INHIBITORY EFFECT OF ESSENTIAL OILS FROM SOME LAMIACEAE SPECIES ON GROWTH OF EUROTIUM SPP. ISOLATED FROM BREAD

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October – November 2018, vol. 8, no. 2
pages: 857-862
Article type: Microbiology of Microbiology
DOI: 10.15414/jmbfs.2018.8.2.857-862
Abstract: Bread is considered an intermediate - moisture food product that is prone to mould spoilage. Growth of spoilage fungi is currently controlled with the addition of chemical preservatives. Consumers demand more natural products and there is a need to reduce the amount of chemical preservatives added to foods. Essential oils (EOs) and their constituents emerged as promising and effective compounds to protect foods from lipid peroxidation and provide microbiological food safety. So the aim of our study was to determinate microscopic fungi involved in contamination of bread target the genus Eurotium and to evaluate the antifungal activity of 5 EOs by vapor contact against the fungal species of this genus. In all samples after three days of cultivation fungi created colonies on the surface of the crust and the pieces of the bread samples stored on DRBC were after three days of cultivation covered with fungi colonies. Altogether 195 isolates were recovered from 8 bread samples and assigned to 10 fungal genera. The most frequently genus of fungi was Penicillium, Aspergillus, Cladosporium, Epicoccum and Eurotium. Based on phylogenetic and morphological studies, five different Eurotium species were identified: E. amstelodami, E. chevalieri, E. herbariorum, E. rubrum and E. repens. In this study, we explored the potential of five essential oils retrieved from some Lamiaceae species, concretely: basil (Ocinum basilicum L.), lavender (Lavandula angustifolia Mill.), oregano (Origanum vulgare L.), mint (Metha piperita L.) and sage (Salvia officinalis L.). Gas chromatography–mass spectrometry (GC-MS) analysis allowed for the identification of 28 compounds as main constituents. Inhibitory activity of EOs was assessed against 5 species of genus Eurotium (2 isolates for each specie). In all studied strains, the essential oils caused significant differences (P<0.05) on the mycelium growth. Our result showed that the best antifungal effect was shown by lavender and oregano (100%) to all species of tested fungi followed by mint (with the best MGI 79.89% to E. rubrum) >basil (with the best Mycelial growth Inhibition (MGI) 93.65% to E. herbariorum) and the low effect had sage essential oil (with the best MGI 68.62% to E. repens). The present study demonstrated the potential food preservative ability of essential oils from some Lamiaceae species.
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INHIBITORY EFFECT OF ESSENTIAL OILS FROM SOME LAMIACEAE SPECIES ON GROWTH OF EUROTIUM SPP. ISOLATED FROM BREAD


AUTHORS

Miroslava Císarová, Lukáš Hleba, Dana Tančinová, Mária Florková, Denisa Foltinová, Ivana Charousová, Kristína Vrbová, Matěj Božik, Pavel Klouček

ABSTRACT

Bread is considered an intermediate - moisture food product that is prone to mould spoilage. Growth of spoilage fungi is currently controlled with the addition of chemical preservatives. Consumers demand more natural products and there is a need to reduce the amount of chemical preservatives added to foods. Essential oils (EOs) and their constituents emerged as promising and effective compounds to protect foods from lipid peroxidation and provide microbiological food safety. So the aim of our study was to determinate microscopic fungi involved in contamination of bread target the genus Eurotium and to evaluate the antifungal activity of 5 EOs by vapor contact against the fungal species of this genus. In all samples after three days of cultivation fungi created colonies on the surface of the crust and the pieces of the bread samples stored on DRBC were after three days of cultivation covered with fungi colonies. Altogether 195 isolates were recovered from 8 bread samples and assigned to 10 fungal genera. The most frequently genus of fungi was Penicillium, Aspergillus, Cladosporium, Epicoccum and Eurotium. Based on phylogenetic and morphological studies, five different Eurotium species were identified: E. amstelodami, E. chevalieri, E. herbariorum, E. rubrum and E. repens. In this study, we explored the potential of five essential oils retrieved from some Lamiaceae species, concretely: basil (Ocinum basilicum L.), lavender (Lavandula angustifolia Mill.), oregano (Origanum vulgare L.), mint (Metha piperita L.) and sage (Salvia officinalis L.). Gas chromatography–mass spectrometry (GC-MS) analysis allowed for the identification of 28 compounds as main constituents. Inhibitory activity of EOs was assessed against 5 species of genus Eurotium (2 isolates for each specie). In all studied strains, the essential oils caused significant differences (P<0.05) on the mycelium growth. Our result showed that the best antifungal effect was shown by lavender and oregano (100%) to all species of tested fungi followed by mint (with the best MGI 79.89% to E. rubrum) >basil (with the best Mycelial growth Inhibition (MGI) 93.65% to E. herbariorum) and the low effect had sage essential oil (with the best MGI 68.62% to E. repens). The present study demonstrated the potential food preservative ability of essential oils from some Lamiaceae species.


