Evaluation of antifungal activity of essential oils against potentially mycotoxigenic Aspergillus flavus and Aspergillus parasiticus

Evaluation of antifungal activity of essential oils against potentially mycotoxigenic Aspergillus flavus and Aspergillus parasiticus

Fernanda C. da Silva^I; Sara M. ChalfounII,^*; Virgínia M. de Siqueira^III; Deila M. dos S. Botelho^II; Nelson Lima^III; Luís R. Batista^IV

^IDepartamento de Biologia, Microbiologia Agrícola, Universidade Federal de Lavras, Brazil

^IIEmpresa de Pesquisa Agropecuária de Minas Gerais, Universidade Federal de Lavras, Brazil

^IIIIBB/Centro de Engenharia Biológica, Universidade do Minho, Braga, Portugal

^IVDepartamento de Ciência dos Alimentos, Universidade Federal de Lavras, Brazil

ABSTRACT

The antifungal activity of essential oils of fennel (Foeniculum vulgare Mill., Apiaceae), ginger (Zingiber officinale Roscoe, Zingiberaceae), mint (Mentha piperita L., Lamiaceae) and thyme (Thymus vulgaris L., Lamiaceae) was evaluated against mycotoxin producers Aspergillus flavus and A. parasiticus. High Resolution Gas Chromatography was applied to analyze chemical constituents of essential oils. The effect of different concentrations of essential oils was determined by solid medium diffusion assay. Mycelial growth and sporulation were determined for each essential oil at the concentrations established by solid medium diffusion assay. At the fifth, seventh and ninth days the mycelial diameter (Ø mm) and spore production were also determined. FUN-1 staining was performed to assess cell viability after broth macrodilution assay. Trans-anethole, zingiberene, menthol and thymol are the major component of essential oils of fennel, ginger, mint and thyme, respectively. The effective concentrations for fennel, ginger, mint and thyme were 50, 80, 50 and 50% (oil/DMSO; v/v), respectively. The four essential oils analysed in this study showed antifungal effect. Additionally, FUN-1 staining showed to be a suitable method to evaluate cell viability of potential mycotoxigenic fungi A. flavus and A. parasiticus after treatment with essential oils.

Keywords: Foeniculum vulgare FUN-1 fungi Mentha piperita Thymus vulgaris Zingiber officinale

Introduction

Aspergillus flavus and A. parasiticus are the predominant species responsible for aflatoxin contamination of crops prior to harvest or during storage (Yu et al., 2004). Many strategies are taken intending to prevent fungal growth and further mycotoxin production and food contamination, including chemical, physical or biological treatments which require sophisticated equipment and expensive chemicals or reagents (Reddy et al., 2010a). The use of natural plant extracts provides an opportunity to avoid chemical preservation, thus the search for new antifungal material natural sources for food preservation has increased (Soliman & Badea, 2002; Irkin & Korukluoglu, 2007).

Mycotoxins, i.e. toxic secondary metabolites produced by filamentous fungi, of most concern are produced by species within the genera Aspergillus, Fusarium and Penicillium, which occur in major food crops in the field and continue to contaminate during storage (Reedy et al., 2010b). Aspergillus species are well-known as active agents in decay processes, as human and animal pathogen and as producer of valuable metabolic products (Raper & Fennel, 1965). Aspergillus species are also able to cause diseases in economically important crops, such as maize, corn and peanuts, and to produce potent mycotoxins.

Plants known as condiments are traditionally used to enhance taste or aroma of food, and their essential oils (EO) represent a complex mixture of natural substances. EO are known to possess antibacterial and antifungal activity and have been empirically used as antimicrobial agents (Burt, 2004; Bakkali et al., 2008), but the spectrum of activity and mechanisms of action remain unknown for most of them. For EO of plants used as condiments, the antimicrobial activity seems to be associated with phenolic compounds (Simões & Spitzer, 2000) and the antimicrobial effect is related mainly with changes in the permeability and integrity of cell membrane (Lambert et al., 2001; Carson et al., 2006).

