EFFECT OF ESSENTIAL OILS OF LAMIACEAE PLANTS ON THE PENICILLIUM COMMUNE

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February – March, 2019, vol. 8, no. 4
pages: 1111-1117
Article type: Microbiology of Microbiology
DOI: 10.15414/jmbfs.2019.8.4.1111-1117
Abstract: The aim of this research was to determine the inhibitory effect of vapor phase of eight essential oils (EOs) on the growth of seven strains of Penicillium commune isolated from moldy milk products. Another objective was to determine the minimum inhibitory doses (in vitro and probit analyses) of EOs, which at concentration 625 μL.L-1 of air completely inhibited the growth of all strains. The antifungal activity was evaluated by the micro-atmosphere method. Thyme, red thyme, peppermint, mint, and savory completely inhibited the growth of strains during cultivation at 25 °C and 5 °C. Basil, rosemary, and sage EOs have different effects on the growth of P. commune strains. EOs that completely inhibit the growth of all strains were used to determine their minimum inhibitory doses (MIDs). The best results 62.5 μL.L-1 of air and 125 μL.L-1 of air 7th day at 25 °C of incubation showed red thyme EO. Similar results were also found in thyme EO, but the MID inhibiting the growth of the one strain was 250 μL.L-1 of air. MIDs of savory and peppermint were from 125 to 500 μL.L-1 of air. Mint EO had the highest MID (from 250 to 625 μL.L-1 of air). On the 14th day of incubation we found the same MIDs, respectively higher. It was found that EOs have different effects on individual strains of P. commune. According to probit analyses, the most effective tested EOs were red thyme and thyme, less effective peppermint and savory, and the least effective was mint EO.
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EFFECT OF ESSENTIAL OILS OF LAMIACEAE PLANTS ON THE PENICILLIUM COMMUNE


AUTHORS

Dana Tančinová, Juraj Medo, Zuzana Mašková, Denisa Foltinová, Július Árvay

ABSTRACT

The aim of this research was to determine the inhibitory effect of vapor phase of eight essential oils (EOs) on the growth of seven strains of Penicillium commune isolated from moldy milk products. Another objective was to determine the minimum inhibitory doses (in vitro and probit analyses) of EOs, which at concentration 625 μL.L-1 of air completely inhibited the growth of all strains. The antifungal activity was evaluated by the micro-atmosphere method. Thyme, red thyme, peppermint, mint, and savory completely inhibited the growth of strains during cultivation at 25 °C and 5 °C. Basil, rosemary, and sage EOs have different effects on the growth of P. commune strains. EOs that completely inhibit the growth of all strains were used to determine their minimum inhibitory doses (MIDs). The best results 62.5 μL.L-1 of air and 125 μL.L-1 of air 7th day at 25 °C of incubation showed red thyme EO. Similar results were also found in thyme EO, but the MID inhibiting the growth of the one strain was 250 μL.L-1 of air. MIDs of savory and peppermint were from 125 to 500 μL.L-1 of air. Mint EO had the highest MID (from 250 to 625 μL.L-1 of air). On the 14th day of incubation we found the same MIDs, respectively higher. It was found that EOs have different effects on individual strains of P. commune. According to probit analyses, the most effective tested EOs were red thyme and thyme, less effective peppermint and savory, and the least effective was mint EO.


KEYWORDS

P. commune, essential oils, antifungal activity, vapor phase

INTRODUCTION

Molds are the most common cheese spoilage organisms which can lead to economic loss as well as raising public health concerns due to the production of mycotoxins (Cheong et al., 2014). According to Pitt and Hocking (2009), cheese is very susceptible to mold growth. Spoilage is generally confined to molds which are psychrotolerant. Lund et al. (1995) reported that 91 % of isolates from cheeses from Denmark, France, Greece, UK and other countries were Penicillium species. Penicillium commune was the most widespread and most frequent occurring (42 %) species. P. commune is one of the most important cheese contaminant (Kure and Skaar, 2000; Kure et al., 2001, Garnier et al., 2017).  The growth of P. commune on cheese results in discoloring of the surface and production of off flavors. In spite of the fact that the dairy staff daily clean and disinfect the production plants and environments, periodical growth of P. commune on cheese is a major problem (Lund et al., 2003).

