21].

Moisture Content and Water Activity of Bread
The moisture content (MC) and water activity (a w ) of bread were measured using the Lab Master a w Standard (Novasina AG; Lachen, Switzerland) and the Kern DBS 60-3 moisture analyzer (Kern and Sohn GmbH, Balingen, Germany), respectively, after the bread cooling [20,21].

In Situ Antifungal Analyses on Bread Model
First, the bread samples were cut into slices with a 150 mm height and placed into 0.5 L sterile glass jars (Bormioli Rocco, Fidenza, Italy). A fungal spore suspension of each strain (in final concentration of 1 × 10 6 spores/mL) was diluted in 20 mL of sterile phosphatebuffered saline with 0.5% Tween 80 by adjusting the density to 1-1.2 McFarland; 5 µL of inoculum were used for bread inoculation. The EOs in concentrations of 125, 250, and 500 µL/L (EOs + ethyl acetate) were evenly distributed in a volume of 100 µL on a sterile paper-filter disc (6 cm), which was inserted into the cover of the jar, except for the treatment of the control group. The jars were hermetically closed and kept at 25 • C ± 1 • C for 14 days in the dark. The size of the microfungal colonies with visible mycelial growth and visible sporulation [20,21] was evaluated using stereological methods. In this concept, the volume density of the colonies was firstly assessed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), counting the points of the stereological grid hitting the colonies and those falling to the reference space (growth substrate used, i.e., bread). The antifungal activities of the EOs were expressed as the percentage of mycelial growth inhibition (MGI), which was calculated using the formula: MGI = [(C − T)/C] × 100 [71], where C = volume density of the fungal colony in the control group and T = volume density of that in the treatment group.

Statistical Analysis
The data from the analyses was statistically evaluated using Prism 8.0.1 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey's test was used to evaluate the statistical significance of differences between the analysed groups of samples. The level of significance was set at p < 0.05. MIC50 and MIC90 values (i.e., concentration causing 50% and 90% reduction of microbial growth) were estimated by the logit analysis.

Conclusions
The current study evaluated the chemical composition, antioxidant, antibacterial, and antifungal activities of rosemary, lavender, and mint EOs (125, 250, and 500 µL/L concentrations) against selected microorganisms. Our results revealed that MEO possessed the highest DPPH radical-scavenging activity, which was even significantly (p < 0.05) different from that of LEO and REO. Considering the antimicrobial activity, the EOs exhibited the strongest inhibitory efficiencies against the growth of P. aeruginosa and C. tropicalis (LEO), S. enterica (MEO), and S. aureus (REO). From the fungi strains, MEO (in all concentrations) was able to totally inhibit the growth of P. expansum and P. crustosum, whilst the growth of P. citrinum was completely suppressed only by its lowest concentration. From in situ analysis, REO (125 µL/L) exhibited the lowest MGI of P. citrinum, and 500 µL/L of MEO caused the highest MGI of P. crustosum. Our results suggest the analysed EOs have promising potential as innovative agents for use in the storage of bakery products to extend their shelf-life. Thus, their combination with other preservatives or modified atmosphere packaging could be a valuable alternative in the food industry. Moreover, our results complement our previous studies, thus creating a comprehensive view of the biological activities of various commercially available EOs obtained from the same company, Hanus s.r.o. (Nitra, Slovakia).  Acknowledgments: This work has been supported by the grants of the VEGA no. 1/0180/20.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Essential oils samples are available from the authors.