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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 #430287

Research Project: Improved Analytical Technologies for Detection of Foodborne Toxins and Their Metabolites

Location: Mycotoxin Prevention and Applied Microbiology Research

2020 Annual Report

The goals of this project are to enhance food safety through the development of tools to more effectively monitor for natural toxins, and to reduce exposure to such toxins. The first goal will be addressed by improving methods for toxin detection, in particular methods to detect multiple toxins and their metabolites simultaneously. To meet this goal requires the development of materials with performance characteristics capable of being used in multiplexed assays and materials that can detect toxins that are currently poorly detected (such as the masked mycotoxins). Development of these materials is integrated with a second goal, the removal of toxins from foods, thereby reducing exposure. To meet these goals we have four objectives. Objective 1. Improve detection of foodborne toxins through development of novel technologies based upon biosensor platforms and new component materials. Sub-objective 1.1. Development and evaluation of multiplexed assay platforms. Sub-objective 1.2. Development and evaluation of materials that can function in multiplexed assays. Objective 2. Improve detection of foodborne toxins through development of direct detection technologies based upon novel mass-spectrometric platforms. Sub-objective 2.1. Develop novel ambient ionization mass spectrometric techniques for detecting single or closely related groups of foodborne toxins. Sub-objective 2.2. Expand techniques such that they can detect multiple toxins simultaneously (multiplexed assays). Objective 3. Improve the ability to detect and measure “masked” mycotoxins and biomarkers of mycotoxin exposure in commodities and foods. Sub-objective 3.1. Application of novel mass spectrometric tools to detect masked toxins. Sub-objective 3.2. Development of novel bio-based materials for masked mycotoxin detection. Objective 4. Improve toxin detection methods and reduce exposure through the development and application of synthetic materials. Sub-objective 4.1. Develop synthetic receptor materials using computational methods and materials science/synthetic strategies. Sub-objective 4.2. Characterize and apply synthetic receptor materials in methods to reduce exposure to toxins.

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. 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 seeks to address the need for improved toxin detection by developing rapid, multi-toxin detection methods. Two major approaches will be used: development of advanced biosensor techniques and development of novel chemical methods based upon mass spectrometry. In support of the former, novel biological binding materials will be developed. 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.