Today’s consumers demand food that is minimally technologically processed and without synthetic preservatives or additives, because of the possible adverse health effects. Therefore, the food industry is now focused on finding solutions that fully satisfy the criteria of consumers while retaining the food safety. Application of natural antimicrobial agents such as extracts, essential oils and components of spices, and other aromatic plants could be significant in resolving these problems. These agents may be useful as additives in limiting or preventing the development of harmful fungi in food, as food surface protectants, or in modified atmosphere packaging of food (Kocic-Tanackov et al., 2014). Essential oils have deserved much attention in the past decades for their antimicrobial activity, since many of them have demonstrated efficacy against food-borne pathogenic and spoilage microorganisms (Bassanetti et al., 2017).

The aim of the present research was to determine the inhibitory effect of vapor phase of eight essential oils on the growth of seven different strains of P. commune isolated from moldy milk products. Another objective was to determine the minimum inhibitory doses of essential oils, which at a concentration 625 μL.L-1 of air completely inhibited the growth of all the strains.

MATERIAL AND METHODS

Plant essential oils

The essential oils used in this study were extract of basil (Oscimum basilicum L.), rosemary (Rosmarinus officinalis L.), thyme and red thyme (Thymus vulgaris L.), peppermint (Mentha piperita L.), mint (Mentha crispate L.) savory (Satureja hortensis L.), and sage (Salvia officinalis L.). Essential oils were commercially produced.

Chemical composition of essential oils

Semi-quantitative composition of the essential oil samples was determined by gas chromatography coupled with mass spectrometry (GC-MS) using an Agilent 7890B oven coupled with Agilent 5977A mass detector (Agilent Technologies Inc., Palo Alto, CA, USA) and CombiPal autosampler 120 (CTC Analytics AG, Zwingen, Switzerland). Prior to the analysis, essential oil samples were diluted in hexane (HPLC ≥ 97 %, Sigma Aldrich GmbH, Germany) to a concentration of 10 µL mL-1. One microliter of diluted sample was injected in inlet operated in split mode (1:10; 250 °C). Separation was achieved using a ZB-WAXplusTM capillary column (10 m × 0.1 mm × 0.10 µm) (Phenomenex Inc., Torrance, CA, USA) and the following oven temperature programme: 50 °C for the first 5 minutes, increased to 240 °C at the rate of 3 °C min-1, and it was kept constant for 2 minutes. Helium was used as carrier gas at the constant flow (1.2 mL min-1). The mass detector parameters were as follows: ionization energy of filament: 70 eV, transfer line temperature: 250 °C, MS source temperature: 230 °C, quadrupole temperature: 150 °C. The mass spectrometer was programmed under electron impact (EI) in a full scan mode at m/z 40-400. The identification of compounds was carried out by comparing of mass spectra (over 80 % match) with a commercial database NIST® 2014 and the retention times of reference standards (nerol, linalool, geraniol, citral, α-pinene and β-pinene). Semi-quantitative content of determined compounds was calculated by dividing individual peak area (excluded by solvent peak area) by total area of all peaks. Peaks under 0.1 % were not counted.

Fungal culture

Seven strains (Table 1) from different moldy milk products were used. These strains belong to the Collection of Fungi of Department of Microbiology; Faculty of Biotechnology and Food Sciences SUA in Nitra, Slovakia. 5 day old cultures cultivated on Czapek yeast extract agar (CYA) at 25 ± 1 °C were used for each experiment (CYA, Pitt et Hocking, 2009).

