Growth of Penicillium commune is affected by essential oils of family Myrthaceae.

The essential oils used in this study were extracts of plants from family Myrtaceae. Specifically, we used clove essential oil (from Syzygium aromaticum L.; leaves), eucalyptus essential oil (from Eucaliptus globulus leaves), Tea Tree essential oil (from Melaleuca alternifolia Cheel; leaves) cajeput essential oil (from Melaleuca leucadendra L.; leaves and twigs), niaouli essential oil (from Melaleuca quinquenervia (Cav.) S. T. Blake; leaves). Essential oils were commercially produced.

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 hexan (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, where it was kept constant for 2 minutes. Helium was used as carrier gas at the constant flow (1.2 mL.min^-1 – constant flow). 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 retention times of reference standards (nerol, linalool, geraniol, citral, α-pinene, and β-pinene). The 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 0.10% were not counted.

Seven strains from different moldy milk products were used. Specifically: Penicillium commune KMi 177 (from moldy 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). These strains belong to the Collection of Fungi of Department of Microbiology; Faculty of Biotechnology and Food Sciences SUA in Nitra, Slovakia. Five-day old cultures cultivated on Czapek yeast extract agar (CYA) at 25 ±1 °C were used for each experiment (CYA) (Pitt and Hocking, 2009).

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 40 mm) was placed in the lid of Petri dish, and 50 μL of essential oil was pipetted by micropipette on 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 with a digital caliper on the 3^rd, 7^th, 11^th, and 14^th day – the strains cultivated at 25 ±1 °C and on 3^rd, 7^th, 11^th, 14^th, 21^st, 28^th, and 35^th day – the strains cultivated at 5 ±1 °C.

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

Essential oils that completely inhibit the growth of all strains at both temperatures were used to determine their minimum inhibitory doses (MIDs). EOs dissolved in dimethyl lsulfoxid (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. The EVE^TM Automatic cell counter (NanoEnTek, Korea) was used to determine the number of spores. Petri dishes (Ø 90 mm, two-sector, three replicates) containing 15 mL of CYA were inoculated by 5 μl 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.

The ability of the 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.

The statistical data evaluation was performed using SAS 9.3 package. The glimmix was used to test the effect of strain, essential oil, day, and the interaction of the strain, essential oil, and day on the mold growth with repeating random-residual-option and unstructured covariate structure. Ranked data were analysed. Holm-Tukey post hoc test was applied to performe for multiple comparison. Results were considered as significant at p value ≤0.05.

All strains of P. commune without essential oil in the atmosphere – controls (without essential oil at the atmosphere), were grown on the first measurement (3^rd day of incubation) at 25 ±1 °C. The intensity of the growth of the strains at 25 ±1 °C is shown in Figure 1. Dairy products are stored at low temperatures, so an additional cultivation temperature of 5 ±1 °C was used. At 5 ±1 °C (controls), the growth of two strains (KMi 270 and KMi 276) was recorded on the 3^rd day and of other five strains on the 7^th day. The intensity of the growth of the strains at 5 ±1 °C is shown in Figure 2.

Plant oils obtained from plants of three genera of family Myrthaceae were used in the research: Syzygium, Eucaliptus, and Melaleuca. The growth of the strains of P. commune was affected by all the essential oils used (Table 2 and Table 3). Only clove (from Syzygium aromaticum L.) essential oil, as the only one, completely inhibited the growth of the strains of P. commune at 5 ±1 °C, respectively 25 ±1 °C, throughout the experiment.

Guynot et al. (2003) demonstrated the potential of clove essential oil aganist species belonging to Eurotium, Aspergillus and Penicillium genera. According to Císarová, Tančinová and Medo (2016) clove essential oil (500 μL.L^-1 of air) completely inhibited growth of isolates of Aspergillus flavus and Aspergillus parasiticus.

Eucaliptus (from Eucaliptus globulus) essential oil only partially inhibited the growth of strains of P. commune at 25 ±1 °C and 5 ±1 °C. Even the growth stimulation of the strain P. commune KMi 277 was observed (at 25 ±1 °C). The influence of the eucalitptus esseniatial oil is shown in Figure 3 and Figure 4. Unlike our results using another type of volatile essential oil, Ramezani et al. (2002) showed a strong fungicidal activity of the volatile oil of Eucaliptus citriodora.

Three essential oils were used from genus Melaleuca: tea tree, cajeput, and niaouli. Tea tree (from Melaleuca alternifolia Cheel) essential oil showed a strong inhibitory effect on the strains of P. commune. The growth of six strains at 25 ±1 °C, respectively five at 5 ±1 °C was completely inhibited throughout all cultivations. Only one strain (P. commune KMi 403) in the presence of tea tree oil grew at 25 ±1 °C, but its growth was small (84.8 – 99.5% of inhibition). The influence of tea tree essential oil on the growth of the strains P. commune at 25 ±1 oC is shown in Figure 5. Two strains of P. commune (KMi 276 and KMi 403) were able to grow in the presence of tea tree essentail oil at 5 ±1 °C from the 28^th day of the experiment, but growth inhibition was significant (Figure 6). A weaker inhibitory effect was found in cajeput (from Melaleuca leucadendra) essential oil (Figure 7 and Figure 8) and the weakest in niaouli (from Melaleuca quinquenervia (Cav.) S. T. Blake) essential oil (Figure 9 and Figure 10). Stević et al. (2014) also reported excellent antifungal activity of tea tree essential oil against the most of tested fungi (species of genera Fusarium, Aspergillus, Alternaria, Penicillium and others).

In this study clove essential oil (625 μL.L^-1 of air) was able to inhibit the growth of all strains at all days of cultivation on the Czapek yeast extract agar at 5 ±1 °C and 25 ±1 °C also. Clove essential oil was used for the determination of MIDs. The results are shown in Table 4. The MIDs were 125 μL.L^-1 of air for the strains P. commune KMi 177 and KMi 277 and 250 μL.L^-1 of air for the strains P. commune KMi 270, KMi 276, KMi 370, KMi 402 and KMi 403 on the 7^th day of cultivation. On the 14^th day of incubation, the same values of MIDs were determined. In some strains, the number of growing colonies increased, but this did not affect the MIDs. Císarová et al. (2016) determined lower MIDs of clove essential oil in vapor phase against the tested aspergilly strain 62.5 μL.L^-1.

Using probit analysis, predicted MIDs90 and MIDs50 were calculated. The results are shown in Table 5. The highest MIDs90 were determined for strain KMi 403 – 301.37 μL.L^-1 of air on 7^th day and 292.56 μL.L^-1 of air on 14^th day of cultivation. The lowest MIDs90 were determined for the strains KMi 177 and KMi 277 – 104.93 μL.L^-1 of air on the 7^th day and on the 14^th day of cultivation.

In our research, we have confirmed the ability of five essential oils from plants family Myrtaceae to inhibit (partially or completely) the growth of the strains of P. commune. Testing should be supplemented by testing the effects of essential oils on the sensory properties of foods. Our tested strains were obtained from the moldy dairy products whose sensory properties could be affected by essential oils.