Progress Report
Progress was made on all four objectives and associated subobjectives. The objectives fall under National Program 108, Component 1, Foodborne Contaminants. This project is directed towards Problem Statement 4, chemical and biological contaminants: detection and characterization methodology, toxicology, and toxinology. The project is part of a larger management unit that deals with the problems associated with fungi that infest plants and the toxic metabolites that such fungi can produce (mycotoxins). Research conducted under Objective 1 of this project uses novel technologies and materials to improve detection of such toxins. Objective 3 of this project targets modifications to toxins that can make them undetectable by commonly used testing methods (i.e. masked or hidden). Significant progress was made in both objectives through the development of antibodies directed against two groups of mycotoxins: citreoviridin and roquefortine C. The first of these, citreoviridin, has been associated with a disease known as Cardiac Beriberi” (Shoshin beriberi). The disease was extremely important in Japan in the early 20th century but was almost completely eliminated through a combination of better food hygiene and supplementation of foodstuffs with vitamin B1. However, the potential for the causative fungus to grow on polished rice remains a concern, particularly in areas of the world where supplementation with vitamin B1 is not practiced. Previous screening methods based upon immunoassays have been insufficiently sensitive to screen for citreoviridin in polished rice. A monoclonal antibody was developed against citreoviridin and used to develop a rapid screening assay (enzyme linked immunosorbent assay: ELISA). The ELISA was used to detect the toxin in spiked white rice. Progress was also made on the development of a monoclonal antibody to detect roquefortine C (ROQC), a toxin that has been associated with several intoxications in dogs and humans, following consumption of nuts and other foodstuffs molded by Penicillia. A monoclonal antibody and associated immunoassay were developed to detect this toxin in nut ‘milks’ and in the sera of dogs. Results of a small survey suggested that ROQC was present in nut creamers in very small amounts (< 0.6 ng/g) that do not present a hazard. Because it is very sensitive the screening assay may be most useful in the diagnosis of intoxications of dogs where the dog has consumed moldy foods or where poisoning with mycotoxins is suspected. Improved methods for detecting mycotoxins based upon mass spectrometry (MS) are a central component of Objective 2 of this project. Paperspray ionization MS was utilized to simultaneously quantify several mycotoxins, including aflatoxins and fumonisins from maize. Paperspray ionization is an ambient ionization mass spectrometry technique that has recently been introduced as a modification of desorption electrospray ionization (DESI). We have previously used DESI-MS for analysis of several mycotoxins. We found that careful control over the geometry of the ion source allowed quantitative analysis of mycotoxins. Calibration curves were developed for mycotoxins, indicating that for several toxins the paperspray provided superior analytical performance, as compared to other ambient ionization mass spectrometry methods, including direct analysis in real time-mass spectrometry (DART-MS). Especially in the case of the fumonisins, the paperspray technique performed much better than the DART-MS technique used for many of the mycotoxins. The paperspray method was further applied to masked forms of the fumonisins, again yielding quantitative information on presence of the modified toxins. Extension of this work to incorporate isotopically labelled forms of toxin standards should markedly improve the performance. Successful development of these methods may offer grain processors the potential of high sensitivity and extremely rapid methods for highly specific determination of toxins in grain, contributing to the assurance of the safety of the U.S. food supply. Fumonisins are mycotoxins that inhibit formation of a class of lipids, sphingolipids, that are essential for normal functioning of human and animal cells. The fumonisins are members of a class of compounds known as sphinganine-analog metabolites (SAMs). This year, we surveyed over 300 species of fungi for genes involved in SAM production. This led to the identification of genes that are likely responsible for SAM production in 96 species of Fusarium and 37 species of other fungi. The results will help to identify novel SAMs and the roles that they play in fungal biology. The resulting knowledge will enable new strategies to eliminate fumonisins in crops and, thereby, improve food and feed safety. Fusarium head blight (FHB) is a disease of cereals crops worldwide. FHB is caused by a wide variety of fungi, which makes it difficult to determine which fungal toxins (mycotoxins) might also be present in FHB-affected grain. As part of an international project to improve food safety through enhanced mycotoxin monitoring, we determined the prevalence of FHB pathogens and toxin types associated with wheat and barley in Brazil. Greater pathogen and toxin diversity were discovered than had been previously documented in Brazil. In growing seasons that were less conducive to FHB epidemics, substantial shifts in pathogen and toxin prevalence were also found. This indicated that rarer toxin types may be more prevalent in grain when environmental conditions are less favorable to the dominant FHB pathogen. These results provide new information on the prevalence of FHB pathogens and mycotoxins, which can be used to develop regionally targeted disease and mycotoxin control programs that improve crop production and food safety. Objective 4 of the project is the development and application of synthetic materials for improved toxin detection and reduction of toxin exposure. Progress has been made on the design of detection components for additive manufactured materials to detect mycotoxins (3D printed materials). Computational simulations between potential additive manufacturing components and important mycotoxins were evaluated and favorable components were identified to include in materials. Mycotoxins investigated include fusaric acid, citrinin, and ochratoxins. Results of this study will be used to create additive manufactured materials using 3D printing technology.

1. A rapid test for the fungal toxin citreoviridin in rice. Shoshin beriberi, a disease also known as heart attacking paralysis, was a significant contributor to mortality in Japan in the early twentieth century. It is now rare in developed countries but is still a problem in parts of the world. Shoshin beriberi results from thiamine (vitamin B1) deficiency but has also been linked with citreoviridin, a toxin produced in rice contaminated with the mold Penicillium citreonigrum. Citreoviridin has also been found in maize, pecan nuts, and wheat products. An ARS scientist in Peoria, Illinois, in collaboration with scientists at Azabu University, Kanagawa, Japan, developed an assay for rapid screening of citreoviridin in polished white rice. This test helps improve food safety by providing a tool for diverting contaminated rice from both human food and animal feed supplies. This tool can be used to monitor for the presence of citreoviridin, which is essential for developing a risk assessment for this toxin.