FUN-1 is a halogenated asymmetric cyanine compound, virtually no fluorescent in aqueous solution. FUN-1 is permeable to cell membrane and flows through freely into the cell and initially appears in the cytoplasm as a bright diffuse green/yellow stain. In normal fungal cells, FUN-1 is metabolically converted into orange/red cylindrical intravacuolar structures (CIVS). Adenosine triphosphate (ATP) is required for CIVS formation. However, in non-metabolic active cells, the dye remains in the cytoplasm in a diffuse pattern, thus indicating a disorder in the viable state of the cells (Millard et al., 1997).

The aim of this study was to investigate inhibitory concentrations of fennel (Foeniculum vulgare Mill., Apiaceae), ginger (Zingiber officinale Roscoe, Zingiberaceae), mint (Mentha piperita L., Lamiaceae), and thyme (Thymus vulgaris L., Lamiaceae) against mycotoxin producers Aspergillus flavus and A. parasiticus. Mycelial growth and sporulation inhibition effects and broth macrodilution assay for cell viability test using FUN-1 staining were also studied.

Materials and Methods

Essential oils

Foeniculum vulgare Mill., Apiaceae, Zingiber officinale Roscoe, Zingiberaceae, Mentha piperita L., Lamiaceae and Thymus vulgaris L., Lamiaceae essential oils were purchase from Ferquima Industry and Trade Ltda, Vargem Grande Paulista-SP, Brazil, and stored in hermetically sealed flasks at 4 ºC. Samples of the essential oils as a reference are stored in the Agricultural and Livestock Minas Gerais State Research Institution - EPAMIG, Lavras-MG, Brazil. Analytical gas chromatography was carried out on a Varian CP-3380 chromatograph equipped with a flame ionization on Chromatography Laboratory of the Department of Chemistry, Federal University of Minas Gerais, Belo Horizonte/MG, Brazil.We used a column HP-5 (HP) 30 m x 0.25 mm with a gradient of temperature: 50 ºC, 3 min, 3 ºC/min to 170 ºC; injector (split 1/200) to 200 ºC and detector 200 °C. N₂ as carrier gas (2 mL/min) and injection volume 1 mL. Samples were diluted to 0.5% in chloroform. The identification of peaks was made by calculation of retention index standards for hydrocarbons C10 to C18 linear and comparison with literature data (Adams, 2007). The EO were tested pure and dissolved in DMSO (dimethyl sulfoxide) at concentrations of 90/10, 80/20, 70/30; 60/40; 50/50 (v/v; oil/diluent).

Fungal isolates and culture conditions

Aspergillus flavus (CMEcoCentro 00062) and Aspergillus parasiticus (CMEcoCentro 00040) isolated from peanuts and coffee, respectively were used for EO antifungal assay. The fungi were maintained in tubes with potato dextrose agar (PDA: 4 g/L potato, dextrose 20 g/L, agar 15 g/L) at 4 °C.

Antifungal activity of essential oils

Effect of different concentrations of essential oils

A spore suspension (10⁶ spores/mL) was prepared and 120 µL of it was spread on dishes with MEA. After absorption, a sterile filter paper disc (Ø 6 mm) was added to the center of the plate and 10 µL of different dilutions (100, 90, 80, 70, 60 and 50%) of EO were added on it. Filter paper disks impregnated with DMSO only were used as control to confirm no solvent effect on bioactivity. The dishes were incubated for five days at 25 °C. The lowest concentration able to inhibit each fungus (visible inhibition halo around the disk of filter paper) was used in the following test. The tests were performed in triplicate.

