Location: Mycotoxin Prevention and Applied Microbiology Research
2024 Annual Report
Objectives
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.
Approach
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: “Greener” plant-based natural products were investigated for their use as safer antifungal compounds to reduce levels of fungi and associated mycotoxins in post-harvest commodities. To examine the potential for safer antifungal treatments, biosynthesized compounds were produced for further study and modelled for properties related to their potential antifungal capabilities. Analyses with chemical predictive modeling tools developed by researchers at the Environmental Protection Agency, indicated that the new compounds are safe alternatives for limiting the growth of mycotoxin producing fungi associated with food decomposition. We modelled 49 antifungal candidates for predicted toxicity and identified five of these candidates for further testing. Validations of these chemical predictive modelling results are now being completed using antimicrobial assays against Fusarium fungi and other plant pathogens.
In further support of Objective 1, commercially available agricultural bioproducts were evaluated for their ability to bind two common mycotoxins to reduce levels of these toxins from processes related to the utilization of corn for ethanol production and in vitro animal digestion models. “Biochars” produced from bamboo, pine and coconut waste were evaluated for their ability to sequester the toxins, including fumonisin B1. Toxin sequestration capacities of eight different commercially available biochars were evaluated in artificial stomach and ethanol fermentation experiments. Performance of biochar toxin sequestration properties were compared to conventionally used montmorillonite clay in conditions related to corn ethanol production.
Objective 2: Oats harvested in crop year 2023 and directed to the U.S. food and feed supply were surveyed for fungal and mycotoxin contamination. A liquid chromatography – tandem mass spectrometry method for determination of several mycotoxins and their masked counterparts was utilized for analysis of representative samples of oat grain obtained from the survey. Fusarium species-specific primers were optimized for polymerase chain reaction-based analysis to identify and estimate the quantity of seven different fungal species potentially contaminating the sampled oats. While some difficulties persist in demonstration of specificity of some fungal assays in differentiation between closely related Fusarium species responsible for production of trichothecene mycotoxins, the results of this multi-year survey will be used to assure consumers of the low mycotoxin risks associated with the utilization of oat products in the U. S. food and feed supply. Utilization 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 support Objective 2 and to 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 2023-2024. 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. During the 2023 crop year the use of in-field spore collection devices in 20 central Illinois locations allowed sampling of air-borne fungal inoculum in corn fields, along with sampling of harvested corn adjacent to spore collection devices. Results of this sampling of air-borne inoculum, along with determination of fungal and mycotoxin levels in harvested grain associated with spore collection sites, should aid in the development and refinement of predictive tools for cereal grain mycotoxin contamination.
To further support Objective 2 and to develop the ability to characterize toxin and fungal contamination in U.S. crops, a study was initiated with the assistance from the North American Millers’ Association to conduct a survey of mycotoxin and fungal contamination in wheat destined for domestic milling and processing. 750 samples from 22 states were collected from local elevators and processed for mycotoxin and fungal analysis. Preliminary results confirm that most U.S. wheat is free from high levels of aflatoxins, fumonisins and trichothecene mycotoxins.
Objective 3: Predictive models for classification of maize samples. Fungal contamination of crops, such as maize, causes physical alterations in the composition of the maize and can lead to the production of mycotoxins. Attempts were made to determine whether the changes in composition of the maize could be correlated with mycotoxin content, using a technique known as ambient ionization mass spectrometry. Samples of maize were ‘fingerprinted’ using ambient ionization mass spectrometry and machine learning was applied to determine if significant differences could be observed between contaminated and non-contaminated maize. The efficacy of such models was evaluated with a ‘correctness score’, which was the number of samples correctly classified by the method relative to the total number of samples tested. Correctness scores of greater than 95% are widely regarded as acceptable. In testing of maize samples, correctness scores of 95% were achieved, with most models yielding scores of 85% to 90%. However, despite the apparently accurate prediction, the method was found to be unacceptable. Ideally, predictive models are trained with sample sets that mimic what will be encountered in the real world. For this test, 253 samples of Illinois maize were used to train the model. The samples were collected as part of a survey program conducted by the Illinois Department of Agriculture and therefore were a good representation of what the model would be expected to encounter in the real world. Most of the samples did not have detectable mycotoxins. The models correctly classified the negative samples as negative. However, many of the ‘positive’ samples were also classified as negative. Therefore, despite a high correctness score, the screening method was not acceptable for prediction of the presence of mycotoxins. This result is likely based in the inability of the technique to identify features within the samples that were specific to mycotoxin contaminated maize.
