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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Mycotoxin Prevention and Applied Microbiology Research » Research » Research Project #438643

Research Project: Innovative Approaches to Monitor, Predict, and Reduce Fungal Toxins

Location: Mycotoxin Prevention and Applied Microbiology Research

2022 Annual Report

The goals of this project are to reduce exposure to such toxins and to enhance food safety through the development of tools to more effectively monitor for natural toxins. The first goal will be addressed by development of materials capable of being used in the removal of toxins from foods, thereby reducing exposure. The second goal involves surveying one important commodity, oats; improving methods for toxin detection; and, in particular, developing methods to allow for the prediction of toxin contamination. To meet these goals, we have three objectives. Objective 1. Develop materials and methods to maintain commodity value and safety by eliminating mycotoxin contamination. Sub-objective 1.A. Develop antifungal compounds. Sub-objective 1.B. Remediation. Objective 2. Determine the occurrence of mycotoxins in alternative grain commodities, for example the U.S. oat supply. Determine the fungi and oat cultivars associated with reduced mycotoxin levels and/or disease. Sub-objective 2.A. Survey U.S. oats for mycotoxin contamination. Sub-objective 2.B. Survey U.S. oats for fungal contamination. Objective 3. Develop analytical tools to predict and evaluate the presence of natural toxins (mycotoxins) in grain commodities and their related foods. Sub-objective 3.A. Develop tools to predict the presence of toxins in commodities and foods. Sub-objective 3.B. Develop tools to detect emerging toxins, their metabolites and masked forms.

Food crops are commonly infested with fungi, both in the field and in storage. Certain fungi produce toxins (mycotoxins) that can adversely affect human health and the health of domestic animals. Certain toxin-binding materials may also have the potential to be used to remove toxins from foods, and this will be approached through the development and application of novel synthetic materials to reduce exposures. By permitting the timely diversion of contaminated ingredients from the food supply, detection of foodborne toxins can directly improve food safety and the safety of animal feed. Monitoring for the presence of such naturally occurring toxins is widespread and occurs at many of the stages between the producer and the consumer. Increasing the efficiency and improving the accuracy of monitoring results in more appropriate and efficient diversion of contaminated products. The need to monitor for greater numbers of mycotoxins is a trend that will continue, in particular because of recent concern over the so called “masked” mycotoxins. This project will put a particular focus on the food safety of a commodity important to U.S. consumers, oats. A survey of both toxin contamination and fungal contamination in U.S. oats will aid in the assurance of a safe supply of the key ingredient in human diets. Further, this project seeks to address the need for improved toxin detection by developing rapid detection methods leading to the prediction of toxin contamination in a variety of food / feed ingredients.

Progress Report
Objective 1: Goals of this objective are to develop materials and methods to maintain commodity value and safety by eliminating mycotoxin contamination of corn and processed corn products that are used as food and feed. Improper storage can promote mold growth and results in harmful levels of mycotoxins. Grain tainted with mycotoxigenic fungi can continue to accumulate mycotoxins during downstream processes. Safer compounds with antifungal properties can be more widely used to reduce levels of harmful fungi and mycotoxin levels. Safer compounds found in common foods that have broad antimicrobial activities were identified using models that predict favorable antifungal properties based on characteristics related to chemical structures. Artificial intelligence methods enabled the rapid identification of the chemical properties that are related to the favorable antifungal properties. Identified antifungal compounds included several food compounds with predicted broad antimicrobial activity that are also designated as “Generally Recognized as Safe” by the U.S. Food and Drug Administration. The identified lead compounds exhibit antifungal activity against fungi harmful to agriculture in inhibition assays conducted with ARS researchers in Peoria, Illinois. These models are publicly available in research publications. In 2022, agriculturally derived materials and biopolymers were evaluated for their ability to remove the Fusarium mycotoxins deoxynivalenol and zearalenone from processes related to the utilization of corn for ethanol production. Agriculturally derived materials are value-added renewable bioproducts and their use promotes values of agricultural commodities. Deoxynivalenol is a Fusarium mycotoxin that is associated with feed refusal. Zearalenone is a prevalent estrogenic mycotoxin that harms swine production. Use of the developed materials has the potential to reduce human and animal exposure to mycotoxins and to reduce levels of mycotoxins in food and feed products. Objective 2: Goals of this objective are to determine the occurrence of mycotoxins in oats, and to determine the fungi and oat cultivars associated with reduced mycotoxin levels and/or disease. A variety of fungi can infest developing grains and, ultimately, contaminate grains with mycotoxins. A comprehensive survey of contamination of U.S. oats with harmful fungi and their associated fungal toxins should aid in the development of strategies to assure the safety of the U.S. oat supply. Sampling plans are being refined with cooperators to effectively sample fungal and mycotoxin contamination in U.S. oats. During the 2021 crop year survey samples were obtained representing oats harvested and directed to the U.S. food and feed supply. A liquid chromatography – tandem mass spectrometry method for determination of several mycotoxins and their masked counterparts was developed for oat grain. Methods for polymerase chain reaction-based determination of several fungal species were developed and optimized for determination of levels of contamination of harvested oats with mycotoxin producing fungi. Development of these chemical and microbiological tools should allow the characterization of pre-harvest and post-harvest fungal and mycotoxin contamination of oats destined for U.S. processing and prompt diversion of contaminated grain from the food supply. To further develop the ability to sample fungal contamination in developing crops, funding was secured from the National Predictive Modeling Tool Initiative to conduct the project “Quantitative monitoring of mycotoxin - producing fungi in corn production fields” over the years 2022-2023. The project supports the development and implementation of remote sensing devices for the identification and quantitation of air-borne fungal spores in agricultural fields. A goal of the study is to develop tools for the prediction of pre-harvest and post-harvest fungal and mycotoxin contamination of a variety of food / feed grains. Objective 3: Fungi from the genus Penicillium are used in the ripening of two popular types of cheese: soft-ripened cheeses (Brie, Camembert, etc.) and blue-veined cheeses (Roquefort, Stilton, Gorgonzola, DanaBlu, etc.). However, certain strains of these fungi produce toxins (mycotoxins). One such mycotoxin, a-cyclopiazonic acid, is a neurotoxin. Data on the levels of cyclopiazonic acid in cheeses marketed in the United States are extremely limited. To address this, an antibody-based assay (enzyme-linked immunosorbent assay) was adapted for measuring cyclopiazonic acid in 254 samples of soft-ripened and blue-veined cheeses. Cyclopiazonic acid was detected in 46% of soft-ripened cheeses and in 24% of blue-veined cheeses, generally at low levels, however higher levels were occasionally found. The impact of the observed levels upon consumers is likely low. However, the results of this study provide data needed to perform risk assessments for human exposure from the consumption of such cheeses.