Mycelial growth and sporulation assay

The effect of each EO (i.e., at the EO concentration determined by solid medium diffusion assay) on mycelial growth and sporulation were performed using the in vitro bioanalytical method. For 250 mL of culture medium Malt Extract Agar (MEA: 20 g/L malt extract, 5 g/L peptone, 20 g/L agar) was added 250 µL of each EO. 10 mL of culture medium containing EO was added to Petri dishes (Ø 90 mm). After solidification, inoculation was made of each fungus at a central point of the dishes. The dishes were incubated at 25 °C for nine days. At the fifth, seventh and ninth days, it was measured the diameter of the colony (Ø mm) and the produce of spore in a Neubauer chamber. The tests were performed in triplicate.

Broth macrodilution test using hyphae as inoculum

Fungi were tested by the broth macrodilution method for antifungal activity of EO described previously (Espinel-Ingroff et al., 1995). The spore suspension was harvested by flooding each colony with 2 mL of sterile 0.85% saline and further diluted to in RPMI 1640 medium to obtain 10⁶ spores/mL. Spore suspension was previously incubated at 35 °C for 24 h for the formation of hyphae. A total of 100 µL of the fungal suspensions was inoculated into 24-well plates containing 800 µL of RPMI medium and 100 µL of each EO. Final concentrations, i.e., 50%, 50%, 50% and 80% for fennel, mint, thyme and ginger, respectively were used. EO antifungal activity was evaluated by FUN-1 viability test as further described. RPMI medium added with DMSO alone was used as control to confirm no solvent effect on bioactivity.

FUN-1 viability staining of hyphae

At each EO concentration tested, fungal hyphae were used for FUN-1 staining (Molecular Probes, The Netherlands) at 48 h of incubation. For FUN-1 staining, 50 µL of hyphae suspension plus 50 µL of FUN-1 were added on a Ependorff tube, homogenised, following incubation in the dark at 30 ºC during 30 min. 20 µL of Calcofluor White M2R (CW: 4,4'-bis[4-anilino-6-bis(2-ethyl)amino-s-triazin-2-ylamino]-2,2'-disulfonic acid, Molecular Probes, The Netherlands) (25 µM) which stains fungal cell walls was also used as counterstain. The excitation/emission wavelength for CW was 346/433 nm and the signal acquired was blue. 20 µL of each sample were pipetted on glass slides and observed under an Olympus BX51 epifluorescent microscope using UV light equipped with 40x/0.30 and 10x/0.65 objectives. FUN-1 stains (EX 450-490 nm, EM 520) the dead cells with a diffuse yellow-green fluorescence and the metabolically active cells with red Cylindrical Intra-Vacuolar Structures (CIVS). The images were acquired with a colour camera Zeiss AxioCam HRc using the software CellB^®. Storage and handling of reagents were performed as recommended by the supplier. Two samples from each well were assayed and examined for staining with FUN-1.

Statistical analysis of experiments

The data were analyzed by the statistical program Analysis of Variance System for Balanced Data-SISVAR (Ferreira, 2000).

Results

Essential oils and major constituents

The major components of EO are listed in Table 1. Trans-anethole (51.5%), zingiberene (32.7%), menthol (55.4%), and thymol (54.9%) were the main components of fennel, ginger, mint, and thyme, respectively.

Effect of different concentrations of essential oils

Inhibition zones diameters in solid culture of A. flavus and A. parasiticus are listed in Table 2. The same concentration of each EO was established for both A. flavus and A. parasiticus, i.e. 50% for fennel, mint, and thyme EO and 80% for ginger EO (Table 2). For both fungi the EO of ginger a halo of inhibition was observed at a concentration of 80%.

Mycelial growth and sporulation assay

The effects of EO on mycelial growth and sporulation of A. parasiticus are reported in Table 3. Throughout the study it was found that each EO showed different effect on mycelial growth and fungal sporulation. All essential oils showed inhibitory effect on mycelial growth of A. parasiticus at the seventh and ninth days of analysis (Table 3). For fennel and thyme EO, similar results were observed at the fifth day. Ginger EO showed effect in inhibition of mycelial growth at seventh day (44.66 mm). For sporulation was observed that thyme EO showed inhibitory effect on all analysed days. At the seventh day, the EO of fennel caused a considerable increase in number of spores (4.10x10⁶ spores/mL). Same effect was observed for the EO of mint (2.5x10⁶ spores/mL) and ginger (2.18x10⁶ spores/mL) on nine days. The EO of thyme showed the best inhibitory effect on mycelial growth and sporulation of A. parasiticus (Table 3).