KEYWORDS

antifungal activity, bread, Eurotium, essential oils, vapor

INTRODUCTION

 Baked goods are considered to be one of the most important products of the food industry. Bread is a staple food in many countries and consumed daily all over the world. Within the European Union (EU) the production of bread is relatively stable showing low growth in most western countries (Elsanhoty et al., 2013). However, like many processed foods, bakery products, are subject for physical, chemical and microbiological spoilage (Saranraj and Sivasakthivelan, 2016). The ingredients contained in bread are supportive to growth of microorganisms and multiplication at different stages of bread production, slicing and wrapping. The main microbial spoilage types of bread are moldiness and ropiness which are troublesome for bakers. Mold growth often begins within a loaf of sliced bread, where is a moisture more available than on the surface, especially in the crease (Salim-ur-Rehman et al., 2007). Mould spoilage is a serious and costly problem for bakeries. Filamentous fungi involved in bread spoilage include Rhizopus sp., and Mucor sp., Penicillium sp., Eurotium sp., Aspergillus sp. and Monilia sitophilia. Storage of bread under low humidity conditions retards mould growth. However, fungi such as  Eurotium spp. are the most common causes of spoilage of dried cereals capable of growth at 0.75 aw (Dao and Dantigny, 2011). Eurotium species are usually the first fungi to colonize improperly water allowing other species (Saranraj and Sivasakthivelan, 2016) and losses of bakery products due to mould spoilage vary between 1-5 % depending on seasons, type of products and methods of processing. Nowadays, storage fungi are commonly controlled by synthetic chemicals; however, these agents usually impact the human health and the environment adversely (Nakahara et al., 2013). In bakery products, preservatives (salts of propionic and sorbic acids) are added to prevent growth of spoilage fungi. However, in recent years, there has been consumer pressure to reduce using of preservatives (Saladino et al., 2017). Furthermore, the use of fungicides is more harmful in the post-harvest period because of the short time between treatment and consumption. Moreover some fungi have shown resistance against broad spectrum fungicides such as benzimidazoles, imazalil and prochloraz due to repeated usage (Lima et al., 2015; Farzaneh et al., 2015). Consequently, interest in more natural, non-synthesized, antimicrobials as potential alternatives to conventional antimicrobials for extending shelf life and combat foodborne pathogens has increased. Aromatic plants and their components have been examined as potential inhibitors of bacterial and fungal growth and most of their properties have been linked to essential oils and other secondary plant metabolites. Historically, essential oils from different sources have been widely promoted for their potential antimicrobial capabilities (Calo et al., 2015). The essential oils from Lamiaceae species are accredited with antioxidant, antiseptic, anti-inflamatory, antimicrobial and fungicidal properties (Burt, 2004; Kumar et al., 2014). For all these reasons, research has focused on finding natural alternatives to traditional solutions (Cherrat et al., 2014). The good natural antimicrobial has to fulfil some requirements such as: (a) to be active in low concentrations in its natural form, (b) to be inexpensive, (c) not to generate sensorial changes in the product, (d) to inhibit a wide range of spoilage and pathogenic microorganisms and (e) not to be toxic. EOs have been identified as natural food additives which can find useful application in food preservation (Davidson et al., 2013). The object of our study was to determinate microscopic fungi involved in contamination of bread with the target the genus Eurotium and to evaluate the antifungal activity of 5 EOs by vapor contact against the fungal species of this genus.

