INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Aflatoxins are mycotoxins with highly toxic and carcinogenic properties produced by some strains of Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius. These fungi are frequently found in foodstuffs and animal feeds. However, not all strains are able to produce aflatoxins, and this has encouraged the use of screening for their aflatoxin production abilities. The methodology commonly used for this survey involves the culture of strains in a suitable liquid or solid medium and their later extraction and analysis for the presence of aflatoxins by chromatographic techniques. Yeast extract-sucrose (YES) medium (3) and natural media with wheat, rice, or peanut (9) have been used for this purpose. Testing large numbers of isolates on a variety of substrates with this procedure is tedious and time-consuming. For this reason, several screening methods for direct visual determination of aflatoxin production have been developed. These methods use more or less complicated culture media containing additives to enhance the production of aflatoxins in order to achieve direct visual determination of a bright blue or blue-green fluorescent area surrounding colonies under UV radiation. Thus, a complex agar medium containing sucrose, various salts, and an aqueous extract of aflatoxin-free peanuts (5); a modified Czapek agar medium containing corn steep liquor, named aflatoxin-producing ability (APA) medium (9); media containing coconut, named coconut agar medium (4, 13), coconut extract agar (11, 12), and coconut cream agar (6); the synthetic liquid medium of Adye and Mateles (1); and a silica gel medium (15) are currently in use.

The natural fluorescence of aflatoxins arises from their oxygenated pentaheterocyclic structure. The cyclodextrins (cyd) are molecules formed by the action of the enzyme cyd-transglycolase on dextrans and have different sizes [they contain from six to eight units of glucose in an (1-4) configuration, according to which they are called -, -, or -cyd]. They are available commercially, and their physical and chemical properties have been described in the literature (14). These oligomers are able to include a large number of organic and inorganic species in their cavities, and in this work, excitation of the natural fluorescence of aflatoxins has been accomplished using a derivative of these molecules (2, 8, 16, 17).

Experimental results obtained in previous work (2, 16, 17) revealed that -cyd and its methylated derivatives have excellent cavities for exciting the fluorescence responses of aflatoxins B₁ and G₁ through the formation of inclusion complexes. In this work, we have exploited this property for the direct visual measurement of aflatoxin production by A. flavus and A. parasiticus strains. The enhancement phenomenon was optimized for representative aflatoxigenic or nonaflatoxigenic Aspergillus strains directly in a culture medium traditionally used for fungal isolation from foods (Sabouraud dextrose agar [SAB]) and in a culture medium used for mycotoxin production (YES).

In the present paper we propose the use of a methylated -cyd derivative, the -W7M 1.8-cyd (M-cyd), as an additive to enhance the natural fluorescence of aflatoxins in culture media. We report a series of experiments that led us to the development of a rapid and simple method for the detection of aflatoxin-producing strains using common fungal culture media (7a).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Strains used. The representative strains of A. flavus, A. parasiticus, Aspergillus ochraceus, and Aspergillus versicolor used in our experiments are listed in Table 1 and were supplied by the Spanish Type Culture Collection (Burjassot, Valencia, Spain). The method was also evaluated with 24 additional A. flavus group strains belonged to the culture collection of our laboratory. Twelve of these strains were isolated from foods (18), and the rest were isolated from feed. Of these 24 strains, 20 are nonaflatoxigenic.

Media. The culture media used were Czapek agar, SAB, YES agar, and APA medium. All were supplied by Difco (Detroit, Mich.). cyd added to the media tested for aflatoxin detection were -cyd and a methylated -cyd derivative, pharmaceutical-grade M-cyd from Wacker (Munich, Germany).

Cultivation and observation of fluorescence. All strains had been tested previously for aflatoxin production in APA medium (incubation was for 10 days at 28°C). These strains were also cultivated in YES medium under the same conditions used for APA medium, and chloroform extracts (7) of all them were obtained and analyzed by high-pressure liquid chromatography (HPLC) with fluorescence detection (2) to confirm the presence of aflatoxins.

Extraction and quantitation of aflatoxin. To confirm the correlation between fluorescence and aflatoxin production, all colonies, whether or not they showed fluorescence, were extracted according to the method described by El-Banna et al. (7) with some modifications. The colonies and the surrounding agar were weighed and introduced into stomacher bags. Extraction was carried out using 20 ml of chloroform (twice with 10 ml each), and homogenization for 3 min in a stomacher. The chloroform phase was filtered through Whatman no. 3 filter paper and concentrated to dryness under a nitrogen steam. The residue was redissolved in 200 µl of mobile phase, and volumes of 50 µl of extract were injected into the HPLC system. The HPLC method used in this study was a modification of the method of Cepeda et al. (2). The apparatus and conditions used were as follows: column, Tracer Inertsil ODS2, 5-µm particle size (150 mm by 4.6 mm [inner diameter]) from Teknokroma (Barcelona, Spain); mobile phase, methanol-water (45:55, vol/vol); and flow rate, 0.6 ml/min. The pump was a PU-l580 from Jasco (Tokyo, Japan), and the injector was a Rheodyne (Cotati, Calif.) model 7125 equipped with a 50-µl loop. The fluorescence detector, an RF-535 (Shimadzu, Kyoto, Japan) was set up with a _ex of 365 nm and a _em of 418 nm.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