There was no difference in the effect of EO on the A. flavus on the fifth, seventh and ninth days of analysis. The EO of thyme highlighted in the evaluation of mycelial growth and sporulation of A. flavus. The EO of fennel presented similar to sporulation control, it is not considered effective in this study.

FUN-1 viability staining

In this study, positive control of A. flavus and A. parasitucus (i.e. fungi without the addition of EO) showed green fluorescent hyphae with clearly CIVS (Figures 1A and 2A). Thus, fungi were classified as viable. This same feature was observed with ginger EO, and CIVS were also observed within both A. flavus and A. parasiticus hyphae after treatment (Figures 1B and 2B). These result correlate to results found in growth effect assay in which a high ginger oil concentration (80%) did not considerably affect fungal growth. The presence of CIVS after treatment with ginger oil classifies both fungi as viable. On the other hand, only bright diffuse yellow/green cytoplasm was observed after treatment with fennel, mint, and thyme (Figures 1C-E and 2C-E). Thus, in this case fungi were classified as non-viable. These same EO showed inhibition effects in both fungi; this feature was also confirmed by FUN-1 staining results.

Discussion

Recently, the scientific interest in biological properties of EO has been increased. New researches about biological active secondary compounds present in EO of plants have been seen as a potential way to control fungal contamination (Burt, 2004; Soliman & Badea, 2002; Tajkarimi et al., 2010). The results obtained on the chemical characterisation of the fennel, ginger, mint, and thyme EO used in this study is according to the large chemical polymorphism for the majority of EO (Sridhar et al., 2003; Gulfraz et al., 2008; Matan et al., 2009; Razzaghi-Abyaneh et al., 2009).

Thyme EO showed better antifungal effect against Aspergillus flavus and A. parasiticus. This antifungal activity is reported in several studies. Soliman & Badea (2002) reported that thyme EO (≤500 ppm) completely inhibited the growth of A. flavus and A. parasiticus. This same effect was observed by Nguefack et al. (2004), that the thyme OE at a concentration of 200 ppm reduced the radial growth of A. flavus by 81%. Complete inhibition of conidial germination of A. flavus was achieved by 1000 ppm. Among various plants tested, thyme EO inhibited growth and aflatoxin production of A. parasiticus (Razzaghi-Abyaneh et al., 2009). The same effect was observed for A. flavus (Kumar et al., 2008). Lis-Balchin & Deans (1997) reported that strong antimicrobial activity could be correlated with essential oils containing high percentage of monoterpenes, eugenol, cinnamic aldehyde, and thymol. It seems possible that phenol components may interfere with cell wall enzymes like chitin synthase/chitinase as well as with the α- and β-glucanases of the fungus (Adams et al., 1996). Thymol, the major constituent of the Thymus vulgaris, in a study by Zambonelli et al. (2004) was correlated with damage to the cell, as increase in the vacuolization of the cytoplasm and an accumulation of lipid droplets, ripples in the plasmalemma and changes in the mitochondria and endoplasmic reticulum of Colletotrichum lindemuthianum and Pythium ultimum. Rasooli et al. (2006) observed severe hyphae collapsing, plasmatic membrane rupture and destruction of mitochondria in Aspergillus niger treated with essential oils of Thymus eriocalyx and T. xporlock.

Antifungal activity of fennel and mint EO is reported in other investigations (Duarte et al., 2005; Özcan et al., 2006; Sokovic & Van Griensven, 2006; Gulfraz et al., 2008; Freire et al., 2011). The effect antifungal of fennel EO is directly related to its main component trans-anethole (Patra et al., 2002; Muckenstrum et al., 1997) and the effect of mint EO can be attributed to menthol and 1,8-cineole, which exhibited very good antifungal properties (Griffin et al., 2000).

