Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: February 27, 2004
Publication Date: May 24, 2004
Citation: MAHONEY, N.E., MOLYNEUX, R.J., CAMPBELL, B.C., CARY, J.W. WALNUT CONSTITUENTS INHIBIT AFLATOXIN FORMATION BY ASPERGILLUS FLAVUS. AMERICAN SOCIETY FOR MICROBIOLOGY ANNUAL MEETING. 2004. Technical Abstract: Tree nuts are susceptible to aflatoxin contamination and therefore subject to regulatory restrictions both domestically and abroad. In order to identify natural sources of resistance to aflatoxin accumulation, tree nuts were screened for their ability to support aflatoxin production by Aspergillus flavus using agar-based media composed of almond, pistachio, or walnut cultivars. A. flavus grown on walnut cultivars produced significantly less aflatoxin than on pistachio or almond cultivars. Walnut cultivars were not uniform in their ability to inhibit aflatoxin; 'Tulare' completely inhibited the production of aflatoxin, while 'Chico' was the most susceptible. The aflatoxin inhibiting property of 'Tulare' kernels was found to reside in the seed coat and bioassay-directed chemical fractionation of the seed coat resulted in the identification of hydrolyzable tannins with potent aflatoxin inhibiting properties. 'Tulare' tannin inhibited aflatoxin production 100% when incorporated at 0.5% into an agar-based medium consisting of 'Tulare' kernels without seed coat. In order to elucidate the mode of action it was first confirmed that our A. flavus isolate produced a previously reported extracellular tannase. This tannase produced gallic acid, ellagic acid, and glucose upon hydrolysis on walnut tannin. Gallic acid incorporated at 0.2% in Vogel's medium N reduced aflatoxin production 95%, while ellagic acid had little effect at the same level. Using chemical hydrolysis, gallic acid levels in 'Tulare' and 'Chico' seed coat were measured on a biweekly basis throughout the 2002 and 2003 growing seasons. Levels in 'Tulare' were significantly higher and were maintained throughout the growing season, while 'Chico' steadily declined as the kernels matured. At maturity 'Tulare' had a gallic acid content 1.5 ' 2.0 times higher than 'Chico'. This evidence indicates that the gallic acid moiety produced by the action of fungal tannase on hydrolyzable tannins is responsible for the aflatoxin inhibiting properties of walnut. Gallic acid levels should be amenable to enhancement with the goal of reducing aflatoxin contamination in tree nuts.