Table 1 Origin of the strains Penicillium commune

Strain Origin
P. commune KMi 177 cheese flavored with pepper
P. commune KMi 270 smoked cheese (block)
P. commune KMi 276 smoked cheese (slices)
P. commune KMi 277 smoked cheese (slices)
P. commune KMi 370 sour cream
P. commune KMi 402 sour cream
P. commune KMi 403 parenica (pasta filata)

Antifungal activity of essential oils

The antifungal activity of selected essential oils was evaluated by the micro-atmosphere method. The test was performed in sterile plastic Petri dishes (Ø 90 mm) containing 15 mL of CYA. The evaluation by filter paper was made by the adapted method from Guynot et al. (2003). Essential oils were tested in concentration 625 μL.L-1 of air. A round sterile filter paper (Ø 9 cm) was placed in the lid of Petri dish and 50 μL of essential oil was pipetted by micropipette to the paper. Dishes were kept in inverted position. Filter paper discs impregnated with sterilized distilled water were used as a control. Each strain was inoculated in the center of Petri dishes with sterilized needle. Dishes were tightly sealed with parafilm and incubated for 14 days at 25 ±1 °C and for 35 days at 5 ±1 °C (four replicates were used for each treatment). The diameters (Ø mm) of the growing colonies (from the reverse side) were measured at the 3rd, 7th, 11th, and 14th day – strains cultivated at 25 ±1 °C and 3rd, 7th, 11th,14th, 21st, 28th and 35th day – cultivated at 5 ±1 °C  with a digital caliper.

Inhibition of mycelial growth

According to Cakir et al. (2005) and Kordali et al. (2008), growth inhibition of treated samples (T) against control (C) was calculated by the percentage of growth inhibition using the following equation:

% of inhibition =(C-T)/C x 100

where, C is the mean of six replicates of hyphal extension (mm) of controls and T is the mean of six replicates of hyphal extension (mm) of plates treated with either essential oil.

Minimum inhibitory doses (MIDs)

Essential oils that completely inhibit the growth of all strains were used to determine their minimum inhibitory doses (MIDs). EOs dissolved in DMSO were prepared at different concentrations (625, 500, 250, 125, 63, 31.25, and 15.63 μL.L-1 of air). For each fungal strain, a conidial spore suspension of 106 spore’s ml-1 was prepared. Petri dishes (Ø 90 mm, two-sector, three replicates) containing 15 mL of CYA were inoculated by 5µl spores suspension. Cultivation was carried out at the 25 ± 1 °C and measured after 7 and 14 days. The MID (expressed as microliters of EOs per volume unit of atmosphere above the organism growing on the agar surface) was defined as the lowest concentration of the oil which did not permit any visible growth after 7 or 14 days in comparison with control sets.

Probit analyses

The ability of strains to grow in the presence of EO was coded to binomial scale (1 – growth observed, 0 – without growth). Such data were processed by probit analysis in Statgraphics Centurion XV (Statgraphics) software. Doses that inhibit the growth in 50 % respectively
90 % of cases (MID50 and MID90) were reversely predicted from regression equation.

Statistical analysis

Average diameters of developed colonies were compared using 2-factor ANOVA with interaction. Average values for strains as well as essential oils were compared using Tukey post hoc test. Analysis was performed in Statgraphics.

RESULTS AND DISCUSSION

In this study, evaluating the antifungal properties of eight essential oils from family Lamiaceae. Essential oils are complex mixtures of low molecular weight compounds extracted from plants by steam distillation and various solvents. Terpenoids and phenylpropanoids are the major constituents, which provide characteristic aroma and biological properties to essential oils (Raut et Karuppayil, 2014). According to authors (Ben Farhat, et al., 2016; Méndez-Tovar et al., 2016; Dušková et al., 2016) the growing seasons, different growth stage of plants, and climatic conditions of each year in terms of the essential oil content and composition were proven. Based on the above, we also focused on the composition of the essential oils used. The GC-MS analyses of the essential oils led to identification of 98 compounds, 34 from them are presented in ≥1 percentage amount in minimal one essential oil. The identified compounds (34) are listed in Table 2. The major components according to the concrete essential oil were: basil – Estragole (84.98 %); red thyme – Thymol (33.65 %) and o-xylene (43.85 %); rosemary – Eucalyptol (43.17 %), (+)-2-Bornanone  (12.80 %) and α-pinene (10.74 %); thyme – Thymol (40.41 %) and Benzene, 4-ethyl-1,2-dimethyl- (19.45 %); peppermint – Levomenthol (44.94 %) and menthone  (22.51 %); savory – γ-Terpinene (45.09%),  Thymol (20.20 %) and p-Cymene (19.64 %); sage –Thujone (22.37 %), (+)-2-Bornanone (19.65 %) and Eucalyptol (10.84 %); mint – (-)-Carvone (72.62 %) and D-Limonene (15.23 %).