The ginger EO showed low antifungal activity on the mycelial growth of A. flavus when compared to other EO and ineffective inhibitory effect for the other parameters tested and fungi. No inhibitory effect of fungal growth by this EO was reported in studies by Sridhar et al. (2003) and Thanaboripat et al. (2007). Holley & Patel (2005) reported that spices, such as ginger, black pepper, red pepper, chili powder, cumin and curry powder showed lower antimicrobial properties.

The differences between the antifungal activity of essential oils studied against A. flavus and A. parasiticus and permanence of the inhibitory effect over time are directly related to chemical composition of the EO, the microbial species, the mechanism of action and the method used to analyze the antimicrobial activity of the EO (Bakkali et al., 2008; Pinto et al., 2009; Tajkarimi et al., 2010).

In this study, the method used to evaluate the antifungal activity of EO was solid medium diffusion. Research has shown that this method has limitations. The use of the method is limited to fast-growing microorganisms, aerobic or facultative aerobic (Ostrosky et al., 2008). Another problem noted is the uneven distribution lipophilic components of EO which results in unequal concentrations of EO in the agar causing the formation of regions with varying antimicrobial activity which can lead to misinterpretations (Lambert et al., 2001; Suhr & Nielsen, 2003). Ostrosky et al., 2008 also report that the agar diffusion method, the antimicrobial activity of oil can be influenced by the culture medium (agar concentration and origin), pH, oxygen availability, amount of inoculum and incubation conditions. Studies to compare methods of application of EO with antimicrobial activity have been performed (Hammer et al., 2003; Suhr & Nielsen, 2003).

To corroborate the result of in vitro and verify the feasibility of the method for testing antifungal with EO, cell viability associated FUN-1 staining was performed. This test showed to be a suitable method to evaluate cell viability of potential mycotoxigenic fungi A. flavus and A. parasiticus after treatment with EO. Studies show that the applicability of fluorescent dyes as a reliable and fast alternative to assess fungal cell viability in environmental and medical fields has been increasingly used (Pina-Vaz et al., 2001; Bowman et al., 2002; Wierzchos et al., 2004). In a study with A. fumigatus, FUN-1 and CW were apply to document patterns of structural changes and its relationship with MIC after following antifungal treatment (Gangwar et al., 2006). CW binds to glucans and chitin in the cell walls and septa of fungi and it has been useful to analyse fungal morphology and as a counterstaining (Agger et al., 1998). Antifungal activity analyses of EO against pathogenic fungi such as A. fumigatus and Candida spp. has been carried out using FUN-1 as well (Pinto et al., 2009). Although the interest in discover new antifungal agents which activity against mycotoxigenic fungi has been increased, a lack of studies in this area still remains and more efforts to establish standardized methods in this area is needed.

Already published results of studies which evaluate antifungal agents indicate that FUN-1 method correlates closely to colony counts method. FUN-1 staining is as a rapid and sensitive method for assaying the viability of fungi and may be applied as an alternative for antifungal tests using EO. In addition, essential oils showed inhibitory effect on fungi tested being, the thyme EO more effective. However, it is necessary to standardize the method for testing antifungal agents with essential oils and more detailed studies on the mechanism of action of these substances in the fungal cell.

Acknowledgements

The authors are grateful to the colleagues from Laboratory of EPAMIG and Microbiology DEB/UFLA and the Micoteca da Universidade do Minho, Center for Biological Engineering, UMINHO for their support to perform this work. A special aknowledgment is also due to the FAPEMIG and MUM-UMINHO for the financial support of bench work and to CAPES for granting the first author with a PhD scholarship.

Received 28 Nov 2011

Accepted 6 Feb 2011

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Publication Dates

• Publication in this collection
  17 Apr 2012
• Date of issue
  Oct 2012

History

• Received
  28 Nov 2011
• Accepted
  06 Feb 2011