Introduction fluorescence detection has become the method of choice due to the high resolution and sensitivity of the
1. Introduction fluorescence detection has become the method of choice due to the high resolution and sensitivity of the
The genus Aspergillus fungi are commonly found in
fluorescence detectors [6, 7].
food and feed, and considered potential producers of Because of their high toxicity they can cause
mycotoxins [1]. These substances are quite common serious problems for human health, and as a result the
in peanut kernels, and have a high incidence of maximum tolerated levels in food have been
aflatoxins [2]. established in several countries, but they are still very
Aflatoxins are mainly secondary metabolites produced by strains of Aspergillus parasiticus and
common in peanuts [8].
Aspergillus flavus [3]. The most commonly found
2. Materials and Methods
substances in peanut kernels are aflatoxins B1, B2, G1 and G2 [2]. 2.1 Isolation of Fungi
The aflatoxins have been primarily identified by The fungi were isolated from five samples of column chromatography [4], revealing compounds
peanuts marketed in the city of Fortaleza (Ceará, with fluorescence blue and green. High performance
Brazil), and the seeds were surface disinfected in liquid chromatography (HPLC) has been shown to be
sodium hypochlorite 0.4% for 2 min, washed in sterile the best method of identification [5]. HPLC with
water and spread on water-agar Petri plates with 10 seeds per plate with a total of 10 plates per sample.
Corresponding author: Maria Edite Bezerra da Rocha, The plates were kept at room incubation with 12 h of M.Sc., professor, research field: micotoxins. E-mail:
light and 12 h dark with temperatures ranging from [email protected].
Production of Aflatoxins from Aspergillus flavus in Liquid Medium
25 °C to 32 °C [8]. After a week of incubation, the chloroform/methanol (2:1) for a few seconds and fungi were then identified according to their pressed against the surface of a 20 cm × 20 cm silica morphological characteristics, using taxonomic gel plate (Merck, Darmstadt, Germany) 2 µL of a keys [9].
standard suspension of aflatoxins B1, B2 and griseofulvin was added to the same plate. Using
2.2 Production and Extraction of Aflatoxins toluene/ethyl acetate/formic acid (5:4:1, v/v/v) as a From the strains obtained from the Aspergillus
mobile phase, the plate was then visualized using flavus peanuts, five discs with a size of 7 mm were
ultraviolet light with a wavelength of 365 nm. The removed and were inoculated into 100 mL of liquid
quantitative determination was performed by medium of extract of malt Erlenmeyer flask of 250
comparing the fluorescence ratios and the frequency mL. After 2 days the medium was inoculated into a
(Rfs) of samples with the standards. second medium (1L) containing 5% sucrose,
2.3.2 Quantitative Analysis
The concentration of aflatoxin in the liquid medium 0.0176 g, adapted [10] in a 2 L erlenmeyer flask and
MgSO 4 ·7H 2 O 0.1%, KH 2 PO 4 1%, ZnSO 4 ·7H 2 O
was determined in a high performance liquid cultured for 3 more days. The media were kept at
chromatograph (Knauer) with two pumps (model
room temperature varying from 24 o C to 32
Smartline pump 1000) and an automatic gun using agitation of 130 rpm and 4.17 L/min of aeration.
C with an
nucleosil 120-5 C18 column (250 mm × 4 mm) and
After the incubation period, aflatoxins were fluorescence detector (Shimadzu model RF -10 Axl) extracted from the culture medium and the mycelium.
with average sensitivity at a wavelength of 365 nm for The extraction was carried out with 100 mL of
excitation and 450 nm for emission. 20 µL of the chloroform. This procedure was repeated three times
standard was injected and the sample was extracted and then the filtrate was concentrated in a rotary
with chloroform, filtered (membrane, 0.45 mM, vacuum evaporator, after which the extract was
Millipore) was used as mobile phase, water: methanol: diluted in acetonitrile: water (90:10).
acetonitrile (55:30:15) at a flow rate of 1 mL/min. Quantification of aflatoxins in the sample was
2.3 Determination of Aflatoxins performed by interpolation of the areas of the
2.3.1 Qualitative Analysis chromatographic peaks obtained from the sample For chromatographic analyses we used standard
compared to the standard, the linear regression aflatoxin B1 and B2 (Sigma-Aldrich) which were
equation of the calibration curve.
dissolved in bezeno:acetonitrile (98:2) and an aliquot