MATERIAL AND METHODS

Fungal isolation

Eight samples of bread (collected from a local markets) were mycological tested. Three samples contained rye/wheat flour (no.1, 2 and 8), two samples (no. 3, 7) contained wheat/potato flour and three breads samples were made from wheat flour only (no. 4, 5 and 6). The aqueous activity (aw) was measured for each sample (Table 1). After analysis the bread samples were three days stored at room temperature in a plastic bag, in the bread box at temperature 25±1°C. After that we detected contamination of samples by microscopic filamentous fungi with a goal to Eurotium spp. occurring in used types of bread in these ways of storage, which are the most used at our homes. Fungi that created visible colonies on the surface of the bread were inserted directly on MEA (Malt Extract agar; Merck, Germany, Klich, 2002). All the bread samples (the middle part) were cut to cubes of sides 1.5 x 1.5 x 1.5 cm and were given in number of five pieces directly on plates with DRBC (Dichloran Rose Bengal Chloramphenicol agar; Merck, Germany) and DG18 medium (Dichloran-Glycerol medium base; Merck, Germany). Cultivation was proceeded for three days in the dark 25±1°C. All experiments were performed in triplicate. Obtained results were evaluated and expressed in relative density (RD) and isolation frequency (Fr) at the genus. The isolation frequency (%) is defined as the percentage of samples within which the species or genus occurred at least once and relative density (%) is defined as the percentage of isolates of the species or genus, occurring in analysed samples (Gautam et al., 2009). These values were calculated according following formula:

Fr (%) = (ns/N)*100                                                                RD (%) = (ni/Ni)*100

Where ns = number of samples with species or genus; N = total number of samples; ni = number of isolates of a species or genus; Ni = total number of isolated fungi (Gonzáles et al. 1996).

Table 1. The flour content (reported by the producer) and aw value of used bread samples

No.  Samples aw
Bread composition reported by the producer (%)
1. Whole grain bread 0.877
47% WF, 2% PF
2. Flax bread 0.901
33% WF, 16% RF
3. Potato bread 0.909
47% WF, 2% PF
4. Wheat bread 0.922
47% WF
5. Wheat bread 0.921
41% WF
6. Wheat bread 0.922
43% WF
7. Potato bread 0.906
47% WF, 2% PF
8. Ray bread 0.895
34% WF, 17% RF

Legend: No.-number of sample, WF-Wheat flour, PF-Potato flour, RF-Ray flour, aw– Water activity

Fungal identification

Isolates of the genus Eurotium were identified to the species level according to morphological characteristics based on microscopy. To determine particular species, diagnostic literature was used as follows: Pitt and Hocking, 1985; Klich, 2002; Samson et al., 2002. Strains were inoculated in three points on the following identification media: CYA (Czapek yeast autolysate agar; Merck, Germany) with 20% and 40% (w/v) sucrose (CY20S, CY40S, respectively) and DG‐18 medium. Inoculated media were incubated at 25, 30 and 37 °C in the dark for 7 days.

Essential oils samples

The essential oils from some Lamiaceae species were used in this study, concretely: basil (Ocinum basilicum L.), lavender (Lavandula angustifolia Mill.), oregano (Origanum vulgare L.), mint (Metha piperita L.) and sage (Salvia officinalis L.). The EOs were supplied by Calendula company a.s. (Nová Ľubovňa, 238 A, Slovakia). All essential oils were extracted by hydrodistillation and stored in air-tight sealed glass bottles at 4°C.

GC-MS analysis of essential oils

Essential oils constituent were identified and the relatively composition of the oil was determined by gas chromatography followed by mass spectrometry (GC-MS). Prior to the analysis, essential oils were diluted in hexane to a concentration of l µL/mL. Analyses were carried out using an Agilent 7890A GC coupled to an Agilent MSD5975C MS detector (Agilent Technologies, Palo Alto, CA, USA) with a HP-5MS column (30 m × 0.25 mm, 0.25 mm film thickness). One microliter of the sample was injected in split mode 1:12, at an injector temperature of 250°C and at electron ionization energy of 70 eV. Analysis were measured in SCAN mode, mass range was 40-400m/z. Starting at 60°C, the oven temperature was increased at a rate of 3°C/min to a maximum of 231°C, where it was kept constant for 10 min. The identification of constituents was based on a comparison of their mass spectra and relative retention indices (RI) against the National Institute of Standards and Technology Library (NIST, USA), as well as authentic analytical standards and data from the literature (Adams, 2007). Relative proportions were calculated by dividing individual peak area by total area of all peaks. The response factor was not taken into account. Only compounds over 1% were included. Peaks under 1% were not counted.

