A polyphasic method for the identification of aflatoxigenic Aspergilla from cashew nuts

Abstract

The invasion of food by toxigenic fungi is a threat to public health. This study aimed at enumerating the microbial profile, detection of aflatoxin producing genes and quantification of the levels of aflatoxin contamination of cashew nuts meant for human consumption. A polyphasic method of analysis using newly formulated β-Cyclodextrin Neutral Red Desiccated coconut agar (β-CDNRDCA) and Yeast Extract Sucrose agar (YES) with Thin Layer Chromatography (TLC), Polymerase Chain Reaction (PCR) and High Performance Liquid Chromatographic (HPLC) method was adopted in determining the aflatoxigenic potential of the isolates, the presence of aflatoxin biosynthetic gene (aflM, aflD, aflR, aflJ omt-A) and estimation of the total aflatoxin content of the nuts. The fungal counts ranged from 2.0 to 2.4 log₁₀cfu/g and sixty-three fungal isolates belonging to 18 genera and 34 species were isolated. The Aspergillus spp. were the most frequently isolated (50.79%) while Trichoderma spp. (1.59%) were the least. and fluorescence production was enhanced on the newly formulated β-CDNRDCA by the aflatoxigenic species. The aflD gene was amplified in all the isolates while aflM, aflR and aflJ gene were each amplified in 77.77% of the isolates and omt-A gene in 70.37%. The aflatoxin content of the nuts ranged from 0.03 to 0.77 µg/kg and were below the 4 µg/kg EU recommended limit for total aflatoxins. The present work confirms that a single method of analysis may not be sufficient to screen for the presence of aflatoxins in foods, as with a combination of different methods.

Keywords: Aflatoxigenic fungi; Aflatoxin; Aspergillus; Biosynthetic gene; Cashew nut; Polymerase chain reaction; Polyphasic.