Detection of aflatoxins


Cultural methods for detection of aflatoxins:

Aflatoxins present important food safely problems in both developed and developing countries. Contamination is monitored in developed countries using enzyme-linked immunusorbent assay (ELISA)- and high-performance liquid chromatography (HPLC)-based assays, both of which may be too expensive for routine use in many developing countries. There is a need for inexpensive alternative approaches to detect aflatoxins in lots of foods and feeds. Reviewed here are culture-based methods that determine if a sample is contaminated with aflatoxigenic fungi. These approaches include (1) blue fluorescence of aflatoxin B1, particularly when enhanced by including ß-cyclodextrin in the culture medium, (2) yellow pigment production, and (3) colour change on exposure to ammonium hydroxide vapour. The presence of aflatoxin B1 can be detected by its blue fluorescence, which is enhanced when the toxin complexes with the hydrophobic pocket of ß-cyclodextrin. The yellow pigment and ammonium hydroxide vapour tests are based on the production of yellow anthraquinone biosynthetic intermediates in the aflatoxin pathway. These compounds act as pH indicator dyes, which are more visible when they have turned red at alkaline pH. Because these tests are based on two different mechanisms, it has been possible to combine them into a single test. In a study of 517 A. flavus isolates from the Mississippi Delta, the combined assay reduced false positives for aflatoxigenicity to 0%, and false negatives to 7%. The increased predictive power of the combined cultural assay may enable its use for inexpensively identifying
potential aflatoxin contamination in feeds and foods

http://www.cababstractsplus.org/google/abstract.asp?AcNo=20043182722


HOW CAN IT BE DETECTED?

Since most samples do not contain a detectable amount of aflatoxin, there is a need for a method which correctly identifies the many negative samples with minimum expenditure of time and money. Such a method is known as the BLACK LIGHT TEST and UVP BLAK-RAY® lamps are recommended for this application. Refer to UVP Brochure 818.
The aflatoxins all have absorption maxima around 360nm with a molar absorptivity of about 20,000: the B toxins are named for their blue fluorescence (425nm) and the G toxins for their green-blue fluorescence (450nm). The B1 toxin is the most common, flowed by B2 toxin, while the G toxins are fairly rare. The fluorescence sensitivity of the G toxins is more than 10 times greater than that for the B toxins. The minimum detectable level is about 100 pgrams for the G toxins and 1 ngram for the B toxins.
Corn is inspected under the BLAK-RAY lamp for a characteristic bright greenish-yellow (BGY) fluorescence in broken and damaged kernels. The test takes 5 minutes or less. If the fluorescence is observed, aflatoxin may be present but not necessarily in appreciable or detectable levels. There are substances in corn and other food that fluoresce under long wave ultraviolet irradiation, but are not associated with aflatoxin. Many other fungi such as Aspergillus niger, various Penicillium species, Aspergillus repens and other species which do not produce aflatoxin may produce fluorescent harmless metabolites so that the fluorescence is not a specific indication of the presence of toxicogenic molds, although it may indicate that conditions have been favorable for growth of the toxicogenic molds.
Additionally, A. flavus, isolated from corn, has displayed a broad spectrum of aflatoxin production, ranging from no detectable yields to high levels (>100 ppm). A probable contributory factor to this is that the fluorescence in naturally contaminated corn is not stable and disappears in 4 to 6 weeks on continuous exposure to visible or ultraviolet radiation although the toxin does not disappear. Fresh samples mush therefore be taken.
The reliability of the method depends on the size of the sample taken for analysis and how it is taken. A sample mush be large enough to be representative of the entire lot of corn and must be taken from all parts of the lot–whether bin, truck or railroad car. One highly contaminated corn kernel may account for objectionable levels of aflatoxin in a 3000-kernel (2 lb.) sample. If only a 1 to 2 lb. sample is collected, the one highly contaminated kernel could be missed. Usually from 5 to 10 lb. samples are collected, but even larger ones would be better. If the sampling is not performed properly, the results obtained by this screening method could be of little value.
The black light test is an excellent screening test for possible presence of aflatoxin, but it does not give quantitative indication. Even with good technique, brightly fluorescing samples may contain less aflatoxin than weakly fluorescing samples. Because of this, additional confirmatory and quantitative measurements are needed to be applied to those samples that reacted positively to the black light test. Non-fluorescing samples need not be subjected to this.