Table 2 Essential oils tested for the fungicidal effect and their compounds [%]* determined by gas chromatography coupled with mass spectrometry (GC-MS)

Compound Essential oils
Basil Red thyme Rosemary Thyme Peppermint Savory Sage Mint
1 α-pinene 0.30 3.49 10.74 1.79 0.73 2.71 6.08 0.61
2 β-pinene 0.34 0.66 7.43 0.17 0.89 3 2.34 0.87
3 (+)-4-Carene 1.02 3.76
4 Camphene 0.96 4.66 1.62 0.23 5.86
5 β-Myrcene 0.17 1.2 0.73 0.52
6 β-Myrcene 0.92 1.48 0.09 2.49
7 D-Limonene 0.28 1.03 2.82 2.03 0.48 1.88 15.23
8 Eucalyptol 4.10 0.9 43.17 1.64 7.01 10.84 0.91
9 γ-Terpinene 0.52 5.67 45.09 0.94 0.08
10 o-Cymene 2.75 1.74 0.32
11 Linalool 1.84 7.12 0.69 5.80
12 endo-Borneol 1.02 3.83 2.25 0.15 4.45
13 cis-α-Bergamotene 2.76
14 Thujone 22.37
15 Thymol 33.65 40.41 20.20
16 (+)-2-Bornanone 0.30 0.41 12.80 2.36 19.65
17 Caryophyllene 0.15 1.43 3.78 6.77 3.33 0.41 5.62 0.63
18 Benzene, 4-ethyl-1,2-dimethyl- 19.45
19 α-Terpineol 1.59 2.31 0.26 0.14 0.11
20 Humulene 0.47 0.12 0.05 6.92
21 Longifolene 0.16 1.62
22 isobornil acetate 1.28
23 terpinen-4-ol 0.13 0.46 2.16 0.44 0.23
24 Estragole 84.89 0.29
25 β-thujone 6.58
26 menthofuran 1.62
27 menthone 22.51
28 p-menthone 4.22 0.65
29 menthol 6.30
30 Levomenthol 0.10 44.94
31 p-Cymene 19.64
32 Bornyl acetate 0.20 0.16 2.29
33 (-)-Carvone 72.62
34 o-xylene 43.85

Legend: *listed are the components that represented min. 1 % in at least one essential oil

Antifungal activity of essential oils

The antifungal activity of eight essential oils against seven strains of P. commune was determined, using micro-atmosphere method (625 μL.L-1 of air). Five essential oils: thyme, red thyme (Thymus vulgaris L.), peppermint (Mentha piperita L.), mint (Mentha crispate L.) savory (Satureja hortensis L.) mint (Mentha piperita L.), completely inhibited the growth of all strains during cultivation at 25 °C and 5 °C. Other essential oils: basil (Oscimum basilicum L.), rosemary (Rosmarinus officinalis L), and sage (Salvia officinalis L.) (Tab 3) have different effects on the growth of P. commune strains. Inhibitory effect (P <0.001) on the growth of the all strains of P. commune was recorded in all essential oils. Significant differences were observed between the individual strains (Table 3), which are documented in Figure 1, too.