Antifungal activity of EOs

The antifungal activity of selected EOs was investigated by the method adapted from Guynot et al. (2003). The test was performed in sterile Petri dishes (Ø 90 mm) containing 15 mL of CYA. Essential oils were diluted in ethyl acetate to give final volume 100 μL. Plates were kept in an inverted position. A sterilized filter paper (square of 1 x1 cm) was placed in the centre of the lid and 100 μl of EOs were added to the paper at concentration 500 μL/L of air. Blank was made by adding 100 μL of ethyl acetate to it. Each fungus was inoculated in the centre on Petri plates with needle-inoculated. Plates were tightly sealed with parafilm and incubated for 7, 11 and 21 days at 25±1 °C (three replicates were used for each treatment). Diameters of the growing colonies were measured. The antifungal activity was expressed in terms of percentage of mycelial growth inhibition and calculated according to the following formula:

Mycelial growth Inhibition: MGI % = [(dc-dt)/dc] *100

Where dc=average (mm) increase in mycelial growth in control, dt=average (mm) increase in mycelial growth in treatment (Marandi et al., 2011).

Statistical analysis

The basic variation-statistical values including means, standard deviation, growth inhibition percentage (%) were calculated from the obtained data using the SAS statistical program (one- factorial variance analysis ANOVA) at P<0.05 level of significance.

RESULTS AND DISCUSSION

Mycological evaluation of bread samples

Bakery products, especially bread, are intermediate moisture content products which are highly perishable. The most common forms of bread deterioration are moisture loss and microbiological spoilage. Spoilage of wheat bread and the other bakery products by colonisation and growth of fungi represents more than 90% of the total microbial contamination (Magan et al., 2003; Arroyo et al., 2008). Moreover, during  bread preparation, such as baking, only the vegetative forms of microorganisms are removed, but bacteria and fungi spores are able to survive (Nowicki et al., 1988). In all samples after three days of cultivation fungi created colonies on the surface of the crust and also the pieces of the bread samples stored on DRBC were after three days of cultivation covered with fungi colonies. Our results showed, that 195 isolates were recovered from 8 bread samples and assigned to 10 fungal genera, including Mycelia sterilia (isolates without sporulation). One of the objectives of this study was to detected contamination of samples by microscopic filamentous fungi with goal to Eurotium spp.. So the isolates of the genera Eurotium have been identified to the species level.

The filamentous fungi isolated from bread samples and their isolation frequency (Fr %) are indicated in Figure 1 and relative density (RD %) in Figure 2. Isolates of the genus Penicillium were found in all tested samples with the highest isolation frequency 100% and higher relative density 50.26% (98 isolates). According to Hocking and Pitt (1980) species of genus Penicillium, Aspergillus, Eurotium, Chrysosporium and Wallemia are the most frequently occurring xerophilic fungi in bread samples. Also Viljoen and Von Holy (1997) recovered 97 isolates from genus Penicillium (37.1%), Aspergillus (18.6%) and Cladosporium (13.4%) as the most frequented in the bread samples. The second highest isolation frequency was detected for the genus Aspergillus (87.5%) with relative density of 16.41% (32 isolates), followed by genus Cladosporium (Fr 75%) (RD 5.64%), Eurotium (RD 5.13%) and Epicoccum (RD 4.62%) with isolation frequency of 62.5%, for both. Similar results were obtained in previous study, but no isolates of the genus Eurotium were occurred in bread samples (Tančinová et al., 2012). Isolation frequency of another four genera [Mycelia sterilia and Trichoderma (RD 1.03%), Rhizopus (RD 5.64%), Alternaria (RD 2.05%)] was less than 40%. Isolates of the genus Mucor was detected in samples with low isolation frequency (Fr 37.5%) but with a higher relative density (RD 8.21%), in comparison with another genus with isolation frequency less than 40%. Vytřasová et al., (2002) in their study isolated W. sebi, E. amstelodami, E. chevalieri, E. herbariorum and E. rubrum from 10 different bread samples. Their also found that during the identification of xerophilic fungi, Eurotium species were often accompanied by Penicillium and Emericella species. These findings correlated with our results (Figure 1).