FURTHER SCREENING
Since the black light test is only a preliminary confirmatory test, another screening test is used in feed mills immediately following the black light test. This test involves the use of a small chromatographic column (mini-column) in the final separation and detection step of the method.
If the black light test gives a positive answer, the 10 lb. sample of cracked corn is ground to pass a twenty-mesh screen and the mixed thoroughly. A 50 g representative subsample of the 10 lb. of finely ground corn is used for the mini-column test. The final separation step of the mini-column method involves transferring 3 ml of chloroform extract to the top of a mini-column. After the chloroform drains into the column, 3 ml of an elution solvent are passed through the mini-column.
The mini-column is then inspected in the dark by shining a long wave black light on it. This, again, should be one of the BLAK-RAY lamps. If a bluish-green fluorescent band is detected at the proper height in the mini-column, the sample is judged to be positive to aflatoxin. In this instance, the property detected by the test is the bluish-green fluorescence.
This test, like the black light test, is a good screening test because it gives few false negative answers. However, this test also has the characteristic of giving only a few false positive answers. This makes it a better screening test, from a scientific viewpoint, than the black light test; however, it is better cost wise to run the two tests one after the other.
After the mini-column test is completed, a judgment can be made as to whether or not a shipment of corn will be accepted by a feed mill. The changes of accepting a truckload of corn that contains more than the 20 ppb aflatoxin is slight, as is the chance of rejected a truckload of corn that does not contain detectable aflatoxin.
If further confirmation is required, a subsample of the ground sample of corn can be sent to a chemical laboratory, or the extract remaining from the mini-column test can be used for a simple confirmatory test.
In this test, a portion of the chloroform extract not used for the mini-column test is evaporated to a small volume and some of this concentrated extract is spotted on a thin layer plate. A small amount of trifluoroacetic acid (TFA) is placed on top of the spot where the sample extract was placed. If aflatoxin is present in the sample spot on the thin layer plate, it will be derivatized to a water adduct of the parent aflatoxin compound. After thin layer chromatography is performed, this plate is inspected in the dark under a long wave black light. If a bluish-green fluorescent spot is detected on the thin layer plate at the same height as a reference spot known to be the derivative of aflatoxin formed by the TFA reaction, this confirmatory test (the TFA test) is positive and the concentration probably greater than 5 to 10 ppb.

QUALITATIVE TESTS
Although the TFA test is a confirmatory test, it is not essentially different from the mini-column test. Both are qualitative tests, that is, both test for qualities or properties of aflatoxin. The final property of aflatoxin involved in the mini-column test is the property fluorescing bluish-green under a long wave black light.
The TFA test involves three additional properties of aflatoxin. One of the properties involved is that aflatoxin will form a derivative in the presence of trifluoroacetic acid while on a thin layer plate at room temperature. Another property is that this derivative will move to a certain height on the thin layer plate using a certain developing solvent. A third property is that this derivative will fluoresce bluish-green under a long wave black light.
However, even if this test also is positive, we are still not 100% certain that aflatoxin has been detected. At this point, the degree of certainty is probably over 99%.
If we desire a further confirmation that aflatoxin is in the sample, a small amount of the underivatized compound can be isolated on a thin layer plate and extracted. This small amount of sample can then be inserted into the entrance of a mass spectrometer. The mass spectrometer is an instrument, which will break the aflatoxin into many fragments. The original mass of the aflatoxin molecule, plus that of each of the fragments produced, will be indicated by the output of the instrument.
The pattern of the various masses is characteristic of the particular aflatoxin involved. If the pattern of the chemical suspected to be aflatoxin is identical with the pattern given by a know aflatoxin, we are about 99.99% certain that the suspect chemical is aflatoxin

