Isolation of microorganisms and substances inhibitory to aflatoxin production

Abstract

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and A. parasiticus. The contamination of crops, feeds, and foods with aflatoxins can have serious effects on the health of humans and animals. Although many studies have been done to develop aflatoxin-control strategies, most are limited in their effectiveness. As part of an effort to develop control procedures, we have devised simple and safe methods that are useful for identifying microorganisms that effectively inhibit aflatoxin production by fungi. These include the microtitre agar plate assay using norsolorinic acid-accumulating mutant fungi, the ultraviolet light photography method using an instant film, the tip culture method, a convenient RNA extraction method for reverse transcription-polymerase chain reaction (RT-PCR) analysis, and other methods. Results of a recent trial have shown that Achromobacter xylosoxidans significantly inhibited aflatoxin production by A. parsiticus, and that the main inhibitory substance produced by the bacterium was cyclo(L-leucyl-L-prolyl). This result confirms that the methods described herein are useful for identifying microorganisms that inhibit aflatoxin production by fungi and could contribute to the development of methods to reduce aflatoxin contamination in commodities.

Keywords:

• inhibition of aflatoxin production
• aflatoxin biosynthesis
• norsolorinic acid-accumulating mutant
• Aspergillus parasiticus
• ultraviolet light photography
• visual agar plate assay
• microtitre agar plate assay
• cyclo(L-leucyl-L-prolyl)

Acknowledgements

The authors are deeply grateful to the late Professor Takashi Hamasaki of Tottori University for his support and encouragement. They thank Dr Ken Voss for reviewing the manuscript. They also thank Masako Kito, Hatsue Arai and Hidemi Hatabayashi for their technical assistance. Homology search of the GenBank database was performed with the assistance of the Computer Centre for Agriculture, Forestry and Fisheries Research, MAFF, Japan. This work was supported in part by the grant Bio-Design Program from National Agriculture and Food Research Organization (NARO), Japan.