Aflatoxin in Corn

Introduction

Aflatoxin is a term generally used to refer to a group of extremely toxic chemicals produced by two molds, Aspergillus flavus and Aspergillus parasiticus. The toxins can be produced when these molds attack and grow on certain plants and plant products. In the United States, aflatoxin production occurs when Aspergillus flavus and Aspergillus parasiticus attack peanuts, cottonseed, white and yellow corn and certain nuts. Most of the aflatoxin problems on corn in the United States are caused by Aspergillus flavus, and the most potent toxin produced by this mold is called aflatoxin B₁ . Drought, extreme heat and corn ear injury from insect feeding stress the corn and create an environment favorable to these molds and to aflatoxin production.

Aflatoxin poses a low level threat to the human food supply in the United States because existing regulations and testing by federal agencies and industry exclude contaminated products from the food chain. Generally, these programs have been successful in protecting U.S. consumers from aflatoxin contaminated food.

Aflatoxin Situation in 2006-2007

Aflatoxin Situation in 1998

Disease Cycle

In the field: Initially, it was believed that Aspergillus flavus was only a problem on corn in storage. But it has since been shown that Aspergillus flavus can also attack corn in the field. Field infection of corn by Aspergillus flavus can result in aflatoxin production in the corn prior to harvest. The fungus is able to invade through the corn silks or in association with insect damage to kernels and ears.

Temperatures ranging from 80 degrees F to 100 degrees F and a relative humidity of 85% are optimum for aspergillus flavus growth and aflatoxin production. Periods of drought and heat stress during the growing season, especially during pollination and as kernels mature, favor Aspergillus flavus infection. Corn damaged by insects or weather factors such as hail, early frost that cracks the pericarp and wind storms, may also be predisposed to infection by Aspergillus flavus.

On corn in the field Aspergillus flavus is evident as a greenish-yellow to yellowish-brown, felt-like or powdery mold growth on or between the corn kernels. Mold growth is more likely to develop adjacent to or in insect damaged kernels on ears (see figures 1 and 2).

Figure 1. Aspergillus flavus in standing corn.

Figure 2. Close up of Aspergillus flavus on corn.

In storage: Aspergillus flavus can also develop or continue to develop on corn in storage (see figure 3). The extent and severity of both invasion by Aspergillus flavus and the production of aflatoxin in the stored grain are influenced by several factors including moisture content and temperature of stored grain, condition of grain going into storage and length of storage.

Figure 3. Aspergillus flavus in stored corn.

Aspergillus flavus grows best on corn at 18.0-18.5 % moisture. Moisture content below 13% prevents invasion by Aspergillus flavus. Fungal growth may begin on corn at a moisture content lower than 18.0%. Then as the fungus grows, respiration occurs releasing heat and moisture into the surrounding environment in the grain mass. This results in an increase in the moisture content and temperature of the surrounding corn, causing a hot spot. If moisture content and temperature continue to rise, the environment for Aspergillus flavus becomes more favorable. Fungal growth is best at 18% moisture. At 20% moisture content and above, other fungi grow better and crowd out Aspergillus flavus.

Aspergillus flavus grows best at high temperatures. The fungus will grow slowly in grain between 40-50 degrees F but will grow rapidly in grain at 80-90 degrees F.

Corn contaminated with Aspergillus flavus going into storage will deteriorate at a lower moisture content, at a lower temperature and in a shorter time than grain that is free or almost free of Aspergillus flavus as it goes into storage. Corn with cracks or breaks in the pericarps or seed coats, broken kernels or other physical damage is more subject to invasion by Aspergillus flavus.

It is important to note that the presence of Aspergillus flavus on corn does not necessarily mean that aflatoxin is also present in that corn. Circumstances that favor mold growth may also favor mycotoxin production but mold growth may also occur with little or no mycotoxin production.

Aflatoxin

Aflatoxin is a term generally used for a group of toxins produced by Aspergillus flavus and Aspergillus parasiticus. These toxins are named for the fungus producing them, e.g. "A" from the genus name Aspergillus, "fla" from the species name flavus added to toxin to give the name aflatoxin. There are several different toxins in the aflatoxin group. They are designated aflatoxin B₁ and aflatoxin B₂ (because they are blue under UV light ), aflatoxin G₁ and G₂ (because they are green under UV light) and aflatoxin M₁ which may be found in milk of cows fed aflatoxin contaminated feed.

Although it has been known for more than 100 years that some kinds of moldy grains when eaten by animals or humans could cause illness, intensive study of mycotoxins and the illnesses caused by mycotoxins only dates from the 1960's. In 1960 scientists determined that the deaths of more than 100,000 turkey poults in England were due to a toxic substance in the peanut meal ration fed to the birds. The toxin was a product of the mold Aspergillus flavus growing in the meal. The toxin was soon purified, chemically characterized and named aflatoxin. Feeding tests with laboratory animals showed that aflatoxin in amounts of a few parts of toxin per billion (ppb) parts of feed could cause serious injury, including fatal liver cancer, to animals.