Accomplishments
1. Developed a rapid test to detect a novel group of fungal toxins and keep infected wheat-based commodities from entering the human food chain. New fungal toxins (mycotoxins) continue to be discovered. One such group of toxins are the NX-toxins, which are produced by certain strains of a group of fungi that cause Fusarium Head Blight, an economically important disease of cereal crops. The toxins are similar to the better-known "vomitoxin" and the strains that produce them have been isolated in the northeastern United States and in Canada. There are currently no rapid screening tests for this group of toxins. ARS researchers in Peoria, Illinois, developed several antibody-based screening assays for the NX toxins in wheat. These are the first rapid screening tests for this group of toxins and will be important tools for the diversion of infested commodities from the human food and animal feed supply chains.
2. Remediation of toxin contaminated grain by insects. Insect upcycling of toxin contaminated grain into a safe alternative feed source. Fungal toxin contamination of the U.S. corn supply continues to be a concern for consumers of the commodity. Diversion of contaminated corn from feed and food use creates a waste stream of limited utility. In collaboration with ARS researchers in Stoneville, Mississippi, ARS researchers in Peoria, Illinois, optimized conditions for the cricket consumption of fumonisin-contaminated corn and examined the ability of crickets to consume fumonisin toxin contaminated corn. Insects, including crickets, are increasingly used as an ingredient in livestock feed rations. It was shown that crickets can readily grow on a ration of corn and that the crickets do not retain fumonisin and can be harvested as a safe product of the remediation of the contaminated corn. Use of contaminated corn for rearing insects can upcycle the waste commodity while producing a valuable and safe ingredient for livestock feeds, providing grain processors with an avenue for utilization of a toxic waste commodity and livestock producers with a safe, high protein feed ingredient.
3. Development of biochar materials for the removal of aflatoxin B1, ochratoxin A, and zearalenone mycotoxins from food and feed. Fungal toxin binding materials are important tools to reduce illness from consumption of toxin- contaminated food and feed products. ARS researchers in Peoria, Illinois, evaluated four biochar materials for their ability to sequester the common fungal toxins aflatoxin B1, ochratoxin A, and zearalenone. Benchtop simulated stomach fluid studies indicated that certain pine biochars and activated coconut charcoals could bind high amounts of the fungal toxins. Other biochar materials with lower surface areas had lower binding capacities. Use of biochars to remove mycotoxins allows utilization of contaminated commodities, providing a means for the reduction of human and animal exposure to toxins found in food and feed. This provides a benefit to grain processors, livestock producers and human consumers of grain-based products.
Review Publications
Appell, M., Wegener, E.C., Sharma, B.K., Eller, F.J., Evans, K.O., Compton, D.L. 2023. In vitro evaluation of the adsorption efficacy of biochar materials on aflatoxin B1, ochratoxin A, and zearalenone. Animals. 13(21). Article 3311. https://doi.org/10.3390/ani13213311.
Mandap, J.A.L., Hellmich, R.L., Busman, M., Maier, D.E., Munkvold, G.P. 2024. Protection from stored grain insects using transgenic maize hybrids and implications for Aspergillus flavus and aflatoxin contamination. Journal of Stored Products Research. 106. Article 102258. https://doi.org/10.1016/j.jspr.2024.102258.
Tittlemier, S.A., Cramer, B., DeRosa, M.C., Dzuman, Z., Malone, R., Maragos, C., Suman, M., Sumarah, M.W. 2024. Developments in analytical techniques for mycotoxin determination: an update for 2022-23. World Mycotoxin Journal. https://doi.org/10.1163/18750796-bja10002.
Paulk, R.T., Abbas, H.K., Rojas, M.G., Morales Ramos, J.A., Busman, M., Little, N., Shier, T.W. 2024. Evaluating Acheta domesticus (Orthoptera: Gryllidae) for the reduction of fumonisin B1 levels in livestock feed. Journal of Economic Entomology. https://doi.org/10.1093/jee/toae025.
Maragos, C.M., Vaughan, M.M., McCormick, S.P. 2024. Monoclonal-antibody-based immunoassays for the mycotoxins NX-2 and NX-3 in wheat. Toxins. https://doi.org/10.3390/toxins16050231.