Figure 1. Isolation frequency (Fr) (%) isolated genus from bread samples (n = 8)

Figure 2. Relative density (RD) (%) isolated genera from bread samples (n = 8)

In our study were isolated the genus Eurotium from five bread samples (no. 1, 2, 4, 6 and 7). Based on phylogenetic and morphological studies 10 isolates of the genus Eurotium were identified to the species. In total, five different Eurotium species were identified: E. amstelodami, E. chevalieri, E. herbariorum, E. rubrum and E. repens. For each species two isolates were detected. The characteristics of the strains did not differ significantly from known species described in the literature (Figure 3). The colonies of all isolates had relatively slow growth on CY20S and MEA with a diameters between 20-32 mm after 7 days of incubation; were usually plane, low and voluminous with different colours (from pale to brown yellow, greyish greeny); reverse were pale, olive, orange or brown. All strains were able to developing cleisthothecia after 10 days of cultivation. Conidiophores borne from aerial hyphae; ascospores yellow, ellipsoidal, with rough walls and with two conspicuous longitudinal ridges (E. amstelodami) (Figure 3 X,Y); smooth walled, with two prominent, parallel flanges (E. chevalieri) (Figure 3 C,D,E,F) and with a shallow longitudinal furrow and roughened ridges (E. herbariorum) (Figure 3 CH,I,J); conidia on CY20S and MEA from ellipsoidal to spherical.

Figure 3. Eurotium spp.-10 days of incubation, colonies on CYA20S: E. chevalieri (A-top, B-reverse), E. herbariorum (G-top, H-reverse), E. repens (K-top, L-reverse), E. rubrum (P-top, R-reverse) and E. amstelodami (U-top, V-reverse); microstructures (scale bar = 10, 20 µm): E. chevalieri (C- heads, conidiophore, D-conidia, E,F-ascospore), E. herbariorum (CH, I-conidia, J-head, conidiophore), E. repens (M-head, conidiophore, N-cleisthothecium, O-conidia), E. rubrum (S-head, conidiophore, T-conidia) and E. amstelodami (X-head, conidiophore, Y-cleisthothecium)

Essential oils analyses by using GC-MS

Qualitative and quantitative analysis of the essential oils is listed in Table 2.

Table 2. Qualitative and quantitative analysis (%) of the used essential oils by GC-MS

RIb Component Basilc Lavender Oregano Mint Sage
938 α-pinenea 1.20 5.10
981 β-Pinenea 1.00 2.30
993 β-Myrcene 1.30
1029 p-Cymenea 9.80 1.80
1031 D-Limonenea 1.90 1.90
1034 1.8-Cineole 3.50 1.10 7.20 11.00
1043 β-trans-ocimene 1.60
1053 β-Ocimene 1.40
1062 γ-Terpinene 5.50 1.1
1101 (-)-Linalool 1.20 35.50
1107 α-Thujone 23.00
1119 Fenchyl alcohol 6.00
1148 (−)-Isopulegol 20.10
1158 (±)-Citronellal 23.10
1168 Borneola 8.50 4.20
1177 (−)-Menthol 40.80
1185 Menth-1-en-4-ol 2.50 1.20
1192 α-Terpineol 1.20
1200 Estragol 88.6
1259 Geraniala 35.10
1287 (−)-Bornyl acetate 2.30
1296 Thymola 2.2
1297 2-Undecanone 3.10 5.8
1306 Carvacrola 71.10
1419 β-Caryophyllene 3.90 1.4 3.40 5.60
1437 α-Bergamotene 2.00  
1455 α-Caryophyllene 2.40 7.00
1574 Caryophyllene oxide
total 95.30 88.80 92.50 91.90 91.40

Legend: a – Identification confirmed by co-injection of authentic standard; b – RI: identification based on Kovatʼs retention indices (HP-5MS capillary column) and mass spectra; c – relative proportion were calculated in % by dividing individual peaks area by total area of all peaks.

The main components of basil essential oil (Ocinum basilicum L.) were estragol (88.06%). Carvacrol was identified and determined as the major component of oregano (Origanum vulgare L.) (71.10%) oil. Lavender (Lavandula angustifolia Mill.) oil was comprised mainly of geranial (35.10%) and (-)-Linalool (35.50%) while sage (Salvia officinalis L.) constituted mainly α-Thujone (23.00%), (-)-Isopulegol (20.10%) and 1.8-Cineole (10.00%). In mint (Mentha piperita L. L.) essential oil, the main components were (-)-Menthol (40.80%) and (±)-Citronellal (23.10%).

 Antifungal activity of EOs on the growth of Eurotium spp.