Aflatoxin is extremely durable under most conditions of storage, handling and processing of seeds or in foods or feeds made from contaminated seeds. It is very heat stable and will withstand temperatures up to boiling. Toxin levels in corn may decline in storage, but may still be present after 7 years.

Aflatoxin becomes more prevalent, and therefore more of a food safety concern, during a drought because low rainfall and high temperatures encourage the growth and survival of the molds that produce the toxins. Also, crops stressed by drought and high temperatures and/or weakened by insect or other damage, (i.e. hail or frost) are more susceptible to mold growth and subsequent aflatoxin contamination. The aflatoxin-producing molds can grow on crops in the field, poorly dried harvested crops in storage and processed food and feed products.

Alternative Uses for Aflatoxin Contaminated Corn

Sampling and Testing for Aflatoxin

Field sampling is difficult. If a corn combine is used, make one or more passes the entire length of the field. As the hopper is being emptied, pass a cup through the moving stream at 30 second intervals until the collected volume totals 10 pounds. Mix this well and save for testing.

Sampling from loads of grain or stored grain must be done carefully to insure that a representative sample is taken. The recommended procedure is to sample during loading or transfer of grain by passing a cup through a moving stream of grain at frequent intervals (for example, every minute) and combining the samples for a representative, composite sample. Probe sampling is acceptable if grain is not being moved or transferred. Again, it is important to take a number of probes and combine these into one representative sample. The initial sample should be about 10 pounds. This sample should be mixed thoroughly and then a subsample tested using one of the methods described below.

There are several methods of checking corn for the presence of aflatoxin. The use of a blacklight is a screening technique that indicates potential aflatoxin problems but does not give quantitative results. Various rapid test kits may be used in the field, on the farm or at the elevator to detect the presence of aflatoxin and, in some cases, quantify the level present. Finally, laboratory tests such as the minicolumn, thin layer chromatography and gas chromatography can give quantitative results.

Blacklight: Corn kernels infected with Aspergillus flavus will often produce a characteristic bright greenish-yellow fluorescence (BGYF) when examined in a darkened room under long wave ultraviolet light, a blacklight. This fluorescence is the result of properties of kojic acid, a compound produced by Aspergillus flavus but not related directly to aflatoxin. Bests results are obtained when the blacklight test is done on cracked kernels. The blacklight test is a presumptive test. Blacklight positive samples usually contain some aflatoxin but this method cannot determine if the load exceeds the FDA Guideline of 20 ppb. If a load of corn is found to be positive with a blacklight test, it is recommended that a representative sample of this lot be taken and a determinative test such as a minicolumn or other test be performed. The blacklight test may result in false positives and is not the method of choice for most grain buyers.

Minicolumn Test: The minicolumn test is a more determinative test for aflatoxin. It is rapid, relatively inexpensive and can be performed at the buying point. This test is commonly employed to determine if corn exceeds the FDA Guideline of 20 ppb. A concentrated corn extract is passed through a minicolumn tube that contains layers of alumina and silica gel. After treatment with a developing solution, a fluorescent blue band appears. The color intensity of the blue fluorescence is directly proportional to the level of aflatoxin, but in most cases the minicolumn test is used only to identify lots with concentrations in excess of 20 ppb. If the sample used for analysis is representative of the entire load, it is an acceptable method for determining whether to accept or reject loads.

Thin Layer Chromatography (TLC): Thin layer chromatography is a more precise measure of determining aflatoxin concentrations in corn. This method involves extraction with chloroform followed by purification on a silica gel column for quantitative thin layer chromatography. If this method is coupled with Association of Official Analytical Chemists approved extraction methods, it is superior to other methods of quantifying aflatoxin. This method is often used by testing laboratories.

Rapid Test Kits (ELISA Kits): Recently, several commercial firms have marketed rapid test kits based on enzyme-linked immunosorbent assays for use in determining the presence of aflatoxin in corn samples. These test kits are self contained and provide all necessary instructions to complete analysis on-farm, at the elevator or at the buying point. Several kits are available at varying prices (ranging from $5-15/ test depending on quantity ordered).

Testing Laboratories: Samples for aflatoxin testing can be submitted to the Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, P.O. Box 6023, Columbia, MO 65205. The sample should be a half pound sample that is representative of the entire load or volume of grain. There is a $15.00 fee for aflatoxin analysis.