Table 3 Effect of essential oils (treatment) and strains on the growth of Penicillium commune

  Temperature of incubation
25 °C 5 °C
Average diameter (in mm)
3rd day 7th day 11th day 14th day 3rd day 7th day 11th day 14th day 21st day 28th day 35th day
Essential oil
Basil 2.7 d 16.0 d 28.7 d 36.6 d 0.0 a 0.0 a 0.0 a 0.0 a 1.0 c 3.2 c 7.1 c
Thyme 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
Rosemary 2.1 c 11.7 c 22.2 c 30.0 c 0.0 a 0.0 a 0.2 a 1.6 b 4.4 d 7.3 d 11.1 d
Red thyme 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
Peppermint 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
Savory 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
Sage 1.0 b 7.0 b 19.4 b 26.5 b 0.0 a 0.0 a 0.0 a 0.0 a 0.7 b 2.4 b 4.8 b
Mint 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
control 15.3 e 30.2 e 40.0 e 45.7 e 0.4 b 4.8 b 10.2 b 14.9 c 24.8 e 31.2 e 37.8 e
P <0.001 <0.001  <0.001 <0.001 <0.001 <0.001  <0.001  <0.001 <0.001  <0.001 <0.001
Strain
KMi 177 2.2 bc 6.0 b 10.5 c 12.6 b 0.0 a 0.7 cd 1.5 d 1.9 c 3.6 c 4.8 c 7.0 b
KMi 270 3.8 d 6.9 c 10.7 c 14.0 c 0.1 b 0.3 a 0.8 a 1.3 a 3.4 c 5.0 cd 6.9 b
KMi 276 2.4 c 4.6 a 9.0 a 13.0 b 0.2 c 0.7 d 1.2 bc 2.1 c 2.9 b 5.1 d 6.9 b
KMi 277 2.1 b 8.8 d 11.5 d 14.3 c 0.0 a 0.8 d 1.3 c 2.4 d 5.0 e 6.9 f 10.0 c
KMi 370 1.2 a 5.8 b 10.0 b 12.7 b 0.0 a 0.5 bc 1.0 b 1.6 b 2.2 a 3.2 a 4.4 a
KMi 402 1.1 a 4.1 a 8.6 a 11.3 a 0.0 a 0.4 ab 1.0 b 1.5 ab 2.7 b 3.5 b 4.8 a
KMi 403 3.7 d 14.4 e 25.6 e 29.9 d 0.0 a 0.4 b 1.2 bc 2.0 c 4.3 d 5.7 e 7.4 b
P <0.001 <0.001  <0.001 <0.001 <0.001 <0.001  <0.001  <0.001 <0.001  <0.001 <0.001

Legend: Averages followed by the same letter (in columns) is not statistical different at α = 0.05 (ANOVA, Tukey test) small letters – difference within treatment, capital letters – different within strains.

Figure 1 Growth of strains of P. commune on the 35th day of incubation at 5 °C in the presence/absence without of essential oil

