Aflatoxin Assays Research Articles

Mycotoxins in Australia: biocontrol of aflatoxin in peanuts

The major mycotoxin problem in Australia is the formation of aflatoxins in peanuts by Aspergillus flavus and A. parasiticus. This is controlled by good farm management practice, segregation into grades on aflatoxin content at intake to shelling facilities, colour sorting and aflatoxin assays. A second problem is the potential presence of ochratoxin A in grapes and grape products, resulting from infection by Aspergillus carbonarius. Good quality control before and during wine making ensures ochratoxin A is kept to very low levels, but in dried vine fruit, ochratoxin A levels may be higher. Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. Some details of the process are given, including some basic laboratory experiments.

Biocontrol of aflatoxins in peanuts

Aflatoxins remain the most important mycotoxin problem in the world, and peanuts are a major source of these toxins. Control of aflatoxin levels in peanuts on a commercial scale is possible by colour sorting and aflatoxin assays on shelled peanuts, and this is widely practised in developed countries. However, this procedure is expensive.

Phage-displayed peptides that mimic aflatoxin B1 in serological reactivity.

To test phage-displayed random peptide libraries as sources of peptides that mimic the binding of aflatoxin B1 to monoclonal antibodies raised against the toxin. For two of the three MAbs tested, clones were obtained by panning, producing phage that bound specifically to MAb 13D1-1D9 (MAb 24; specific for aflatoxins B1 and G1) and MAb 6E12-1E9 (MAb 13; specific for aflatoxins B1, G1 and B2) in ELISA. The amino acid sequences of the binding peptides varied. Those binding to MAb 24 contained the sequence of '...YMD...', and those that bound to MAb 13 contained the dipeptide 'PW'. Mimotope phage was used in a competition ELISA format for assaying aflatoxin concentrations. The results show that mimotope preparations are effective substitutes for pure toxin in these ELISA procedures. These results should contribute significantly to enhancing the safety and diminishing the costs of aflatoxin assays.

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek's-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B1 was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH4)2HPO4 (1.55 gL(-1)) and NaNO2 (1.6 gL(-1)) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B1 production. Zn(2+) and Co(2+) supported significantly both fungal growth, as well as, aflatoxin B1 production at the different tested concentrations. Zn(2+) was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (aw) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum aw for the fungal growth was 0.8 on both coffee beans and rice grains, while aw of 0.70 caused complete inhibition for the growth and aflatoxin B1 production. H2O2 is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO3 and C6H5COONa converted aflatoxin B1 to water-soluble form which returned to aflatoxin B1 by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B1 production. Stearic acid supported the fungal growth and decreased the productivity of AFB1 gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B1 production. Vitamins C and D2 were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B1 production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B1 synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B1 production. All the tested enzymes as well as aflatoxin B1 production were inhibited by either catechol or phenol.

Degradation of Aflatoxin by Poultry Litter

Two trials were conducted to determine whether deep stacking of contaminated corn with poultry litter destroys aflatoxin. Contaminated corn was ground and mixed with litter to carbon:nitrogen ratios of 30:1. Moistures were adjusted by adding tap water just prior to incubation or stacking. The initial laboratory trial included only broiler litter at 40% moisture, whereas the subsequent field trial involved a 2 × 2 factorial design with litter type (turkey or broiler) and moisture (20 or 40%) as main effects. Aflatoxin assays were reduced in the laboratory trial from 433 and 402 to 54 and 8 ppb in Containers 1 and 2, respectively, after 35 d of incubation at 28 C. In the field trial, aflatoxin disappeared from broiler and turkey litter mixtures with projected moistures of 20% after 10 and 6 wk of storage, respectively, whereas disappearance in mixtures containing projected moistures of 40% required 5 and 3 wk, respectively. Differences in moisture appear to account for differences in the ability of turkey and broiler litter to detoxify aflatoxin. Hence, turkey and broiler litter would appear equal with respect to the ability to detoxify aflatoxin-contaminated corn. Disappearance of aflatoxin during storage with litter could have occurred as a result of ammonia release during storage or microbial detoxification mechanisms. However, nitrogen values suggest that microbial action was responsible for much of the detoxification, as aflatoxin disappeared from mixtures with little apparent ammonia release.