The Missouri Department of Agriculture is also offering aflatoxin testing for $20.00 per test. Those wanting grain tested should provide the department with approximately two pounds of grain. The grain sample should be representative of all the grain stored, so grain should be pulled from more than one area to make the sample. Each sample must include some type of identification along with the name, address and telephone number of the person to receive the test results.

The department is providing aflatoxin testing from its New Madrid office. Samples can be sent directly to the Grain Inspection Services Laboratory, 718 U.S. Highway 61, New Madrid, MO 63869, (573-748-5526).

For each sample tested, the department will supply results and information on the amounts of aflatoxin acceptable in grain used for human consumption and livestock feed.

Acceptable Tolerances of Aflatoxin in Corn for Food and Feed Uses

(Normally interstate shipment does not allow blended corn. This no-blending policy may be relaxed by the FDA in response to specific state requests in years when drought damaged corn is testing high in aflatoxin. See section immediately following for further information on this)

Present acceptable levels of aflatoxin in corn used for food and feed as established by the United States Food and Drug Administration (FDA) are as follows:

1. Corn containing no more than 20 ppb of aflatoxins when destined for food use by humans, for feed use by immature animals (including immature poultry) and by dairy animals, or its destination is not known.

2. Corn containing no more than 100 ppb aflatoxins when destined for breeding beef cattle, breeding swine or mature poultry (e.g. laying hens).

3. Corn containing no more than 200 ppb aflatoxins when destined for finishing swine (e.g. 100 lbs. or greater).

4. Corn containing no more than 300 ppb aflatoxins when destined for finishing (i.e. feedlot) beef cattle.

Methods of Reducing Aflatoxin in Corn

Blending: The physical mixing of contaminated (greater than 20 ppb) corn with uncontaminated (less than 20 ppb) corn is not usually a recommended practice. Blending corn lots to reduce the level of aflatoxin in corn going into interstate trade is prohibited by the FDA. In 1998 special permission was obtained to allow the "blending" of aflatoxin adulterated corn with non-contaminated corn. This special permission has expired.

Detoxifying: Currently, there is no FDA-approved nor sanctioned method for "detoxifying"- through ammoniation or other means- corn that contains aflatoxin. FDA has approved ammoniation as a method for detoxifying cottonseed.

Several states, including Texas, have implemented state policies that allow ammoniation of corn as well as cottonseed. Corn ammoniated under these specific state policies is prohibited from being distributed in interstate commerce and is subject to labeling and feeding restrictions. This policy is not approved in Missouri.

Another approach to detoxifying aflatoxin contaminated corn is the use of dietary additives which bind aflatoxins and prevent their absorption from the gut. These dietary additives include various clay minerals such as bentonites and hydrated sodium calcium aluminosilicates (HSCA). Research has shown that at a concentration of 0.5% of the diet, the aluminosilicates are very effective at binding aflatoxins and preventing their absorption in both ruminants and nonruminants. These dietary additives are sold as anti-caking agents and their use in detoxifying mycotoxins has not yet been approved by the Food and Drug Administration.

Alternative Uses for Aflatoxin Contaminated Corn

• Use as a component in fertilizers.
• Ethanol or gasohol production.

Management Practice to Minimize Aflatoxin Problems in Field Corn

1. Plant regionally adapted hybrids.

2. Use a balanced fertility program designed for optimum yields.

3. Select planting dates appropriate for your area.

4 Follow recommended management practices to limit damage by ear feeding insects.

5. Attempt to best utilize your irrigation practices to deliver optimum water from silking stage to late dough stage.

6. Make adjustments in combine ground speed and cylinder speed to minimize trash and broken kernels in hopper. Aflatoxin is often associated with broken or light weight kernels.

7. If drought has occurred during the season, consider harvesting irrigated or high yielding fields separately from dryland or poor yielding fields.

8. Begin corn harvest when grain moisture is about 24% and dry the grain to 15% moisture within 24 hours or as soon as possible.

9. Corn which collects in auger wells and pits around dump stations frequently contains the mold or aflatoxin. Thoroughly clean all such areas before and after use. Remove leftover grain from trucks, trailers, holding bins, drying facilities and storage bins before beginning a new lot of grain.

Management Practices to Minimize Aflatoxin Problems in Stored Corn

1. Thoroughly clean bins, areas around bins and all grain handling equipment before putting any grain in storage.

2. Clean grain going into storage to remove light weight and broken kernels as well as foreign material and fines.

3. Moisture content is by far the most important factor affecting the growth of microorganims in stored corn. After harvest, corn should be dried to 15% moisture content within 24 hours. Grain going into long term storage should be dried to 13% moisture.

4. Aerate grain to safe and equalized temperatures through the grain mass.

5. Protect grain from insects.

6. Check stored grain on regular basis and aerate as needed to maintain low moisture and proper temperature.