All strains of P. commune, without essential oil at the atmosphere – controls, were grown on the first measurement (3rd day of incubation) at 25 °C. Dairy products are stored at low temperatures, so an additional cultivation temperature of 5 °C was used. At 5 °C (controls), the growth of two strains (KMi 270 and KMi 276) was recorded on the 3rd day and other five strains on the 7th day. Inhibition of mycelial growth (percentage of inhibition) is shown in Figure 2. Thyme, red thyme, savory, peppermint, and mint essential oils completely inhibited the growth of the strains of P. commune at 5 °C, respectively 25 °C, throughout the experiment.  Basil, rosemary, and sage essential oils inhibited the growth of the strains of P. commune, but their inhibition effects were depending on the individual strains. Sage essential oil 100 % inhibited the growth of the strain P. commune KMi 402 at 5 °C, for example. The stimulation effect was observed in one case. Basil essential oil stimulated growth of P. commune KMi 277 (Fig 2g) at the 14th day of incubation at 25 °C. Strong inhibition effect (100 %) of thyme and mint determined Císarová et al. (2016b) on Aspergillus flavus and Aspergillus parasiticus. Thyme oil (625 μL.L-1 of air) totally inhibited growth of Aspergillus niger and Aspergillus tubingensis (Císarová et al. 2016a). The 100 % inhibition effect of thyme, red thyme, mint, and savory essential oils (625 μL.L-1 of air) against Rhizopus spp. has shown Tančinová et al. (2018). According Elshafie et al. (2015), thyme essential oils can be utilized against Monilinia laxa, Monilinia fructigena, and Monilinia fructicola. Alizadeh-Saltech et al. (2010) tested the influence of vapor phase of sage, savory, and zataria essential oils on the growth of Rhizopus stolonifer. Sterile filter paper discs soaked with 3, 6, 12, 24 or 48 µl pure essential oils were placed on the inner surface of the Petri dish lid. Sage oil did not have an acceptable inhibitory effect on the R. stolonifer. Savory, and zataria, but showed strong antifungal activity against R. stolonifer. The 100 % inhibitory effect of the savory essential oil and the partial inhibitory effect of sage essential oil have been observed in our experiment, too. Servili et al. (2017) recommended exposure to volatiles of the rosemary and peppermint essential oils an innovative method to control the postharvest gray mold of table grapes. Sarkhosh et al. (2017) tested the inhibitory properties of five essential oils: mint, savory, thyme, cinnamon, and lavender against anthracnose (Colletotrichum gloesporioides Penz) of avocado fruit. Authors report the potential of using savory and thyme essential oils as biological fungicides for increasing the storage time of avocado fruit. Thyme oil highly reduced 64 % of Botrytis cinerea colonization on pretreated detached leaves of tomato compared to untreated control (Ben-Jabeur, et al., 2015). Combrinck et al. (2011) tested antifungal properties of eighteen essential oils (including peppermint and thyme oils) on the fungal growth of five plant pathogens (Lasiodiplodia theobromae, Colletotrichum gloesporioides, Alternaria citri, Botrytis cinerea, and Penicillium digitatum). Thyme oil proved to be the most effective inhibitor, totally inhibiting all of the pathogens tested at concentrations of 1000 µl.l-1 (medium) and lower, with the exception of resistant Penicillium strains. The concrete essential oil can have different influence on the growth of species of molds. It is very individual. In our research, we noticed differences between the tested strains within P. commune. Therefore, it is necessary to use more strains of the same species in the trials so that the results are not influenced by the individual attributes of the concrete strain.

Figure 2 Inhibition of Penicillium commune strains growth causing by tested essential oils

In this study essential oils were able to inhibit growth of all strains at all days of cultivation at the highest concentration (625 μL.L-1 of air) and they were used for determination of MIDs. The results are shown in Table 4. The best results 62.5 μL.L-1 of air for strain KMi 177, KMi 276, KMi 277, KMi 370 and 125 μL.L-1 of air for KMi 270, KMi 402 and KMi 403 at 7th day of incubation showed red thyme essential oil. Similar results were also found in thyme essential oil, but the MID inhibiting the growth of the strain KMi 403 was 250 μL.L-1 of air.  MIDs of savory and peppermint were from 125 to 500 μL.L-1 of air depending on the strains. Mint essential oil had the highest MID (from 250 to 625 μL.L-1 of air). On the 14th day of incubation, we found the same MIDs, respectively higher. It was found that EOs have different effects on individual strains of P. commune.

Table 4 The inhibitory effect (in %) of essential oils on the growth of colonies (n = 6) of Penicillium commune on CYA at 25 ° C after 7 and, respectively, 14 days of cultivation