In this study was evaluated the antifungal activity of 5 essential oils to inhibit bakery spoilage fungi – species of genus Eurotium by vapour contact. The effect of EOs basil, lavender, oregano, mint and sage on fungal growth of Eurotium spp. is showed in Table 3. In all studied strains, the essential oils caused significant differences (P<0.05) on the mycelium growth. Among the EOs, the ones that totally inhibited growth of all isolates were lavender and oregano with mycelial growth inhibition of 100% after 21st days of cultivation (Table 4). Basil, sage and mint were also able to inhibit the growth of mould, but depending of the isolates and time of cultivation. The most sensitive isolate was Eurotium amstelodami (2 isolates). Oregano and lavender EOs had antifungal activity against this strain at all days (7th, 11th and 21st) of the cultivation. The essential oil of mint inhibited its growth until 7 days completely with mycelial growth inhibition of 60.37% and basil EO was able to inhibit its growth until 11days of cultivation completely with mycelial growth inhibition of 93.65% after 21 days of cultivation.

Table 3. Antifungal activity of selected essential oils to Eurotium spp.

Species Days Essential oils (mean colony diameter in mm ± SD) (500µL/L of air) Control
Basil Lavender Oregano Mint Sage
Eurotium chevalieri

(n=2)

7th 10.50b ± 3.32 0a ± 0 0a ± 0 5.77 b ± 6.31 7.42 b ± 1.80 25.42± 1.51
11th 15.67c ± 1.8 0a ± 0 0a ± 0 10.83b ± 1.47 20.33d ± 3.95 32.33e ± 3.20
21st 22.00c ± 1.67 0a ± 0 0a ± 0 16.25b ± 2.27 27.40d ± 3.04 41.17± 2.32
Eurotium herbariorum

(n=2)

7th 5.75a ± 1.60 0a ± 0 0a ± 0 3.17a ± 3.48 6.25a ± 7.02 27.08b ± 6.81
11th 10.42± 2.11 0a ± 0 0a ± 0   6.25b ± 0.75 11.25c ± 2.82 39.67d ± 3.44
21st 17.42± 3.00 0a ± 0 0a ± 0 10.83b ± 1.47 15.00c ± 1.55 45.33d ± 0.82
Eurotium repens

(n=2)

7th 5.00± 1.09 0± 0 0a ± 0 2.67ab ± 3.33 0a ± 0 27.00c ± 5.08
11th 10.67b  ± 3.70 0a  ± 0 0a ± 0 7.50ba  ± 3.44 4.67ab ± 5.28 34.83c ± 7.80
21st 17.50± 6.20 0± 0 0a ± 0 19.08± 9.75 13.10b ± 7.17 41.75c ± 5.13
Eurotium rubrum

(n=2)

7th 9.25b ± 7.75 0a ± 0 0a ± 0 1.00a ± 1.67 7.00ab ± 8.07 26.92± 2.05
11th 16.92± 9.84 0a ± 0 0a ± 0 5.00± 3.90 20.17± 4.20 35.25± 4.37
21st 26.67± 9.70 0a ± 0 0a ± 0 8.58± 5.10 34.30bc  ± 4.69 42.67± 1.97
Eurotium amstelodami

(n=2)

7th 0a ± 0 0a ± 0 0a ± 0 0a ± 0 5.17a ± 3.06 26.17b ± 7.52
11th 0a ± 0 0a ± 0 0ab ± 0 8.83b ± 2.54 13.17b ± 4.87 38.50c ± 5.92
21st 2.75± 2.22 0a ± 0 0a ± 0 17.17b ± 7.25 20.20b ± 3.54 43.33c ± 4.92

Legend: Data in the column followed by different letters are significantly different in 95.0% Tukey HSD test, n= number of isolated tested, SD-standard deviation

Species Eurotium repens (2 isolates) was sensitive to sage until 7 days of cultivation. Moreover sage EO had higher mycelial growth inhibition (68.62%) after 21days of cultivation in a comparison with basil (58.08%) and mint (54.30%) essential oils. The best antifungal effects were shown after 21days of cultivation by mint with mycelial growth inhibition for Eurotium chevalieri (2 isolates) 60.53%, E. herbariorum (2 isolates) 76.11% and E. rubrum (2 isolates) 79.89%, respectively. These species were sensitive to oregano and lavender at all days of the cultivation, as well as other tested species of genus Eurotium. Our result showed that the best antifungal effect was shown by lavender and oregano (100%) to all species of tested fungi followed by mint (with the best MGI 79.89% to E. rubrum) >basil (with the best MGI 93.65% to E. herbariorum) and the low effect had sage essential oil (with the best MGI 68.62% to E. repens). Also authors Kocić-Tanackov et al. (2012) demonstrated that antifungal activity of oregano extract completely inhibited the growth of  Aspergillus wentii and growth of A. carbonarius and A. niger was reduced.

