Aflatoxin extraction

Figure 5.9. Structure of aflatoxins extracted from corn and peanuts.

Test preparation for both methods is similar. It comprises an extraction by a water-acetone mixture with its further degreasing protein isolation and redistribution of aflatoxin B into chloroform, concentrating by means of evaporation of the dissolvent on the rotary evaporator (t = 40°C). 

Palmitic and stearic acids aflatoxins Bi and Bi 15 to 35% unseparated fatty acids intensified the fluorescence when investigating com extracts [224]... 

This sample preparation involved, firstly, an extraction and the elimination of the solid matrix by filtration and, secondly, a concentration procedure employing a solid phase extraction cartridge. The compounds of interest were separated solely by dispersive interactions with the reversed phase. In the example given, the corn meal was spiked with the aflatoxins. 

Sophisticated and veiy sensitive methods have been developed in the food industry for detecting many other microbial toxins. For example, aflatoxin deteetion in seedstuffs and their oils is performed by solvent extraction, adsorption onto columns containing selective antibodies for them, and detected by exposure to ultraviolet light. 

Antimutagenic activity. Water extract of the dried gum, on agar plate at a concentration of 2 mg/plate, was inactive on SalmO nella typhimurium TAIOO vs aflatoxin Bl-induced mutagenesis and a concentration of 10 mg/plate, was inactive on SalmO nella typhimurium TA98 " . Asafoetida, on agar plate at a dose of 0.5 (Xg/plate was active on Salmonella typhimurium TA98 and... 

Initial attempts to purify enzymes from A, parasiticus mycelial extracts that catalyze aflatoxin synthesis were unsuccessful because these enzymes are present in relatively low concentrations and are extremely short-lived (23). Subsequently, several techiuques were examined for disruption of large quantities of mycelia to obtain active and stable cell-fiee preparations (23, 24) pertinent enzymes were recovered from cell-free extracts after grinding mycelia under liquid nitrogen (24). The optimum age of mycelial cultures for recovery of aflatoxin pathway enzymes was determined to be between 72 and 84 h (24a, 25). 

Inhibition of aflatoxin biosynthesis by neem extracts in fungal cells appear to occur in the very early stages of the biosynthetic pathway (i.e., prior to norsolorinic acid synthesis) because after the initiation of secondary metabolism, the inhibitory effect of the neem leaf constituents was lost (84). 

The extraction methods for aflatoxins are based on the solubility of these toxins in organic solvents, mainly chloroform, methanol, acetone, benzene, and acetonitrile. For more complex matrices, the addition of diatomaceous earth or citric acid is required. From matrices of vegetable origin, water is usually added in the extraction step, since it facilitates solvent penetration into substrates, improving the percentage of extraction of the toxin. 

A chloroform-water mixture is the extraction solvent for the contaminants branch (CB) method, proposed by Eppley (36). The CB method has been adopted by the Association of Official Analytical Chemists as the official method for determination of aflatoxins in groundnuts and their byproducts (17). 

Methanol-water is the extraction medium of the method recently tested for validation by the European Commission (39), for the determination of aflatoxins at the European regulatory limits for dried figs, pistachios, peanut butter, and paprika 50 g of the test portion are extracted with methanol-water (80 20) for dried figs and paprika, and methanol-water (80 20) plus 100 ml of hexane for peanut butter and pistachio. After filtration, the filtrate is added to phosphate buffer saline (PBS) for the purification step. 

A mixture of acetone/water is used in the Roch et al. method (40) for aflatoxins in groundnut cake 1 kg of the sample is ground to a fine powder and mixed using a Hobert Vertical Cutter Mill (VCM 25). Sixty g of cake is extracted with 600 ml of acetone water (85 15). The mixture is carefully shaken and filtered through a folded paper 5 ml of the acetone extract is used for the subsequent purification assay. 

Basically the cleanup step consists of one of the two approaches already described in the aflatoxin section (a) the use of solid-phase extraction (SPE) columns, or (b) the use of immunoaffinity (IA) columns. 

T Urano, MW Trucksess, SW Page. Automated affinity liquid chromatography system for on-line isolation, separation, and quantification of aflatoxins in methanol-water extracts of corn or peanuts. J Agric Food Chem 41 1982-1985,1993. 

M Carvajal, F Mulholland, RC Garner. Comparison of the EASI-EXTRACT immunoaffinity concentration procedure with the AOAC CB method for the extraction and quantitation of aflatoxin B, in raw ground unshelled. J Chrom 511 379-383, 1990. 

As sample extraction and sample handling are of general consideration for the more exotic biological matrices often found in food analysis, the application of IPCR in the research project MYCOPLEX [89], founded by the European Union, promises interesting new developments. This project is dedicated to the detection by IPCR of ochra- and aflatoxins in milk and coffee, focusing on sensitivity and simplified antigen extraction by dilution of the samples. 

Jayaraj, A. et al., Metabolism, covalent binding, and mutagenicity of aflatoxin B1 by liver extracts from rats of various ages, J. Natl. Cancer Inst., 74,95, 1985. 

Hajare, S.S., Hajare, S.N. and Sharma, A. (2005) Aflatoxin inactivation using aqueous extract of ajowan (Trachyspermum ammi) seeds, journal of Food Science 70(1), C29-C34. 

Selvi, A.T., Joseph, C.S. and Jayaprakasha, C.K. (2003) Inhibition of growth and aflatoxin production in Aspergillus flavus by Carcinia indica extract and its antioxidant activity. Food Microbiology 20(4), 455 160. 

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