Many agricultural commodities are vulnerable to attack by a group of fungi that are able to produce toxic metabolites called mycotoxins. Among various mycotoxins, aflatoxins have assumed significance due to their deleterious effects on human beings, poultry and livestock. The aflatoxin problem was first recognized in 1960, when there was severe outbreak of a disease referred as "Turkey 'X' Disease" in UK, in which over 100,000 turkey poults were died. The cause of the disease was shown due to toxins in peanut meal infected with Aspergillus flavus and the toxins were named as aflatoxins.

Natural occurrence:

Food products contaminated with aflatoxins include cereal (maize, sorghum, pearl millet, rice, wheat), oilseeds (groundnut, soybean, sunflower, cotton), spices (chillies, black pepper, coriander, turmeric, zinger), tree nuts (almonds, pistachio, walnuts, coconut) and milk.

Physical and chemical properties:

Aflatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive agents, produced as secondary metabolites by the fungus Aspergillus flavus and A. parasiticus on variety of food products. Among 18 different types of aflatoxins identified, major members are aflatoxin B1, B2, G1 and G2. Aflatoxin B1 (AFB1) is normally predominant in amount in cultures as well as in food products. Pure AFB1 is pale-white to yellow crystalline, odorless solid. Aflatoxins are soluble in methanol, chloroform, actone, acetonitrile. A. flavus typically produces AFB1 and AFB2, where as A. parasiticus produce AFG1 and AFG2 as well as AFB1 and AFB2. Four other aflatoxins M1, M2, B2A, G2A which may be produced in minor amounts were subsequently isolated from cultures of A. flavus and A. parasiticus. A number of closely related compounds namely aflatoxin GM1, parasiticol and aflatoxicol are also produced by A. flavus. Aflatoxin M1and M2 are major metabolites of aflatoxin B1 and B2 respectively, found in milk of animals that have consumed feed contaminated with aflatoxins.

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Aflatoxin            Molecular formula       Molecular weight      Melting point

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B₁                     C₁₇ H₁₂O₆                312                               268-269

B₂                     C₁₇ H₁₄O₆                  314                               286-289

G₁                     C₁₇ H₁₂O₇                328                               244-246

G₂                     C₁₇ H₁₄O₇                330                               237-240

M₁                    C₁₇ H₁₂O₇                   328                               299

M₂                    C₁₇ H₁₄O₇                 330                               293

B_2A                    C₁₇ H₁₄O₇                330                               240

 Chemical reactions of aflatoxins

The reaction of aflatoxins to various physical conditions and reagents have been studied extensively because of the possible application of such reactions to the detoxification of aflatoxins contaminated material.

Heat:
Aflatoxins in dry state are very stable to heat up to the melting point. However, in the presence of moisture and at elevated temperatures there is destruction of aflatoxin over a period of time. Such destruction can occur either with aflatoxin in oilseed meals, aflatoxin in roasted peanuts or aflatoxin in aqueous solution at pH 7. Although the reaction products have not been examined in detail it seems likely that such treatment leads to opening of the lactone ring with the possibility of decarboxylation at elevated temperatures.

Alkalis:
In alkali solution hydrolysis of the lactone moiety occurs. This hydrolysis appears to be reversible, since it has been shown that recyclization occurs following acidification of a basic solution containing aflatoxin. At higher temperatures (ca. 100oC) ring opening followed by decarboxylation occurs and reaction may proceed further, leading to the loss of the methoxy group from the aromatic ring. Similar series of reactions also seems to occur with ammonia and various amines.

Acids:
In the presence of mineral acids, aflatoxin B1 and G1 are converted in to aflatoxin B2A and G2A due to acid-catalyzed addition of water across the double bond in the furan ring. In the presence of acetic anhydride and hydrochloric acid the reation proceeds further to give the acetoxy derivative. Similar adducts of aflatoxin B1 and G1 are formed with formic acid-thionyl chloride, acetic acid-thionyl chloride and trifluoroacetic acid.

Oxidizing agents:

Many oxidizing agents, such as sodium hypochlorite, potassium permanganate, chlorine, hydrogen peroxide, ozone and sodium perborate react with aflatoxin and change the aflatoxin molecule in some way as indicated by the loss of fluorescence. The mechanisms of these reactions are uncertain and the reaction products remain unidentified in most cases.

Reduction:

Hydrogenation of aflatoxin B1 and G1 yields aflatoxin B2 and G2 respectively. Further reduction of aflatoxin B1 by 3 moles of hydrogen yields tetrahydroxyaflatoxin. Reduction of aflatoxin B1 and B2 with sodium borohydride yielded aflatoxin RB1 and RB2 respectively. These arise as a result of opening of the lactone ring followed by reduction of the acid group and reduction of the keto group in the cyclopentene ring.

Biology of A. flavus Link ex Fr. and A. parasiticus Spear:

The two fungi A. flavus and A. parasiticus are closely related and grow as a saprophyte on plant debris of many crop plants left on and in the soil. They are distributed worldwide, with a tendency to be more common in countries with tropical climates that have extreme ranges of rainfall, temperature and humidity. Members of the genus Aspergillus are characterized by the production of non-septate conidiophores, which are quite distinct from hyphae and which are swollen at the top to form a vesicle on which numerous specialized spore-producing cells, known as phialides or sterigmata are borne either directly (uniseriate) or on short outgrowths known as metulae (biseriate). Some time difficulty may arise especially to determine because the primary sterigmata are tiny and are easily obscured by spores or other sterigmata. Colonies of A. flavus are green-yellow to yellow-green or green on Czapek's agar. They usually have biseriate sterigmata; reddish-brown sclerotia are often present, conidia are finely roughened, variable in size and oval to spherical in shape. Colonies of A. parasiticus dark green on Czepak's agar, remain green with age. Sterigmata are uniseriate, sclerotia are usually absent; conidia are coarsely echinulate, uniform in shape, size and echinulation.

Terminal portion of a conidiophore of A. flavus showing the basal portion of the vesicle and distribution of radiation phialides (arrows). X 1000.


Phialides and chains of conidia of A. parasiticus illustrating basipetal development of conidia. Those at the base of the chains (arrows) are least mature. X 3000.