Detection and Isolation of Aflatoxin-producing Aspergillus sp. in Chewing and Smokeless Tobacco by Microbial and Molecular Detection Methods and Its Influence on Hepatocellular Carcinomas in Wistar Rats

Abstract

Tobacco contains many harmful chemicals, toxins, and carcinogens which cause serious health hazards. Mycotoxins and Aflatoxins are present in all tobacco including chewing and smokeless tobacco. We detected and isolated the aflatoxins-producing fungi Aspergillus sp. by microbial and molecular-based techniques. 50 samples were collected randomly and grown on SDA media by spread plate method. Isolates of A. flavus were analyzed for Aflatoxin production by Ammonia vapor test and the DNA of isolates was extracted and subjected for further molecular identification. Different fungal genera were observed including the most common Aspergillus while the other were Penicillium, Rhizopus, candida, and Trichophyton. Aspergillus flavus was isolated and characterized as a white soft velvety surface. The diameter of colonies was observed as 50 -70 mm. Coconut-based medium detection by UV light of 365 nm showed the production of blue-green fluorescence on the reverse of colonies on coconut agar medium. DNA extracted from all the strains producing aflatoxins was quantified by VICAM. High levels of Aflatoxin ranging from 21 -47 ug/kg were detected in 10 different samples while the remaining samples had a range of 10 -20 ug/kg. the study showed both quantitative and qualitative techniques for the detection of aflatoxin-producing fungi in chewing tobacco. aflR and aflS were observed as potential candidates for the detection of aflatoxigenic fungi.