Louise S. Lee

Determination of aflatoxins in individual peanuts and peanut sections

Subsamples of a given lot of peanuts may vary greatly in aflatoxin content due to extreme variability in the degree of contamination of individual kernels. A micro method, adapted from the aqueous acetone procedure recently proposed by Pons and Goldblatt for the determination of aflatoxins in cottonseed products, was developed to permit accurate determination of aflatoxins in individual kernels and kernel sections.Use of this procedure permitted the topographic distribution of aflatoxins within single kernels to be mapped and indicated that the toxins are not uniformly distributed within contaminated kernels, even when the kernel contains a high level of aflatoxins. Although wrinkling or discoloration sometimes indicated that a kernel was contaminated, this type of physical damage was not found to be a reliable indication of aflatoxin content. Also it was noted that a few apparently sound and mature kernels contained high levels of aflatoxins.

Assay of aflatoxin in peanuts and peanut products using acetone-hexane-water for extraction

A quantitative method is described for the assay of aflatoxin in peanut products. The procedure involves extraction of aflatoxin from the sample with a homogeneous acetone-hexane-water solvent mixture followed by purification of the extract by phasic extraction of the aflatoxin with aqueous sodium chloride and then with chloroform. The purified chloroform extract is analyzed by thin-layer chromatography by comparison of the intensity of fluorescence of any aflatoxin with the intensity of a known standard. The aflatoxin analyses of peanuts were found to be very variable due to sampling, and this variability has been greatly reduced by finely grinding and thoroughly mixing 2 kg of the sample before removal of an aliquot for assay. The method is sensitive to approximately 2 parts per billion.

Mutagenic potential of ammonia-related aflatoxin reaction products in a model system

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the U.S. Department of Agriculture determined the mutagenic potential of aflatoxin reaction products following ammoniation of aflatoxin B 1 in a pressure chamber used to decontaminate aflatoxin-contaminated cottonseed meal. Uniformly ring-labeled (14C)-aflatoxin B1 was added to nonlabeled B1, distributed on an inert carrier and treated with 4% ammonia at 40 psi, 100 C, for 30 min. Aflatoxin-derived decontamination reaction products were separated, and fractions having a high specific activity were tested for mutagenic activity using the Salmonella/mammalian-microsome mutagenicity assay (Ames test). When concentrations ranging from 3.3 to 100 μg per plate were tested, all fractions exhibited a similar mutagenic response. The observed mutagenic activity was 2,000-20,000 times less than that observed with nonammoniated aflatoxin B1.

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled14C-aflatoxin B1 was added to cottonseed meal that contained ca. 4000 µg naturally incurred aflatoxin B1/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B1 after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B1 was mutagenic at a concentration of ca. 0.005 µg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 µg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 µg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.

Kinetic study of acid-catalyzed conversion of aflatoxins B1 and G1 to B2a and G2a

Adjusting dilute aqueous solutions of aflatoxins B1 and G1 to pH 1, 2 and 3, and heating over a range of 40–100 C resulted in the conversion of B1 to B2a and G1 to G2a as major products. Both B2a and G2a were identified by co-thin layer chromatography with authentic B2a and G2a and M1 on silica gel plates developed in two different solvents. The rate of disappearance of B1 or G1 at given temperature and at constant pH was found to be first order with respect to each aflatoxin. At given temperature the conversion is strongly pH dependent, a 10-fold increase in H+ ion (1 pH unit) producing about a 9-fold increase in the reaction rate, indicating first order dependent of the rate on H+ ion concentration.

Relationship of physical appearance of individual mold-damaged cottonseed to aflatoxin content

Abstract and SummaryOver 700 individual aflatoxin-suspect cottonseed were hand-selected from a heterogenous stockpile of ginned seed. The seed were categorized on the basis of (a) bright greenish-yellow, fluorescence termed cateye, on the linter fibers under ultraviolet light; (b) partially bald seed with part of the linter fibers removed by ginning; (c) a combination of cateye and balding; (d) thin and discolored lint; and (e) bluish, not cateye, fluorescence. Aflatoxin assays on each of the 771 selected seed showed that 142 out of 771 (18%) were contaminated by aflatoxin (B1+B2) in the range of 150 ppb—5.75 million ppb. Some 93% of the aflatoxin-contaminated seed was concentrated in categories (a), (b), and (c), with the highest concentration, 61%, in category (b). Eight seed in these three categories contained over 1 million ppb of aflatoxins. The data suggest that removal of cateye and partially bald seed from contaminated lots of cottonseed should be more effective for controlling aflatoxin contamination in cottonseed than removal of cateye seed alone.

Ammoniation of aflatoxin B1 in a pressure chamber used to decontaminate toxin-containing cottonseed meal

Radiolabelled aflatoxin B1 mixed with non-labelled B1 distributed on an inert carrier was treated in a pressurized ammoniation chamber with 4% ammonia at 40 psi and held at 100 C for 30 min. Twenty per cent of the radiolabel was lost, probably as volatiles. Less than 1% of the original toxin was recovered as B1 indicating that ammoniation altered the structure of essentially all of the B1. Approximately 20% of degraded aflatoxin B1 was accounted for as a 206 MW compound that exhibited properties of a nonfluorescent phenol with a difuran moiety but neither the lactone carbonyl nor the cyclopentenone ring of aflatoxin B1. The remaining degradation products were fragments of B1 having molecular weights less than 200.

Pons scaled-down clean-up column adapted for use in solvent-saving modification of the CB method for aflatoxin

The solvent-saving procedure devised by Pons using a small Chromatographie tube (Bio-Rad Laboratories glass Econo-Column, 10 mm id X 300 mm long) has been adapted and extended for use in modifications of the Official AOAC procedure for quantitative determination of aflatoxins in corn, peanuts, soybeans, coconut and pistachios. Thirty mL of each of 3 solvents for column washes was used instead of the 150 mL specified by the Official CB Method. The analytical aliquot was also reduced 80%, but sample size and extracting solvent volume were not changed, so that there was no loss in sensitivity. Toxins ranging from 3 to over 1,000μg/g of sample were quantitated after clean-up using both procedures with no statistically significant difference between results.

Effect of modular storage of arizona seed cotton on levels of aflatoxins in seed

Samples were drawn from 40 free-standing modules of commercially grown Arizona seed cotton and analyzed for aflatoxin at the time of moduling and after 27 days of field storage. Thirty modules derived from spindle harvested cotton showed no significant increase in aflatoxins following modular storage, while all 10 modules packed with seed cotton derived from ground harvesters yielded significantly higher levels after storage than were detected at the time of moduling. Increases in toxin levels ranged from 12–232% with an average value of 67%. Even under ideal module making conditions significant increases in aflatoxins can be expected in ground-gleaned seed cotton harvested in areas where chronic aflatoxin contamination exists.

Aflatoxin in Arizona cottonseed: Lack of toxin formation following aspergillus flavus inoculation at sutures

Aflatoxin assays were conducted on seeds from cotton bolls inoculated withAspergillus flavus in commercial fields in Arizona. Inoculations were at sutures at the initiation of boll opening either in the morning or evening in August over a three-year period. One morning inoculation was followed by a water treatment that simulated rain. Fully fluffed bolls were harvested after two or four weeks, and lint and seed linters were examined for bright-green-yellow-fluorescence (BGYF). Ginned seed were assayed for aflatoxin. While BGYF of lint was detected, no seed linters exhibited BGYF and no seeds from the 140 bolls examined contained aflatoxin. Results imply that boll infection byA. flavus occurs before the initiation of boll opening, an observation that is in agreement with recent reports ofA. flavus infection but contrary to conclusions made in earlier reports.