Essential oil Strain of Penicillium commune
KMi 177 KMi 270 KMi 276 KMi 277 KMi 370 KMi 402 KMi 403
μL.L-1 of air Day of cultivation
7th 14th 7th 14th 7th 14th 7th 14th 7th 14th 7th 14th 7th 14th
Thyme 625 0 0 0 0 0 0 0 0 0 0 0 0 0 0
500 0 0 0 0 0 0 0 0 0 0 0 0 0 0
250 0 0 0 0 0 0 0 0 0 0 0 0 0 0
125 0 0 0 0 0 0 0 0 0 0 0 0 100 100
62.5 50 50 100 100 66.66 100 100 100 0 0 66.66 83.33 100 100
31.3 100 100 100 100 100 100 100 100 100 100 100 100 100 100
15.6 100 100 100 100 100 100 100 100 100 100 100 100 100 100
Red thyme 625 0 0 0 0 0 0 0 0 0 0 0 0 0 0
500 0 0 0 0 0 0 0 0 0 0 0 0 0 0
250 0 0 0 0 0 0 0 0 0 0 0 0 0 0
125 0 0 0 0 0 0 0 0 0 0 0 0 0 0
62.5 0 50 100 100 0 33.33 0 0 0 0 50 66.66 16.66 16.66
31.3 100 100 100 100 66.66 83.33 100 100 83.33 83.33 100 100 100 100
15.6 100 100 100 100 100 100 100 100 100 100 100 100 100 100
Peppermint 625 0 0 0 0 0 0 0 0 0 0 0 0 0 0
500 0 0 0 0 0 0 0 0 0 33.33 0 0 0 0
250 0 0 0 66.66 0 33.33 0 0 33.33 100 0 83.33 66.66 83.33
125 0 50 100 100 66.66 83.33 0 66.66 100 100 100 100 100 100
62.5 100 100 100 100 100 100 100 100 100 100 100 100 100 100
31.3 100 100 100 100 100 100 100 100 100 100 100 100 100 100
15.6 100 100 100 100 100 100 100 100 100 100 100 100 100 100
Savory 625 0 0 0 0 0 0 0 0 0 0 0 0 0 0
500 0 0 0 0 0 0 0 0 0 0 0 0 0 0
250 0 0 0 0 0 0 0 0 0 0 0 0 33.33 33.33
125 0 0 33.33 66.66 0 33.33 66.66 66.66 100 100 66.66 100 50 100
62.5 100 100 100 100 66.66 66.66 100 100 100 100 100 100 100 100
31.3 100 100 100 100 100 100 100 100 100 100 100 100 100 100
15.6 100 100 100 100 100 100 100 100 100 100 100 100 100 100
Mint 625 0 0 0 0 0 0 0 0 0 0 0 0 0 0
500 0 0 0 0 0 0 0 0 0 0 0 0 50 66.66
250 0 0 0 50 16.66 16.66 0 100 0 16.66 0 33.33 100 100
125 100 100 100 100 100 100 100 100 100 100 100 100 100 100
62.5 100 100 100 100 100 100 100 100 100 100 100 100 100 100
31.3 100 100 100 100 100 100 100 100 100 100 100 100 100 100
15.6 100 100 100 100 100 100 100 100 100 100 100 100 100 100

Legend: CYA – Czapek yeast extract agar

Using probit analysis, predicted MIDs90 and MIDs50 were calculated. The results are shown in Table 5. The most effective tested essential oils were red thyme and thyme, less effective peppermint and savory. MIDs of mint essential oil were the highest. The most resistant strain was KMi 403 with the highest MID90 for thyme, peppermint, savory, and mint essential oils. The highest MID90 of red thyme was determinate for KMi 270. Radaelli et al. (2016) tested antimicrobial activities of six essential oils (basil, rosemary, peppermint, marjoram, and thyme) against Clostridium perfringens.  The essential oil from Thymus vulgaris showed the lowest MIC (minimum inhibitory concentration) against Clostridium perfringens. The authors, as well as our research showed a significant antimicrobial activity of thyme essential oil.

