The inhibitory potential of Zataria multiflora and Syzygium aromaticum essential oil on growth and aflatoxin production by Aspergillus flavus in culture media and Iranian white cheese

Abstract Antifungal activity of essential oils (EOs) of Zataria multiflora (thyme) and Syzygium aromaticum (clove) against Aspergillus flavus growth and aflatoxin B1 production was studied in potato dextrose agar (PDA) and potato dextrose broth (PDB), as well as in Iranian white cheese as a food model. The results showed that the inhibitory potential of EOs in the PDB medium is more than PDA medium. Clove EO on PDB medium prevented fungal growth and aflatoxin B1 production at 300 and 100 ppm, respectively. However, the thyme EO was not able to inhibit fungal growth completely and showed the strongest inhibition effect at 400 ppm. EOs also had more inhibitory activity in laboratory culture media than the food environments. EOs in all concentrations reduced aflatoxin B1 production and fungal growth in cheese, but only the clove EO at 150 ppm was able to prevent the fungal growth and aflatoxin B1 production completely. Thyme EO reduced aflatoxin B1 value to below detection threshold (2 ppb) at 10 μl. Our findings propose EOs as a natural inhibitor to control fungal contamination of foodstuffs such as Iranian white cheese.

Thyme is a spice plant belonging to the Lamiacea family that geographically grows only in Iran, Pakistan, and Afghanistan. It is known as "Avishan-e-Shirazi" in Iran. Carvacrol and thymol are known as main phenolic compounds in its essential oil (EO) composition (Azizkhani et al., 2013). Clove is small shrub with evergreen and sharp leaves belonging to Myrtaceae family that is native to Indonesia and East Pacific.
In this study the inhibitory potential of clove and thyme EOs on growth and aflatoxin B 1 formation by A. flavus ATCC 15546 was evaluated in culture media (potato dextrose agar [PDA] and potato dextrose broth [PDB]) and in Iranian ultrafiltered cheese.

| Preparation of EOs
Using industrial boilers, EOs of thyme leaves was extracted by hydrodistillation method in which the EO extraction efficiency was equal to 1.1%. The extraction efficiency for thyme has been previously reported as 1.66% (Moosavy et al., 2008), which was generally influenced by many factors, such as the plant leaves to water ratio, time, and temperature of extraction process. Initially, thyme was sun dried and the EO was extracted using hydrodistillation method in semi-industrial boilers (manufactured by Kashan-Iran Taghtiran Co.). For extraction of thyme EO, the proportion of water to dry leaves of Zataria multiflora was 800/100 (l/kg). Furthermore, the EO of clove was purchased from a Shargh Osareh Co. (Chenaran Industrial Estate, Mashhad, Iran). Oil extraction efficiency (weight basis) was calculated from the following equation (Shukla, Kumar, Singh, & Dubey, 2009): where A = weight of dry plant, B = weight of essential oil obtained.
For preparing the EOs for experiment, 0.5 ml of the desired EOs was mixed with 9.5 ml of distilled water and three drops of Tween 80 solution. Using a shaker device the reaction mixture was vibrated five times, each time for 30 s.

| Standards
Standard for aflatoxin B 1 were obtained from Sigma Chemical Co.

| Preparation of conidial suspension
Aspergillus flavus (obtained from the Department of Plant Protection, Faculty of Agriculture, University of Shiraz, Shiraz, Iran) was cultured on PDA slope (Merck, Darmstadt, Germany) for 7 days at 28 ± 1°C. Conidia were cultured by adding 0.05% Tween 80 solution (Merck, Darmstadt, Germany) to the culture medium and gently scraping the mycelia with a sterile inoculating loop to release spores. Conidial concentration was determined by a hemocytometer and the suspension was diluted with 0.05% Tween 80 solution to give a final concentration of 10 6 ml (Selvi, Joseph, & Jayaprakasha, 2003).

| Measurement of the EOs effectiveness on the A. flavus growth in PDA culture media
Plates containing any of thyme or clove EOs were prepared. In order to culture A. flavus, one of the fungus 3-day cultures was divided into parts (2 × 2 mm), and each of these parts was placed at the center of the plates and incubated at 28°C. The positive control plate contained fungi with no EO, and negative control plate only contain EOs with no fungi. The plates were checked every day, and the growth of fungus was enumerated as a function of hyphae growth radius. Comparison of this radius in plates containing different concentrations of EO and positive control plate was introduced as inhibitory effect of EO on the fungi growth (Gandomi et al., 2009).
where R0: radius of fungal hyphae growth in the positive control sample, R1: radius of fungal hyphae growth in the test sample.