Performance of Two Immunochemical Assays in the Analysis of Peanuts for Aflatoxin at 37 Field Laboratories

A study was conducted to measure the precision of 2 rapid aflatoxin assay systems in use at 37 peanut buying points during the 1991 harvest season. Aflatoxin laboratories were established at the 37 buying points to analyze peanut samples from all incoming farmers' stock loads as part of a joint project sponsored by various segments of the U.S. peanut industry and the U.S. Department of Agriculture. Eighteen laboratories were equipped with Neogen's veratox FSP rapid assay system, whereas 19 laboratories used Vicam's Aflatest rapid assay system. To monitor the performance of the field laboratories during the project, 3 portions of each of six 27 kg samples of ground peanuts were sent to each laboratory for analysis over a period of 6 weeks. Aflatoxin concentrations ranged from 0 to 300 ng/g when eight 200 g subsamples of each sample were analyzed by liquid chromatography (LC). For the 5 samples contaminated with aflatoxin, relative standard deviations for repeatability (RSDr) for laboratories using veratox FSP ranged from 18.66 to 53.29%, and the relative standard deviations for reproducibility (RSDR) ranged from 22.79 to 59.29%. For laboratories using the Aflatest system, RSDr values ranged from 18.70 to 41.48%, and RSDR values ranged from 23.84 to 47.56%. Horwitz ratios < 2.0 were found for 4 of the 5 contaminated samples for both methods, indicating that the overall precision of the 2 methods used in the project was good. Mean aflatoxin concentrations, as determined with the rapid assay systems, were generally lower than those determined by LC, particularly for more highly contaminated samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Fluorescence Sorting and Color Sorting for the Removal of Aflatoxin from Large Groups of Peanuts

Abstract Fluorescence sorting (by both machine and hand) and color sorting (by both machine and hand) are compared as methods for reducing aflatoxin levels in groups of peanuts. An experimental design is used that allows the four methods to be compared quantitatively. Approximately 1200 photographs of peanut groups were taken, allowing for a more complete understanding of the visual characteristics of contaminated peanuts. Sampling errors are controlled by the experimental design and by including a large fraction of the peanuts in aflatoxin assays. Fluorescence sorting was not effective as an aflatoxin control method. Machine color sorting was shown to be effective in the removal of aflatoxin-contaminated peanuts. Hand sorting was shown to be even more effective than machine color sorting in aflatoxin removal.

Aflatoxin in Arizona cottonseed: Increase in toxin formation during field drying of bolls

Aflatoxin assays and moisture determinations were made on locks from bolls inoculated withAspergillus flavus 30–32 days from flower and harvested after additional periods of 5, 7, 9, 11, 13 and 15 days. Inoculated locks were always tight but had moisture contents comparable to those in non-inoculated locks on the same bolls. Lowest toxin concentrations were in seeds from bolls still green at harvest with moisture contents >50%; highest concentrations were in bolls with fully fluffed locks and moisture contents <10%. The greatest increase in toxin concentrations occurred in bolls following suture opening, at the initiation of boll dry-down. Toxin concentrations were comparable for bolls that fluffed in 11 days from inoculation and those requiring 15 days for fluffing. A boll fluffing 11 days from inoculation had the highest level of toxin detected, ca 400 μg/g. The ripening and drying processes rather than the duration of fungal/plant interactionper se seems critical for maximum toxin formation.

Evaluation of non-polar bonded-phases for the clean-up of maize extracts prior to aflatoxin assay by HPTLC

The ability of a PH non-polar bonded phase to extract aflatoxin from aqueous solutions (1% v/v) containing 5, 10, 20 and 30% acetone, acetonitrile or methanol was determined. The amount of aflatoxin extracted by the cartridge from aqueous solutions remained almost constant (97–102%) as the concentration of methanol increased, but decreased rapidly when the amount of acetone or acetonitrile increased beyond 10%. Examination of the ability of a number of bonded phases (C2, C8, C18, CH and PH) to extract aflatoxin from aqueous methanol extracts of maize revealed that the PH bonded phase was the most efficient. Aflatoxin is eluted from the cartridge by chloroform (7 ml). An additional clean-up step involving bi-directional development was required when quantifying low levels of aflatoxin in maize by HPTLC.

