Food Microbiology

Aflatoxins are toxic substances and are fungal metabolites (Mycotoxins) produced by certain molds of the genus Aspergillus growing on several raw food commodities. Aflatoxins are highly toxic compounds and can cause both acute and chronic toxicity in humans and many other animals. Aflatoxins consist of about 20 similar compounds but only four are naturally found in foods. These are aflatoxins B1, B2, G1 and G2. Aflatoxin B1 is the most commonly found in food and the most toxic e.g. when lactating cattle and other animals ingest aflatoxins in contaminated feed, toxic metabolites can be formed and may be present in milk. These metabolites, aflatoxin M1 and M2, are potentially important contaminants in dairy products. US food safety regulations include a limit of 20 μg/kg for total aflatoxins (B1, B2, G1 and G2) in all foods except milk and a limit of 0.5 μg /kg for M1 in milk. Higher limits apply in animal feeds. If fish or shrimp are fed by contaminated feed, ingestion of such feed can cause toxic metabolites contamination and can be found in liver of the fish and hepatopancreaz of the shrimp. It is very important to assess the quality of these products by monitoring the aflatoxins residue in fish liver and shrimp hepatopancreaz. There are a variety of methods available for aflatoxins determination e.g. by ELISA or by HPLC but the real challenge is to extract the aflatoxins from liver or hepatopancreaz of fish & shrimp. For extraction of aflatoxins from meat matrix, liver, kidneys etc., 70 % methanol is recommended. Helica Total Aflatoxins protocol for ELISA was followed for analysis and validation. Total aflatoxin Assay is a solid phase direct competitive enzyme immunoassay. An aflatoxin specific antibody optimized to cross react with all four subtypes of aflatoxin was coated to a polystyrene micro well of the ELISA plate. Aflatoxins were extracted from sample with 70% methanol. The extracted sample and HRP-conjugated aflatoxin B1 were mixed and added to the antibody-coated micro well. Aflatoxin from the extracted sample and HRP-conjugated aflatoxin B1 compete to bind with the antibody coated to the micro well. Micro well contents were decanted, and non-specific reactants were removed by washing. An enzyme substrate (TMB) was added and color (blue) was developed. The intensity of the color was directly proportional to the amount of bound conjugate and inversely proportional to the concentration of aflatoxin in the sample or standard. Therefore, as the concentration of aflatoxin in the sample or standard increased, the intensity of the blue color decreased. An acidic stop solution was added which changed the chromogen color from blue to yellow. The micro wells were measured optically by a microplate reader (ELISA reader) with an absorbance filter of 450nm (OD450). The optical densities of the samples were compared to the OD's of the kit standards and an interpretative result was determined.