Abstract: The antifungal effect of Na2EDTA on an Aspergillus carbonarius, an A. ibericus, an ochratoxigenic A. niger and a non-ochratoxigenic A. niger strain was studied. Also, the effect of Na2EDTA on the production of ochratoxin A by A. carbonarius and the ochratoxigenic A. niger was evaluated. The poisoned food technique was used with CYA medium supplemented with 0, 1 and 10 mmol L-1 of Na2EDTA. The colony diameters were recorded daily and the amount of ochratoxin A produced was quantified every two days. Significant reductions of growth rates were observed in the presence of Na2EDTA being the calculated EC50 of 2.1 mmol L-1 for A. carbonarius, 0.9 mmol L-1 for A. ibericus, 2.0 mmol L-1 for the ochratoxigenic A. niger and 4.1 mmol L-1 for the non-ochratoxigenic A. niger. Furthermore, 10 mmol L-1 Na2EDTA delayed the production of ochratoxin A and reduced the levels in approximately 99% during 8 days. Na2EDTA is frequently used in the food industry and in agriculture agrochemicals and its effects on ochratoxigenic black aspergilli is not well known. This study showed that Na2EDTA can significantly reduce the growth rates of tested fungi and its ochratoxin A production.
INTRODUCTION
Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species in several agricultural commodities. Therefore, OTA is found in food products such as breakfast cereals, coffee, cocoa products, dried vine fruits, dried figs, beer and wine. The presence of OTA in grapes is due to A. carbonarius and to some ochratoxigenic isolates of the A. niger aggregate (Serra et al., 2003; Gomez et al., 2006). A. carbonarius isolates are producers of higher amounts of OTA and are more frequent on grapes than the ochratoxigenic isolates of the A. niger aggregate (Serra et al., 2006; Esteban et al., 2004). Aspergillus niger aggregate strains are also able to degrade OTA into the less toxic ochratoxin α (Varga et al., 2000; Abrunhosa et al., 2002).
Ethylenediaminetetraacetic Acid (EDTA) is a strong chelating agent that forms several metal and salt complexes. EDTA and EDTA-complexes are widely used in cleaners and detergents, in agriculture or in food processing (Oviedo and Rodriguez, 2003). In the food industry, it is mostly used to remove metallic tastes by sequestering metal ions released during the processing or storage (e.g., in canned beans). In USA, Na2EDTA concentrations of 36 to 500 ppm, which corresponds to 0.1-1.5 mmol L-1 in solution, are allowed in some food products (Heimbach et al., 2000). In agriculture, EDTA is used in agrochemical products to stabilize formulations or to provide micronutrients such as zinc, manganese, iron, copper, magnesium, calcium and potassium. EDTA is also recognized as an antibacterial agent which disrupts the membrane integrity and as a potentiator of other lethal agents (Oita, 2003). EDTA antifungal properties were mainly tested on yeasts (Siqueira and Sen, 2004; Kubo et al., 2005), being nevertheless reported its synergetic effect with another antifungal agent on the reduction of pulmonary aspergillosis (Hachem et al., 2006). Its effect in controlling mildew on tomato leaves was also reported (Ehret et al., 2002).
In this study, the in vitro effect of Na2EDTA on the growth of an A. carbonarius, an A. ibericus, an ochratoxigenic A. niger and a non ochratoxigenic A. niger strains was assessed. All these strains were isolated from grapes.
MATERIALS AND METHODS
Chemical Material
Ethylenediaminetetraacetic acid disodium salt (Na2EDTA)
from Merck commercialized as Titriplex III.
Biological Material
Ochratoxigenic Aspergillus carbonarius strain MUM 03.59, non-ochratoxigenic
A. ibericus strain MUM 03.49, ochratoxigenic A. niger
strain MUM 03.57 and non-ochratoxigenic A. niger strain
MUM 03.58.