First, we assayed the aflatoxin production abilities of all strains used in this experiment by cultivating them in APA medium at 28°C for 10 days (Table 1). Strains NRRL 6540, NRRL 2999, NRRL 3145, 1316, 876, 42, and 19 (the last four are from our laboratory collection) showed fluorescence in this medium after 10 days of incubation. These results were in concordance with those obtained from the chloroform extracts from all strains cultivated in YES medium under the same conditions used for APA medium with analysis by the chromatographic procedure (Table 1). Results with nonaflatoxigenic strains were as expected (they did not show fluorescence in any of the experiments), and only data from a representative negative strain (strain 893) belonging to our collection are shown.

Effect of addition of cyd to the culture media. The effects of the addition of two different cyd to the culture media at different concentrations on rapid visualization of aflatoxin production are shown in Table 2. When these cyd were used as additives for culture media, they did not negatively affect the fungal growth, and in fact an increase in the diameters of the colonies was observed when strains cultivated under the same conditions in media with and without cyd were compared. This experiment was carried out at 28°C.

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Colonies of aflatoxigenic NRRL 2999 (left) and nonaflatoxigenic NRRL 6538 (right) strains observed under UV light. Strains were cultivated in YES medium supplemented with M-cyd and photographed on the third day of incubation at 28°C.

Effect of incubation temperature. The effect of temperature on the fluorescent response was assayed with all aflatoxigenic and nonaflatoxigenic strains grown on the basal media supplemented with 0.3% M-cyd. At 28 and 30°C, no significant differences in the subjective intensity of fluorescence were observed, while at 25°C the fluorescent responses were weak. However, as the sporulation and size of the colonies were greatest at 30°C, we decided to use 28°C as the incubation temperature.

Time of incubation. Using SAB and YES agar supplemented with 0.3% M-cyd at 28°C, we looked for the minimum incubation time necessary to detect a clear fluorescent signal of aflatoxigenic strains. Each day for 10 days, a petri dish of both media assayed (with and without cyd) was examined under UV radiation (365 nm), and the chloroform extract of this agar was analyzed by HPLC. For all aflatoxigenic strains tested, the response was clearly conclusive when fluorescence was visualized after 3 days of incubation (only 2 days for the stronger aflatoxin-producing strains NRRL 2999 and NRRL 3145). Aflatoxins were detected in all chloroform extracts from strains showing fluorescence at the third day by an HPLC method with a detection limit of lower than 5 ppb for each aflatoxin. Strain NRRL 3174, an A. ochraceus ochratoxin A-producing strain, did not show fluorescence until the fifth day of incubation, and the aflatoxin HPLC analyses for this strain at the third and fifth days (data not shown) were negative. Like this mycotoxin, other substances produced by Aspergillus strains, i.e., deoxyhydroxy-aspergillic acid (19), flavocol (19), and asperopterin A or B (10), can exhibit blue fluorescence under UV light, but according to our experiments, only the aflatoxigenic strains give fluorescence at the third day of incubation in SAB and YES agar supplemented with M-cyd (Fig. 2). Therefore, we can conclude that the presence of the fluorescence surrounding colonies in both media proposed by us is indicative of aflatoxin production ability. However, a chromatographic determination is recommended to confirm the presence of aflatoxin. The intensity of fluorescence increased with time; the maximum intensity was observed at 3 to 4 days, and after additional incubation (day 5 or 6), cultures became difficult to evaluate because mycelial growth almost reached the margin of the petri dish and so the agar surrounding colonies in which fluorescence must be observed was very reduced. In other media currently in use for the screening of aflatoxin-producing strains (4-6, 9, 11, 15), from 2 to 10 days are necessary to reach conclusive results. The use of a methylated -cyclodextrin derivative such as M-cyd as an additive for SAB and YES media allows the utilization of a general culture medium for fungal total counts in foods and feeds (SAB) to test the aflatoxigenic ability of Aspergillus strains without the need to use more or less complex media that are exclusive for such purposes. As a basal medium, it is possible to use one that is suitable for the production of mycotoxins in general (YES) and which also allows aflatoxin detection.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   HPLC fluorescence detection chromatograms of chloroform extracts from strains NRRL 2999 (A and B), NRRL 3174 (C), and NRRL 6538 (D) obtained on the third day of incubation at 28°C. Strains were cultivated in SAB supplemented with M-cyd (A) or in YES medium supplemented with M-cyd (B to D).