Table 5 Minimal inhibition doses estimated by probit analyses

Strain of

P. commune

 MID Essential oil
Thyme Red thyme Peppermint Savory Mint
Day of cultivation
7 14 7 14 7 14 7 14 7 14
KMi 177 MID90 74.24 74.94 53.46 74.94 104.85 143.92 104.85 105.03 211.10 211.11
MID50 62.51 63.02 46.93 63.02 94.06 125.00 94.06 94.31 193.74 193.75
KMi 270 MID90 104.85 105.03 104.85 105.03 211.10 294.02 136.48 153.07 211.10 280.37
MID50 94.06 94.31 94.07 94.31 193.74 261.07 119.19 132.06 193.74 250.00
KMi 276 MID90 80.29 105.03 42.57 78.36 153.23 305.97 80.29 153.48 256.22 256.22
MID50 66.99 94.31 34.16 53.70 132.10 213.83 66.99 100.02 230.86 230.87
KMi 277 MID90 104.85 105.03 53.47 53.71 104.85 153.07 153.23 153.07 211.10 393.29
MID50 94.07 94.31 46.93 47.06 94.07 132.06 132.10 132.06 193.74 365.34
KMi 370 MID90 53.47 53.71 46.92 46.76 268.70 524.25 211.10 211.11 211.10 256.22
MID50 46.93 47.06 38.00 37.80 240.54 487.72 193.74 193.75 193.74 230.87
KMi 402 MID90 80.29 89.43 74.24 81.06 211.10 314.06 153.23 211.11 211.10 268.70
MID50 66.99 74.37 62.52 67.56 193.74 277.56 132.10 193.75 193.74 240.54
KMi 403 MID90 211.11 211.11 65.32 65.32 294.02 314.06 295.74 300.70 533.37 540.97
MID50 193.75 193.75 55.95 55.95 261.08 277.56 186.87 240.54 500.00 510.31

Legend: P.Penicillium, MID – minimum inhibitory doses

In our research, we have confirmed the ability of the tested oils family Lamiaceae to inhibit (partially or completely) the growth of P. commune strains. The strains used in our experiment were isolated directly from the moldy dairy products produced in Slovakia. However, the individual strains responded differently on the same oil, and therefore it necessary to use more than one strain in research. Testing should be supplemented by testing the influence of oils on the sensory properties of foods. According to Servilli et al. (2017), the interaction of food matrix components with the essential oils need to be investigated before their application is proposed for commercial practice. A challenge for the application of essential oils is their strong aroma even at low concentrations, which might adversely affect the organoleptic properties of the food being treated. Concentration of these substances applied in cheeses should be considered carefully because of their possible negative impacts on organoleptic properties (Khorshidian et al., 2018).

CONCLUSION

In this study, we evaluated the antifungal properties of basil (Oscimum basilicum L.), rosemary (Rosmarinus officinalis L.), thyme, red thyme (Thymus vulgaris L.), mint (Mentha crispate L.), peppermint (Mentha piperita L.), savory (Satureja hortensis L.), and sage (Salvia officinalis L.)  essential oils. Five essential oils: thyme, red thyme, peppermint, mint, and savory completely inhibited the growth of all strains during cultivation at 25 °C and 5 °C also. Other essential oils: basil, rosemary, and sage have different effects on the growth of P. commune strains. Essential oils that completely inhibit the growth of all strains were used to determine their minimum inhibitory doses (MIDs). The best results 62.5 μL.L-1 of air and 125 μL.L-1 of air 7th day at 25 °C of incubation showed red thyme essential oil. Similar results were also found in thyme essential oil, but the MID inhibiting the growth of the one strain was 250 μL.L-1 of air.  MIDs of savory and peppermint were from 125 to 500 μL.L-1 of air depending on the strains. Mint essential oil had the highest MID (from 250 to 625 μL.L-1 of air). On the 14th day of incubation we found the same MIDs, respectively higher. It was found that essential oils have different effects on individual strains of P. commune. According to probit analyses, the most effective tested essential oils were red thyme and thyme, less effective peppermint and savory. MIDs of mint essential oil were the highest. The most resistant strain was KMi 403 with the highest MID90 for thyme, peppermint, savory, and mint essential oils. The highest MID90 of red thyme was determinate for KMi 270.

Acknowledgments: This work was supported by APVV-15-0543, KEGA 015SPU-4/2018 and Research Center AgroBioTech built in accordance with the project Building Research Centre „AgroBioTech” ITMS 26220220180. The authors thank Eva Čunderlíková for her responsible approach in the preparation and realization of the experiment.

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