Growth Conditions in Presence of Na2EDTA
The antifungal activity of Na2EDTA was evaluated by the agar
dilution method using Czapek Yeast Extract Agar medium (CYA) (Mares et
al., 2004). The medium was supplemented with 0, 1 or 10 mmol L-1
of Na2EDTA (CYA, CYA+1 m and CYA+10 m, respectively). Strains
were first grown in plates with MEA medium (Blakeslee formula), for 7
days, at 25°C, in the dark, for inoculum generation. For each strain,
a spore suspension with 2.5x106 spores mL-1 was
prepared in semi solid agar (0.2% agar and 0.05% Tween 80) using a NeuBauer
chamber (Aberkane et al., 2002). Each strain was centrally inoculated
in triplicate in each media with 10 μL of the respective spore suspension
and incubated at 25°C, in the dark. The colony diameters were recorded
daily and growth rates were calculated by linear regression of colony
diameters against days. The concentration of Na2EDTA at which
survival was 50% (EC50) was determined by fitting the experimental
data to a four-parameter logistic model (Hill equation) using computer
curve-fitting software (Prism 4, GraphPad Software, Inc, San Diego, CA,
USA).
Ochratoxin A Analysis
Every two days, plates were extracted with methanol to quantify the amount
of OTA produced. A modification of the method presented by Bragulat et
al. (2001) was used as follows. The mycelia and media were cut and
transferred to tubes with 20 mL of methanol. The tubes were vigorously
vortexed and allowed to extract overnight. Methanol extracts were filtered
with a 0.45 μm syringe filter of PTFE (Teknokroma) and 1 mL of the
filtrate dried at 50°C with a gentle stream of nitrogen in a clean
vial. Dried residues were resuspended in 1 mL of HPLC mobile phase and
analyzed by high-performance liquid chromatography. The HPLC apparatus
consisted of a Varian 9002 pump equipped with a Jasco FP-920 fluorescence
detector (λex = 333 nm; λem = 460 nm)
and a Marathon Basic autosampler. The analytical column was a C18
reversed-phase YMC-Pack ODS-AQ (250x4.6 mm and 5 μm) fitted with
a precolumn with the same stationary phase. The mobile phase was a mixture
of acetonitrile/water/acetic acid (99/99/2, v/v/v) filtered and degassed.
The flow rate was set to 0.8 mL min-1 and the column temperature
to 30°C. The loop volume was 100 μL. Calibration curves were
prepared with standards of OTA (Sigma).
Statistical Analysis
All statistic analyses were performed with the Statistic Package for Social
Sciences (SPSS) version 15.0. Means were compared by analysis of variance
followed by Duncan`s post-test being the differences considered statistically
significant when p<0.05.
RESULTS AND DISCUSSION
The tested strains were found to be susceptible to the presence
of Na2EDTA in culture media. Na2EDTA produced a
significant increase in the colonies lag phase (Fig. 1)
and a significant decrease on colonies growth rates (Table
1). In the presence of 1 mmol L-1 of Na2EDTA,
the growth rate of A. carbonarius, A. ibericus,
A. niger MUM 03.57 and A. niger MUM 03.58
was reduced in 32, 56, 36 and 16%, respectively. When 10 mmol L-1
of Na2EDTA was used, reductions in growth rate of 88, 89, 87
and 82% were obtained, respectively. The concentration of Na2EDTA
which reduces growth in 50% (EC50) is 2.1 mmol L-1
for A. carbonarius, 0.9 mmol L-1 for A.
ibericus, 2.0 mmol L-1 for A. niger MUM
03.57 and 4.1 mmol L-1 for A. niger MUM 03.58
(Table 2).
