The occurrence and concentration of mycotoxins in U.S. distillers dried grains with solubles.

To provide a scientific sound assessment of the prevalence and levels of mycotoxins in U.S. distillers' dried grains with solubles (DDGS), we measured mainly aflatoxins, deoxynivalenol, fumonisins, T-2 toxin, and zearalenone in 235 DDGS samples collected from 20 ethanol plants in the midwestern United States and 23 export shipping containers from 2006 to 2008 using state-of-the-art analytical methodologies. The results suggested that (1) none of the samples contained aflatoxins or deoxynivalenol levels higher than the U.S. Food and Drug Administration (FDA) guidelines for use in animal feed; (2) no more than 10% of the samples contained fumonisin levels higher than the recommendation for feeding equids and rabbits, and the rest of the samples contained fumonisins lower than FDA guidelines for use in animal feed; (3) none of the samples contained T-2 toxins higher than the detection limit, and no FDA guidance levels are available for T-2 toxins; (4) most samples contained zearalenone levels lower than the detection limit, and no FDA guidance levels are available for zearalenone; and (5) the containers used for export shipping of DDGS did not seem to contribute to mycotoxin production. This study was based on representative DDGS samples from the U.S. ethanol industry, and the data were collected using reference methods. This study provided a comprehensive and scientifically sound assessment of the occurrence and levels of mycotoxins in DDGS from the U.S. ethanol industry.

Some major mycotoxins and their mycotoxicoses--an overview.

Mycotoxins likely have existed for as long as crops have been grown but recognition of the true chemical nature of such entities of fungal metabolism was not known until recent times. Conjecturally, there is historical evidence of their presence back as far as the time reported in the Dead Sea Scrolls. Evidence of their periodic, historical occurrence exists until the recognition of aflatoxins in the early 1960s. At that time mycotoxins were considered as a storage phenomenon whereby grains becoming moldy during storage allowed for the production of these secondary metabolites proven to be toxic when consumed by man and other animals. Subsequently, aflatoxins and mycotoxins of several kinds were found to be formed during development of crop plants in the field. The determination of which of the many known mycotoxins are significant can be based upon their frequency of occurrence and/or the severity of the disease that they produce, especially if they are known to be carcinogenic. Among the mycotoxins fitting into this major group would be the aflatoxins, deoxynivalenol, fumonisins, zearalenone, T-2 toxin, ochratoxin and certain ergot alkaloids. The diseases (mycotoxicoses) caused by these mycotoxins are quite varied and involve a wide range of susceptible animal species including humans. Most of these diseases occur after consumption of mycotoxin contaminated grain or products made from such grains but other routes of exposure exist. The diagnosis of mycotoxicoses may prove to be difficult because of the similarity of signs of disease to those caused by other agents. Therefore, diagnosis of a mycotoxicoses is dependent upon adequate testing for mycotoxins involving sampling, sample preparation and analysis.

A review of rapid methods for the analysis of mycotoxins.

An overview is presented of the analysis of mycotoxins by rapid methods such as: enzyme linked immunosorbent assay (ELISA); flow through membrane based immunoassay; immunochromatographic assay; fluorometric assay with immunoaffinity clean-up column or with a solid phase extraction clean-up column; and fluorescence polarization method. These methods are currently commercially available and are reliable, rapid methods. This review focuses on the basic principle of each rapid method as well as advantages and limitations of each method. Additionally, we address other emerging technologies of potential application in the analysis of mycotoxins.

Validation of an ELISA test kit for the detection of total aflatoxins in grain and grain products by comparison with HPLC.

An ELISA Microtiter Plate, Total Aflatoxin Test called AgraQuant was validated to measure total aflatoxins in a range from 4 to 40 ppb in corn, corn meal, corn gluten feed, corn gluten meal, corn germ meal, corn/soy blend, popcorn, sorghum, wheat, milled rice, soybeans, peanuts and cottonseed. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, aflatoxins are extracted from ground samples with 70% methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 15 min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5 min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability indicate a minimum of 1 year shelf life for the kits; accuracy and precision are comparable to HPLC in the range of 0-320 ppb and limit of detection in corn is 2.5 ppb. Comparison of the method to HPLC, ability to detect individual aflatoxins and ruggedness of the test kits at 18-30 degrees C determined this test to be rugged, sensitive, accurate, precise and effective comparable to HPLC for measuring total aflatoxins ranging from 4 to 40 ppb in the commodities evaluated.