http://www.coleparmer.com/techinfo/techinfo.asp?htmlfile=aflotoxin.htm&ID=6

The Myco4test kit for fumonisin is a 10 minute (two 5 minute incubations) competitive enzyme-linked immunosorbent assay. The microtiter strip well test will detect the quantitative presence of fumonisin in corn and corn products (excluding corn gluten meal and corn gluten feed) in a range of 0 to 6.0 ppm. The principles of the test are based on the ability of free toxin, extracted from the sample, to compete with the enzyme-toxin conjugate (enzyme-labeled toxin supplied in the test kit) for antibody binding sites on the test wells. After a brief washing step, substrate is added to the wells which reacts with the bound enzyme conjugate to develop a blue color. The darker the blue color the lower the levels of toxin. The lighter the blue color the higher the levels of toxin. The test results may be read qualitatively visually or read in a microwell reader at 650 nm to provide quantitative results. The concentration of the toxin is determined by comparison to a standard curve. Up to 38 samples may be run using the 48 well test kit. Due to the speed of the test the maximum amount of wells run at one given time is 24 or two strips of 12. Running the test this way will provide a maximum amount of 19 sample wells. The test kit has been calibrated to a mixture of fumonisin B1, B2 and B3 at 5:2:1 ratios against the kit standard. The test kit and is cross-reactive to fumonisin B1 (97%), B2 (128%) and B3 (83%).

http://www.sdix.com/PDF/Products/User%20Guide%20Myco%20Fumonisin%201.5.doc

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect Fusarium species in foods. Antibodies to proteins extracted from the mycelia of Fusarium graminearum and Fusarium moniliforme (verticillioides) were produced in New Zealand white rabbits. These antibodies detected 13 Fusarium species in addition to the producer strains.

http://www.ingentaconnect.com/content/iafp/jfp/2003/00000066/00000003/art00016

Screening tests for fumonisins are based either on thin-layer chromatography (TLC) separation after appropriate clean-up of maize extracts or on commercially available enzyme-linked immunosorbent assays (ELISAs). Other immunologically based methods, such as dipstick (Schneider et al., 1995) and biosensor methods (Thompson & Maragos, 1996; Maragos, 1997; Mullett et al., 1998), have been described but have not found general use. Immunoaffinity columns have been designed to purify extracts before high-performance liquid chromatography (HPLC) separation and quantification of fumonisins B1, B2, and B3 analogues, and have also been used in a direct fluorimetric method for rapid determination of ‘total fumonisin’ (Duncan et al., 1998).
The TLC and other chromatographic methods for fumonisins have been reviewed (Shephard, 1998). The reversed-phase technique developed by Rottinghaus et al. (1992) has been used in surveys of contamination of maize with fumonisins (Shelby et al., 1994a). When combined with an efficient extract clean-up procedure based on use of immunoaffinity columns and detection by densitometry, TLC can be considered quantitative (Preis & Vargas, 2000).
The performance characteristics of screening tests for fumonisins in maize, based on interlaboratory collaborative studies, have not been reported in the literature. However, in-house comparisons between HPLC methods and the various screening tests have been described. The TLC method of Rottinghaus et al. (1992) has been compared with HPLC over a contamination range of fumonisin B1 of 1–250 mg/kg (correlation coefficient, r = 0.953; p < 0.0005; Schaafsma et al., 1998). The results obtained with a fibre-optic immunosensor in a direct competitive monoclonal antibody format with a fumonisin B1–fluorescein isothiocyanate conjugate compare favourably with those obtained with HPLC (Maragos, 1997).
The commercial availability of ELISA methods has made them popular for screening for fumonisin contamination. Although the antibodies used in ELISAs are raised against fumonisin B1, they generally have significant (but lower) cross-reactivity with fumonisins B2 and B3. The performance of ELISAs is generally assessed by comparison with HPLC determination of fumonisins and has been found to depend on the antibody used (Pestka et al., 1994; Usleber et al., 1994; Sydenham et al., 1996a,b; Kulisek & Hazebroek, 2000). The correlation between the results of HPLC and ELISA for naturally contaminated samples has been reported to vary from 0.51 (p < 0.05; Pestka et al., 1994) to 0.97 (p < 0.001; Sydenham et al., 1996a). However, such comparisons have generally shown an overall trend for the concentrations of ‘total fumonisins’ with ELISA to be greater than those determined in the same samples by HPLC