| Measurement of EOs inhibitory effects on the A. flavus growth in PDB medium
PDB medium was prepared in accordance with Difco method (Difco Laboratories, 1984). Erlenmeyer flasks containing a 3-day culture of fungus were kept in incubator at 28°C for 10 days, and their contents were evacuated in preweighed filter paper that had been put in a desiccator. After complete drawl of fluid, filter paper containing fungal hyphae were transferred to 40°C vacuum oven. Then, the initial weight of filter paper was subtracted from filter paper containing fungus, and dry weight of fungus hyphae was found. Comparison of the hyphae weight of the test sample with the weight of fungus in positive control sample indicated the effect of the tested EOs in inhibiting the fungal growth. After separating the solid part, the permeate was excluded for measuring aflatoxin B 1 tests (Rasooli et al., 2008).

| Determining minimal inhibitory concentration and minimum fungicidal concentration
Suspension spore (0.1 ml) was added to each culture media containing different concentrations of EO. Plates were then incubated at 28°C for 48 hr. The lowest EO concentration which prevented visible microbial growth in that plate was determined as MIC or minimal inhibitory concentration of EO. Some agar pieces were removed from the plates containing MIC of EO and some agar pieces were removed from two plates with higher and lower EO concentration than MIC and finally they were cultured on PDA medium. Moreover, the first concentration at which visible microbial growth was observed was determined as the MFC or minimum fungicidal concentration (Mahboubi & Haghi, 2008).

| Extraction of aflatoxin B 1 from the samples cultured on solid media
Plates containing A. flavus in PDA medium after 5 days of incubation at 28°C were prepared for measuring aflatoxin B 1 test. Culture medium content was completely transferred to 100 ml beaker and 8 ml of chloroform-water mixture with ratio of 1-15 were added to the beakers, and beakers' content was mixed for 20 min. Then the reaction mixture was filtered by filter paper and permeated into second series of beakers. The remaining solids were returned to first series of the beakers, and all steps were repeated with 7 ml of solvent mixture.
Finally, the filter paper was washed with 1-1.5 ml of chloroform, and all permeate obtained were collected in the second series of the beakers. Beakers were completely dried and washed with 3 ml of methanol and the collected solutions were poured in a special container with a lid that prevented the evaporation of methanol (Mayer, Färber, & Geisen, 2003).

| Extraction of aflatoxin B 1 from the samples cultured in PDA medium
First, 15 ml of filtered permeate and 15 ml of water and chloroform mixture were mixed for 20 min, passed through a separator funnel to separate the layers. Then, the lower layer, which contained aflatoxin B 1 , was collected in a 100-ml beaker and completely dried in vacuum oven at 40°C. Beakers were completely dried and washed with 3 ml of methanol and the collected solutions were poured in a special container with a lid that prevented the evaporation of methanol (Razzaghi-Abyaneh et al., 2008).

| Measurement of aflatoxin B 1 in food samples
In this study, high-performance liquid chromatography was used to determine the level of aflatoxin B 1 in each treated samples of EOs in PDA and PDB medium. For this purpose, 50 μl of condensed sample and various dilutions of the standard solution were spot on the chromatography plates coated with silica (G-HR type, 20 × 20 cm and unsaturated with a thickness of 25 mm). The solvent system composed of ether, methanol, and water with volume ratios of 96, 3, and 1, respectively, were used to separate all four components of aflatoxin B 1 . The solvent system was poured in the tank and the door was closed. After development of the plates to 1.5 cm from the edge of top of the plate, chromatography plates were removed from the tank, and dried under the hood. To confirm the presence of aflatoxin B 1 , trifluoroacetic acid was sprayed on the plates. The spots were examined under ultraviolet light and their exact location was marked with a needle.
The marked locations were completely scraped and transferred to test tubes. To each tube, 2 ml of methanol was added and kept in the refrigerator. The tubes were then removed from the refrigerator and after mixing for 5 min they were centrifuge at 330g.