Evaluation of a Testing Program for Aflatoxin in Corn

A computer model that accounts for sampling and analytical variability was developed to simulate the aflatoxin testing program administered by the North Carolina Department of Agriculture (NCDA) to regulate aflatoxin in corn meal. Monte Carlo solution techniques were employed to account for conditional probabilities that rise from multiple samples being used in the testing program. The NCDA testing program was then evaluated by applying the computer model to a hypothetical group of 1000 corn meal lots with the same distribution of aflatoxin concentrations as was observed among aflatoxin assays made by NCDA on commercial lots of corn meal from 1977 to 1980. The average of the 1000 lots assayed was 17.7 parts per billion (ppb). The model predicted that 79.5% of the lots would be accepted and 20.5% of the lots would be rejected by the NCDA testing program. The accepted and rejected lots contained an average of 5.7 and 64.2 ppb aflatoxin, respectively. The testing program accepted 7.3% of the lots with more than 20 ppb aflatoxin (consumers' risk) and rejected 1.0% of the lots with 20 ppb or less (processors' risk). A correct decision was made 94% of the time.

Estimation of the Distribution of Lots of Shelled Peanuts According to Aflatoxin Concentrations1

Abstract The distribution of shelled peanut lots according to their true aflatoxin concentrations is different than the distribution of aflatoxin assays made on those lots. This difference is due to assay errors associated with sampling, subsampling and analysis. A method was developed to estimate the distribution of the lots according to their true aflatoxin concentration based on aflatoxin assays. Estimates of the distribution of the 1973, 1974, and 1975 peanut lots were computed from their aflatoxin assays which were recorded by the Peanut Administrative Committee (PAC). Fifty-five percent of the PAC assays for the 3 crop years were less than 3 parts per billion (ppb) aflatoxin, whereas the estimated lot distribution indicated that only 48% of the lots were less than 3 ppb. On the other hand, only 94% of the assays compared to an estimated 96% of the lots were less than 25 ppb.

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.

Use of zinc acetate in extract purification for aflatoxin assay of cottonseed products.

AbstractThe official Association of Official Analytical Chemists’ and American Oil Chemists’ Society’s methods for aflatoxin assay of cottonseed products utilize 20% lead acetate solution to remove gossypol, fatty acids, and traces of lipids as insoluble lead derivatives. The substitution of a 20% zinc acetate solution containing a trace of a trivalent metal chloride to precipitate zinc derivatives yields, in some cases, superior cleanup of interfering compounds of primary extracts.

Aflatoxin Occurrence in Some White Corn Under Loan, 1971: IV. Mold Flora

SUMMARY An extensive, as well as an intensive, study was made of white corn under government loan as it was delivered in 1972 to the Agricultural Stabilization and Conservation Service at Diehlstadt, Missouri. The corn (450,000 bu) came from 77 loans and had been stored on farms. From each of the 1,283 trucks representative samples of corn were taken and ground for aflatoxin assay; a small portion was used to streak plates of yeast extract agar containing tetracycline. After incubation for 5 days at 28 C, the plates were read under a dissecting microscope for the common types of molds with special emphasis on the presence of the Aspergillus flavus group. A rough estimation was made of the A. flavus colonies per plate. Other molds recorded were A. niger, A. fumtigatus, A. terreus, A. glaucus group; species of Trichoderma, Penicillium, Fusarium, Rhizopus, Mucor, and Absidia. Of the 1,283 samples, each representing a truckload of corn, 394 contained aflatoxin, and of these, A. flavzs group was present in all but seven. The molds most common in the 1,283 samples were Penicillium sp. (94%), Fusarium sp. (89%), A. flazus (82%), and A. niger (71%). Aspergillus parasiticus was seen in only 15 times. No correlation seemed to exist between the grade of corn and the presence of A. flavus. However with some molds, a higher percentage occurred in the poorer grades. When actual counts of A. flavus were made on corn from three loans, the occurrence of A. flavus closely paralleled the aflatoxin content of the corn samples.

Control of aflatoxins in the food industry

Abstract

Use of Tracheal Organ Cultures for Bioassay of Aflatoxins

Abstract Tracheal organ cultures from day-old chicks were used for the assay of aflatoxins. Ciliary epithelium in the organ culture was found to be highly sensitive to aflatoxins B1 and G1. Anatoxin B1, incorporated into the medium in which the organ cultures were established, readily caused distinguishable eytomorphological changes in 48 hr at a concentration of 0.2 μg/ml and complete cessation of ciliary movement at a concentration of 0.5 μg/ml. Anatoxin G1 produced the same effects, but at higher concentrations (0.5 μg/ml and 2.0–2.5μg/ml). The degree of cytomorphological changes of the ciliary epithelium in the organ cultures was proportional to the concentration of aflatoxins and the period of time exposed. A dose-response curve was developed for the period of observation. The technique is simple, reproducible, and inexpensive. The feasibility of developing this test system as a biological assay for the presence of aflatoxins and other mycotoxins in feeds and foodstuffs is being explored.

