New Additive for Culture Media for Rapid Identification of Aflatoxin-Producing Aspergillus Strains

Abstract

A new reliable, fast, and simple method for the detection of aflatoxigenic Aspergillus strains, consisting of the addition of a cyclodextrin (a methylated ␤-cyclodextrin derivative) to common media used for testing mycotoxin production ability, was developed. We propose the use of this compound as an additive for fungal culture media to enhance the natural fluorescence of aflatoxins. The production of aflatoxins coincided with the presence of a bright blue or blue-green fluorescent area surrounding colonies when observed under longwavelength (365-nm) UV light after 3 days of incubation at 28°C. The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by high-pressure liquid chromatography with fluorescence detection.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 fluorescen...

Anti-aflatoxigenic effect of essential oils on Aspergillus Spp. isolated from pistachio in Saudi Arabia

“…In addition, the table shows that all isolates of A. niger were negative for fluorescence. These results agree with those of Fente et al (2001).…”

Molecular characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates collected from corn grains

“…A new, reliable, fast, simple method for the detection of aflatoxigenic Aspergillus strains involves the addition of a cyclodextrin to the common media used for testing isolates for AF production to enhance the natural fluorescence of AF. Colonies of Aspergillus produce a bright yellow-orange pigment, and blue (B1, B2) or green (G1, G2) fluorescent halos appear around aflatoxigenic colonies upon exposition to UV light (Fente et al, 2001).…”

Molecular characterization of Aspergillus flavus and aflatoxin contamination of wheat grains from Saudi Arabia

“…In this method, aflatoxigenic strains produced a blue fluorescence on the reverse side of the colony under UV light, whereas non-aflatoxigenic ones did not produce fluorescence (Davis et al, 1987). This method was improved by adding cyclodextrin (a methylated β-cyclodextrin derivative) to common media used for testing AF production, to enhance the natural fluorescence of AFs (Fente et al, 2001).…”