http://www.inchem.org/documents/jecfa/jecmono/v47je03.htm#3.0

THE TIMES THURSDAY DECEMBER 2 1999
GM crop toxin is leaking into the soil
BV NICK NUTTALL ENVIRONMENT CORRESPONDENT
SOME genetically modified crops are leaking powerful toxins from
their roots into the soil, scientists have found.
Researchers described the findings as "surprising and unexpected",
raising fresh fears about the environmental impact of such crops.
Companies have modified plants to produce poisons or toxins to combat
the pests that eat their stems and leaves. But the discovery that the
same plants are also leaking toxins into the soil has not, until now,
been considered an issue.
It will raise fears among some scientists, regulators and
environmental groups that beneficial soil organisms might be killed
and that in- sects living in the soil might become resistant to the
poisons.
The findings, published to- day in Nature, have been re- leased by a
team at the Univer- sity of New York that has been studying the roots
of GM maize.
Several crops, from maize, to corn and potatoes, have been genetically
modified to kill insect pests using a gene derived from a bacterium
called Bacillus thuringiensis (BT). In the United States 15 million
acres of corn modified with the BT gene were planted in 1998 or just
under 20 per
cent of the total crop. GM maize has also been planted in Europe
although the acreage is far smaller.
Concerns about the impact of such crops on the environ- ment were
triggered earlier in the year when it was found that monarch
butterflies had died after feeding on milk- weed dusted with pollen
from GM corn.
Other research found that lacewings that had fed on corn borers reared
on BT corn had also died, raising concerns that such crops are harming
more than just pests.
Professor Guenther Stozky, of New York University's labo- ratory of
microbial ecology, who has led the research, said yesterday that the
monarch re- search showed that the toxin was released from the pollen.
"Now we have found it is also continuously released from the roots
into the soil. The fact that the toxin is re- leased from the roots
was unex- pected," he said.
Professor Stozky said that the BT toxin was a large pro- tein molecule
which they had considered too large to cross the root membrane.
During the research, the team grew GM seedlings in the laboratory for
25 days. Each plant produced on average 105 microgrammes of pro- tein
and this was tested against larvae of the tobacco hornworm. Up to 95
per cent of the larvae died after five days with 50 per cent killed at
a dose of just 5.2 micro- grammes of protein.
Because the roots are constantly leaking the toxin, there is also the
risk that pests in the soil might rapidly become immune to the poison
triggering new, resistant, strains.
Biotechnology companies are likely to claim that, because the
bacterium from which the BT gene is taken, is found in the soil the
toxin is naturally part of the environment underground. But Professor
Stozky challenged such assertions, claiming that the bacterium was not
prevalent in the soil.
Dr Doug Parr, of Greenpeace said that the findings underscored the
"ability of GM crops to wrong-foot their creators and produce
unexpected and unwanted effects".
Dr Penny Hirsch, a soil expert at lACR-Rothamsted in Harpenden,
Hertfordshire, said yesterday that the findings were "interesting" but
added that field tests were needed to see whether the effects in the
laboratory were happening in the real world.
Taken from: http://www.gene.ch/gentech/1999/Dec/msg00018.html