| Estimation of A. flavus growth in Iranian white cheese
For estimation of A. flavus growth, using a homogenizer, 20 g of cheese samples were mixed with 90 ml of distilled water for 30 s.

| Statistical analyses
SPSS Software (Version 8.2; SAS Institute, Cary, NC, USA) was used to conduct statistical analyses. These data were analyzed by one-way analysis of variance (ANOVA). Comparison of means was performed by Duncan's multiple range tests and a p < .05 was considered statistically significant.

| Mold inhibitory potential of EOs in PDA medium
The results of the inhibitory potential of thyme and clove EOs against  Kumar et al. (2008) reported that the thyme EO completely inhibit the growth of A. flavus at concentration of 700 ppm.
The results in Tables 1 and 2 also showed that in PDA medium, the inhibitory potential of the two EOs against aflatoxin B 1 production is greater than their inhibitory effect on A. flavus growth; for example, clove EO completely inhibited aflatoxin B 1 production at 100 ppm, while double concentration of clove EO was needed to prevent A. flavus growth completely. The same trend was also observed for the thyme EO. (2) the absence of agar in PDB culture medium and higher water availability of PDB than PDA medium; (3) presence of higher volume of dissolved oxygen in PDB medium than PDA medium; (4) higher incubation time of PDB medium (10 days) than that of PDA medium (3 days) and consequently inability of some inhibitory compounds to maintain their inhibitory effects when incubation time is prolonged. Accordingly, to achieve a correct comparison, the effect of time and amount of culture medium must be omitted for PDA and PDB culture medium. In this regard, all data related to PDA and PDB media were calculated on the basis of medium weight (g) and unit of time (day).

| Inhibitory potential of EOs in PDB culture medium
The results of the inhibitory effects of the EOs against aflatoxin B 1 production in both PDA and PDB media are shown in Figures 1   and 2. It was found that at the same time, the aflatoxin B 1 production in PDA medium was higher than that of PDB medium, which was because of the freely movement of hyphae in the PDB culture medium and greater possibility to access nutrients during incubation. In the case of PDB medium, the hyphae remained at the growth phase for longer time and aflatoxin B 1 production was postponed. Moreover, the pyruvate concentration per unit volume of the culture medium is a critical factor for aflatoxin B 1 production by A. flavus and since in PDB medium the distribution of compounds is more extensive than PDA In each column different superscript letters indicate significant differences (p < .05). EO, essential oil; PDA, potato dextrose agar.
T A B L E 2 Effect of thyme EO on growth and aflatoxin B 1 production by Aspergillus flavus in PDA medium culture medium, the pyruvate concentration in PDB is less than that of PDA and consequently the aflatoxin B 1 production was more restricted in PDB medium. Also, in PDA medium inhibitory effect of EOs was only limited to the location where surface of the hyphae was contacted to the solid media. While in the PDB medium, all of the essential oil had contact to the hyphae.

| Inhibitory potential of EOs in Iranian white cheese as a food model
Results related to the inhibition effects of thyme and clove EOs in Iranian white cheese are presented in  (Bagamboula, Uyttendaele, & Debevere, 2004). Accordingly, the reduction of the inhibitory potential of the EOs against A. flavus growth and aflatoxin B 1 production was ascribed to the created interactions between polyphenols and proteins of cheese (Ultee & Smid, 2001).

| CONCLUSION
In this study it was found that EOs of thyme and clove have antifungal activity, so clove EO in PDA and PDB media and Iranian white cheese completely prevented the growth of A. flavus and aflatoxin B 1 production. EO of thyme also with lower inhibitory potential compared with clove EO decreased A. flavus growth and aflatoxin B 1 production. It was found that the inhibitory potential of each of the EOs in the PDB medium is more than PDA medium, plus deterrence potential of EOs of thyme and clove in a food environment was more than laboratory culture media. Finally, due to strong antifungal effects of EOs of thyme and clove, they can be used as a mold inhibitor in food. However, it is necessary that further studies to be done in the field of economic value and organoleptic effects of each of the EOs in the food.

CONFLICT OF INTEREST
None declared.  In each column different superscript letters indicate significant differences (p < .05). EO, essential oil.