1. Developed a rapid test to measure toxic fungi in cheeses marketed in the United States. Fungi are used to impart desirable flavors and textures to cheeses. However, certain fungi produce secondary metabolites toxic to animals, mycotoxins. Two groups of cheeses where fungi are used for ripening are the blue-veined cheeses and the “soft-ripened” cheeses. The presence of the mycotoxin roquefortine C in blue-veined cheeses has been reported previously, primarily in Europe. The extent to which roquefortine C occurs in cheeses sold in the United States has been largely unreported. An antibody-based screening assay was developed by an ARS researcher in Peoria, Illinois, and used to test for roquefortine C in 202 samples of cheeses sold in the United States. Of the 152 blue cheese samples, 151 contained roquefortine C. The levels found were consistent with the levels found previously in blue-veined cheeses in the United Kingdom and Europe, which have generally been considered non-hazardous for human consumption. An ARS researcher in Peoria, Illinois, developed a method to facilitate the screening of roquefortine C in blue cheeses. The method was able to detect the toxin at levels typically found in blue cheeses, with results that compared well to a standard reference (liquid chromatography – mass spectrometry) method. These results help to establish the technology used (thermal desorption – direct analysis in real time – mass spectrometry) as a more rapid, easier to use, way to measure for this toxin in cheeses. Measuring the levels of such neurotoxins is essential to determining the risk, or lack thereof, for the U.S. population.

2. Developed a cost-effective method for detecting harmful chemicals in food and feed. Raman spectroscopy was utilized for the convenient determination of zearalenone and alpha-zearalanol. Zearalenone and alpha-zearalanol are chemicals monitored by governments to ensure food is safe. Zearalenone is a toxin made by fungi that can taint farm products, such as corn. Swine and humans produce alpha-zearalanol from zearalenone contaminated food and feed. One of the problems with gauging exposure to these toxins is the lack of quick detection methods. ARS researchers in Peoria, Illinois, developed a rapid technique to verify these chemicals using a portable Raman detection device. Raman spectroscopy is a light-based analytical technique that can be used to perform chemical analysis with limited sample preparation. These results show that Raman methods can detect zearalenone and alpha-zearalanol based on biochemical fingerprints. The fingerprints are unique to the toxins and enable clear proof of identity. This work demonstrates a cost- effective way for rapid verification of toxins to help food and feed safety.

3. Identified harmful fungi on corn using chemical vapor fingerprints. Harmful fungi, capable of producing toxins, are found worldwide in harvested corn. The fungal toxins are responsible for a variety of human health problems and have a profound economic effect on producers and processors of corn products. ARS researchers in Peoria, Illinois, developed a technique to determine levels of volatile compounds from such fungi growing on cracked corn. The volatile compounds are produced by a variety of toxin-producing fungi during their growth on any food source. The analysis of the volatile compounds can be used to identify the fungi producing the compounds. The technique is based on drawing an air flow from the area surrounding fungi directly into a highly sensitive mass spectrometer. The procedure allows the rapid and sensitive detection of volatile compounds without extensive sample preparation or chemical separation of the compounds prior to analysis. This work could be used as a basis for development of a rapid, sensitive, and convenient analytical tool for corn processors seeking to assure the safety of corn-based products.

Review Publications
Appell, M.D., Compton, D.L., Bosma, W.B. 2022. Raman spectral analysis for rapid determination of zearalenone and alpha-zearalanol. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 270. Article 120842.
Busman, M., Roberts, E., Proctor, R.H., Maragos, C.M. 2021. Volatile organic compound profile fingerprints using DART–MS shows species-specific patterns in Fusarium mycotoxin producing fungi. The Journal of Fungi. 8(1). Article 3.
Tittlemier, S.A., Cramer, B., Dall'Asta, C., DeRosa, M.C., Lattanzio, V.M.T., Malone, R., Maragos, C., Stranska, M., Sumarah, M.W. 2022. Developments in mycotoxin analysis: an update for 2020-2021. World Mycotoxin Journal. 15(1):3-25.
Maragos, C.M. 2021. Roquefortine C in blue-veined and soft-ripened cheeses in the USA. Food Additives & Contaminants. 15(1):1-9.
Maragos, C.M. 2021. Application of ambient ionization mass spectrometry to detect the mycotoxin roquefortine C in blue cheese. Food Analytical Methods. 15:751-760.