| Fig. 1: | Daily radial growth of A) A. carbonarius MUM 03.59, B) A. ibericus MUM 03.49, C) A. niger MUM 03.57 and D) A. niger MUM 03.58 cultivated in: (-� -) CYA, (-•-) CYA+1 m and (-Δ-) CYA+10 m. Values presented are the mean of three replicates |
| Table 1: | Radial growth rates (cm day-1) of black aspergilli in CYA and in CYA supplemented with 1 and 10 mmol L-1 of Na2EDTA (CYA+1 m and CYA+10 m, respectively) |
| Values are the mean of three replicates±Standard Deviation (SD). Data marked with different letter(s) in each column are significantly different at p<0.05 for the Ducan`s post hoc test | |
| Table 2: | The 50% effective concentration (EC50) of Na2EDTA for the different black aspergilli tested |
| a: EC50 = Concentration of Na2EDTA (mmol L-1) at which survival was 50% as determined by fitting the experimental data to a four-parameter logistic model (Hill equation) using computer curve-fitting software (Prism 4, GraphPad Software, Inc, San Diego, CA, USA), b: 95% Confidence Intervals of fitted EC50, c: Correlation coefficient of fitted curves | |
| Table 3: | Production of ochratoxin A by A. carbonarius MUM 03.59 and A. niger MUM 03.57 in CYA and in CYA supplemented with 1 and 10 mmol L-1 of Na2EDTA (CYA+1 m and CYA+10 m, respectively) |
| Values are the mean of three replicates±Standard Deviation (SD). For each strain and column, data marked with different letter(s) are significantly different at p<0.05 for the Ducan`s post hoc test, nd: not detected | |
The production of ochratoxin A was also affected by the presence of Na2EDTA in culture media. When 1 mmol L-1 of Na2EDTA was used, OTA was detected after 4 days of growth, while when 10 mmol L-1 were used, it was detected after 8 days (Table 3). The accumulation of OTA was significantly lower in most situations. In the presence of 1 mmol L-1 of Na2EDTA and after 8 days of growth, A. niger MUM03.57 produced 7.5 μg OTA/plate. After the same period of incubation with 10 mmol L-1 of Na2EDTA this strain produced only 0.2 μg OTA/plate, which is 98.3% less than the respective control. Under the same conditions, the A. carbonarius strain produced only 0.08 μg OTA/plate, 99.9% less than the control. The reduction in OTA production was not observed when A. carbonarius was grown with 1 mmol L-1 of Na2EDTA (Table 3).
Several studies have previously reported the bactericidal proprieties of Na2EDTA (Oita, 2003; Reidmiller et al., 2006). However, fungi tolerance to Na2EDTA is not well documented despite its utilization by the food and agrochemical industry, being its antifungal effects mainly reported on clinical strains as Candida albicans (Sen et al., 2000) or Aspergillus fumigatus (Hachem et al., 2006).
In this study, it was demonstrated that Na2EDTA can significantly inhibit the growth rate of black aspergilli isolated from grapes and significantly delay and reduce the ochratoxinA produced by the ochratoxigenic strains tested. Namely, 2 mmol L-1 of Na2EDTA were sufficient to reduce the growth rate of ochratoxigenic strains in 50% and 10 mmol L-1 of Na2EDTA to delay the production of OTA in 8 days and reduce the levels produced in approximately 99%. The effect of Na2EDTA on strains growth is probably due to a defective cell wall construction mediated by its zinc binding capacity as presented by Brul et al. (1997) for yeasts. The reductions on OTA amounts produced are probably due to the inhibition of strains growth further than to the inhibition of the mycotoxin synthesis.
In agriculture, Na2EDTA is commonly used on foliar products to supplement micronutrients to plants or to stabilize the formulation of several agrochemical products. It will be interesting to study, in vivo, if the application of products that contain EDTA can contribute to control the presence of black ochratoxigenic aspergilli and so the levels of ochratoxin A in agriculture commodities such as grapes.
SAFETY
Ochratoxin A is a toxic compound that needs to be manipulated with care and with appropriate safety precautions.
ACKNOWLEDGMENT
LuÃs Abrunhosa is grateful for grant SFRH/BD/11228/2002 from Fundação para a Ciência e Tecnologia-FCT, Portugal.