Table 4. Mycelial growth inhibition (%) of Eurotium spp. by tested essential oils after 21st days of cultivation.

Essential oils Species
Mycelial growth inhibition (%)
E. chevalieri E. herbariorum E. repens E. rubrum E. amstelodami
Basil 40.56% 61.57% 58.08% 37.50% 93.65%
Lavender 100% 100% 100% 100% 100%
Oregano 100% 100% 100% 100% 100%
Mint 60.53% 76.11% 54.30% 79.89% 60.37%
Sage 33.45% 66.91% 68.62% 19.62% 53.38%

In case of mint and basil EOs, similar results were obtained in previous studies. The oregano, thyme, clove, lavender, mint sage and eucalyptus essential oils were tested against three strains of A. flavus. The best antifungal activity was shown by oregano, thyme, clove and lavender, but mint essential oil was able to inhibit growth of Aspergillus sp. until 14 days of cultivation. Sage and eucalyptus EOs showed very low or no effect on the growth of tested fungi (Císarová and Tančinová, 2015). Moreover, sage essential oil showed very low inhibitory effect on the growth of tested aspergilli in another study, but was able to inhibit production of mycotoxins by tested fungi. Basil essential oil was able to inhibit growth of all tested strains Eurotium spp. on all days of cultivation or after 14th days (Císarová et al., 2014). The results of authors Alves-Silva et al. (2013) showed that the basil EO showed higher antifungal activity against Mucor racemosus and Penicillium chrysogenum. The present study confirmed the antifungal activity of tested essential oils. Carvacrol and Geranial were the major components of oregano and lavender EOs in this study. These terpenoids are active against a broad spectrum of microorganisms, with Carvacrol (a monoterpenoid phenol) as one of the most active components. Carvacrol and p-Cymene (its monoterpene precursor) are present in oregano and thyme respectively, and they have a promising potential to be used as natural preservatives when both are used together (Gutiérrez-del-Río et al., 2018). The Menthol was the major component of mint EOs. Authors Abbaszadeh et al. (2014) demonstrated that Menthol was found with the most potent inhibitory activity for Cladosporium spp. and Aspergillus spp. (with the MIC of 125 mg/mL), followed by Fusarium oxysporum, Penicillium spp., Rhizopus oryzae, Botrytis cinerea and Alternaria alternata. Estragol (Methyl chavicol) is usually the major component of the basil EOs and these EOs with following chemical composition: 1.8-Cineole, Linalool, Camphor, Methyl chavicol, and Eugenol are often related with greater antifungal activity (Pirbalouti et al., 2013). So the antifungal activity of the essential oils or extract is directly related to chemical composition of the EO, climatic, soil or seasonal variation during growing of plants, extraction used and last but not least to microbial species tested.

CONCLUSION

Nowadays, mould growth is still a cause of high losses to the bread-producing industry. Bread is known as a high moisture product with aw values between 0.96 and 0.98 and is included in commodities with short durability. After three days of storage was found the visible destruction of bread by growing fungi in both: bread samples stored in plastic bag on the surface of the crust and on the pieces of the bread samples stored on DRBC. Our results showed that 195 isolates were recovered from 8 bread samples and assigned to 10 fungal genera. The most frequently genus of fungi was Penicillium, Aspergillus, Cladosporium, Epicoccum and Eurotium. One of the objectives of this study was to detect contamination of samples by microscopic filamentous fungi with goal to Eurotium spp. In total, five different Eurotium species were identified: E. amstelodami, E. chevalieri, E. herbariorum, E. rubrum and E. repens. These species were used in next – essential oils studies. The present study demonstrated the potential food preservatives ability of oregano, lavender, mint, basil and sage EOs. The best antifungal activity was shown by oregano and lavender EOs, but other essential oils also showed inhibition effect on the growth of fungi Eurotium spp. In bakery products, preservatives (salts of propionic and sorbic acids) are added to prevent growth of spoilage fungi. However, in recent years, consumers demand to reduce the use of chemical preservatives. So the essential oils could become an effective modern alternative without health hazards for customers especially for bakery products.

Acknowledgments: This work was supported by project no. FPPV-09-2019 and by Slovak Research and Development Agency under Contract no. APVV-16-0289.

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