Validation of an ELISA test kit for the detection of ochratoxin A in several food commodities by comparison with HPLC.

An ELISA Microtiter Plate, Ochratoxin Test called AgraQuant was validated to measure ochratoxin A in a range from 2 to 40 ppb in corn, milo, barley, wheat, soybeans and green coffee. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, ochratoxin A is extracted from ground samples with 70% methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 10 min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5 min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability indicate a 1 year shelf life; accuracy and precision are comparable to HPLC from 0 to 80 ppb and limit of detection in corn is 1.9 ppb and other food commodities is up to 3.8 ppb. Comparison of the method to HPLC, ability to detect individual ochratoxins, and ruggedness of the test kits determined this test to be rugged from 18 to 30 degrees C, sensitive, accurate, precise and effective comparable to HPLC for measuring ochratoxin A ranging from 2 to 40 ppb in several commodities.

Lower fumonisin mycotoxin levels in the grain of Bt corn grown in the United States in 2000-2002.

Fumonisins were monitored in corn grain collected from Bt hybrids grown in 107 locations across the United States in 2000-2002. Bt corn hybrids contain the Cry1Ab protein from Bacillus thuringiensis that controls European corn borers and other stalk-boring pests. Fumonisin levels were frequently lower in grain from Bt hybrids grown in field trials under conditions of natural (FACT trials) or manual insect infestation (university trials). Over three years of FACT trials, there were 126/210 comparisons when fumonisin levels in grain from control hybrids were >2 ppm, exceeding U.S. FDA guidance levels of 2 ppm for human food. Grain from Bt hybrids was at or below 2 ppm of fumonisins for 58 of the 126 comparisons. The use of Bt hybrids can increase the percentage of corn grain that would be suitable for use in food and feed.

Estimating deoxynivalenol in shelled corn barge lots by measuring deoxynivalenol in corn screenings.

To determine if deoxynivalenol (DON) is concentrated in small corn screenings, fourteen to twenty-three 1.1 kg test samples were taken from each of 10 barges of shelled corn. Each of the 181 test samples was divided into 2 components (fines and clean) using a 5 mm screen. The clean component sample rode the 5 mm screen and the fines component sample passed through the 5 mm screen. The DON concentration in fines component sample was about 3 times the DON concentration in the clean component sample. The DON in the 181 fines and clean component samples averaged 689.0 and 206.1 ng/g, respectively. Regression equations were developed to predict the DON in the barge based upon measurements of DON in the fines component sample. The ratio of DON in the lot to DON in the fines component sample was 0.359. The coefficient of variation (CV) associated with predicting the DON concentration in a lot with 359 ng/g using a 1.1 kg test sample was 47.0%. Increasing sample size to 4.4 kg reduced the CV to 23%.

Supercritical Fluid Extraction of Aflatoxin M1 from Beef Livert †.

Supercritical fluid extraction (SFE) and a pressurized-fluid-extraction process were applied for the removal of aflatoxin M1 from beef liver samples. Various pressures, temperatures, quantity of supercritical carbon dioxide, and organic modifiers were investigated to optimize the extraction methods. Organic modifier was found to be essential for quantitative recovery of aflatoxin M1. Extracts were cleaned up by solid-phase extraction and were analyzed via high-performance liquid chromatography coupled with fluorescence detection of the trifluoroacetic acid derivative. Solvent-modified carbon dioxide SFE achieved recoveries comparable to an AOAC-approved method involving organic solvent extraction. SFE allowed the traditional amounts of sample and organic solvent to be reduced. Also, the supercritical-fluid extraction permitted the use of carbon dioxide modified with acetonitrile: methanol (2:1) to replace methylene chloride as the organic solvent for the extraction step.