Sample preparation for aflatoxin assay: the nature of the problem and approaches to a solution.

AbstractCases have been reported of individual peanuts, cottonseeds or Brazil nuts so highly contaminated with aflatoxin that, for a 50 g portion to be representative of the whole, the sample preparation procedures should grind each unit to a large number of particles and distribute them uniformly throughout the sample. Assuming uniform contamination of the individual kernel, each 50 g sample should contain 1/100 of that kernel. Even though these extreme cases may be encountered only infrequently, the more usual situation still presents difficulties because of great variability in individual kernel contamination. However, if the extreme can be handled, one can expect to handle the more usual situation. Equipment and procedures to achieve this distribution goal are described. The equipment studied includes a food chopper (Hobart), a nut mill (Thomas Mills), a disc mill (Bauer), a hammer mill (Fitzpatrick Model D comminuting machine), a hammer mill designed specifically for peanut samples (Dicken’s subsampling mill), a Polytron homogenizer (Bronwill Scientific), a vertical cutter‐mixer (Hobart), and a sample splitter (Jones riffle). Commodities examined were shelled peanuts and in‐shell Brazil nuts, walnuts, pecans and almonds. Comminution and mixing effectiveness were determined by particle size analysis, by distribution of kernels made radioactive by neutron activation and by aflatoxin analysis of naturally contaminated products. From the results we conclude that the ultimate in sample uniformity can be achieved with a disc mill, solvent addition to obtain a fluid system and mixing and grinding of the fluid with a dispersion mixer‐grinder. A practical uniformity can be achieved in a vertical cutter‐mixer with less expenditure of time and effort for the commodities studied.

The bioassay of aflatoxins and related substances with Bacillus megaterium spores and chick embryos.

AbstractThe “paper disc plate” method of antibiotic assay usingBacillus megaterium spores was tested for the assay of aflatoxins and related compounds. Significant inhibitions were obtained in the range of 1–4 µg/disc for aflatoxin B1 and 4–8 µg/disc for aflatoxin G1. Dicumarol was the most active of the compounds tested and inhibited at 0.1–1.0 µg/disc. Compounds were also tested using nine‐day‐old chick embryos. It was found that embryos incubated in egg cartons were much more sensitive to toxins than eggs incubated on cotton padding or in a commercial egg incubator. The LD100 of aflatoxin B1 to carton‐incubated embryos was 0.01 µg, while to embryos from the commercial incubator the LD100 was &gt;5.0 µg. The results of the two bioassay procedures did not correlate completely with each other.

Fluorodensitometric Measurement of Aflatoxin Thin Layer Chromatograms

Abstract The precision limit of visual comparison procedures used in aflatoxin assay methods can be no better than ± 2 0 % for a single observation and, under operating conditions, it is probably close to ± 2 8 % . Fluorodensitometric procedures described in the literature can improve this precision to ± 9% for the average of multiple observations. A possible source of inaccuracy in the published densitometric procedures is pointed out and a method using internal standards to avoid the inaccuracy is presented. Tested with spiked extracts of "clean" peanut butter, the procedure gave an average 101 ± 3% recovery of added aflatoxin B1 and 89 ± 6% recovery of aflatoxin B2 with a precision for individual assays equal to the precisions reported for the other procedures. The major source of error was shown by elimination to reside in the thin layer chromatography and aflatoxin instability.

Collaborative Study of "A Versatile Procedure for Assay of Aflatoxins in Peanut Products," Including Preparatory Separation and Confirmation of Identity

Abstract collaborative study of the CB procedure for aflatoxin in peanut products was carried out on naturally contaminated peanut butter and peanut meal and on peanut butter to which known amounts of aflatoxin Bx and Gi were added. Part of the study included preparatory isolation of the aflatoxin Bx fovind in the naturally contaminated samples and confirmation of the identity by both chemical and biological tests. The results from 13 collaborators demonstrate both between- and withinlaboratory precision and accuracy equal to the official, first action procedure; advantages in speed and convenience were noted. The preparatory separation was easily accomplished with clear chemical and biological proof of satisfactory isolation by seven of the nine participants in this phase of the study.