2. A rapid test for masked toxins in wheat. Trichothecenes are a group of fungal toxins that can contaminate oats, wheat, barley, and corn, and cause substantial economic losses worldwide. As part of efforts to improve monitoring of these toxins, researchers at the Institute of Sciences of Food Production in Bari, Italy, and ARS scientists in Peoria, Illinois, developed a new method to detect trichothecenes in wheat. Trichothecenes are toxic to plants as well as animals, but plants can protect themselves from the toxins by attaching a sugar residue, which makes them less toxic. The plant toxin derivatives are called masked mycotoxins. There is concern that during digestion, the original toxin may be released from the masked mycotoxin. The new method is rapid, sensitive, and convenient and will be used to monitor trichothecenes and their modified forms in wheat. Improved monitoring for the trichothecenes and their masked forms can be used to reduce exposure to these toxins by diverting them from the food supply.

3. Predictive models to identify antifungal compounds. Antifungal chemicals are often used to reduce agricultural commodity spoilage and reduce the occurrence of mycotoxins. However, there is a need for safer, better antifungal agents. Some potential antifungal agents are phenolic compounds that have many uses due to their consumer-friendly properties. To aid in the selection of better antifungal compounds, ARS scientists in Peoria, Illinois, applied computational artificial intelligence and machine learning methods to develop mathematical models that identified chemical properties of phenolic compounds that reduce contamination by mycotoxin-producing fungi. Two of the antifungal compounds evaluated, thymol and caracrol, are components of essential oils of many plants, including the popular culinary herb thyme. These models will help toxicologists, microbiologists, and chemists discover better antifungal agents to benefit the food industry.

4. Natural antifungal compounds. Ferulic acid, a natural antioxidant found throughout the plant kingdom, is usually bound to other plant components, such as sugars and oils. These ferulic acid compounds are of special interest due to their health benefits and antimicrobial properties, as well as their use as cosmetic ingredients, but methods to determine their structures are lacking. To assist in the identification of these compounds, ARS scientists in Peoria, Illinois, developed a rapid method to generate their structural fingerprints. This method can easily differentiate between different ferulic acid-containing compounds that are produced industrially with vegetable oils. The transfer of this method to industry supports expanded, value-added markets for agricultural materials and byproducts.

5. Guide to selecting appropriate tests to screen for aflatoxins. Because of the potential role that for aflatoxins have in the development of human liver cancer, substantial efforts are made worldwide to keep these toxins out of the human food and animal feed supplies. There are a wide variety of screening tests available from commercial laboratories that are critical to filling the needs of producers, handlers, and processors. An ARS scientist in Peoria, Illinois, prepared a guide to commercially available aflatoxin test kits. The guide provides background on the aflatoxins, their chemical properties, and the wide variety of means with which they can be detected. The guide focuses on criteria relevant to selecting appropriate screening tests, including cost, accuracy, cross reactivity, applicability to the desired commodity or food, speed, format, sensitivity, and third-party validation. Because users, including producers, storage facilities, and processors, have very different needs, there is no single best test or format. The guide serves as a resource to help users navigate among the many tests and make a selection based upon their unique needs, facilitating the most efficient use of limited testing resources.

6. Risk assessment tool for grain-based products containing masked mycotoxins. Plants are able to modify mycotoxins, creating masked forms that are often less toxic, but not detected by normal toxin assays. Scientists at the University of Aberdeen, Aberdeen, Scotland, in collaboration with ARS scientists in Peoria, Illinois, used a simulated gut to assess the metabolism of masked mycotoxins by gut microbes. Microbes found in the human gut converted masked forms of trichothecene and fumonisin mycotoxins into their parent compounds at rates that were dependent on the specific masked toxin and the human volunteer. The results suggested that gut metabolism of grain contaminated with masked toxins may result in exposure to the parent toxins, potentially increasing risk to consumers. This work provided a tool for regulators to conduct effective risk assessments for grain-based products contaminated with masked mycotoxins and offers toxicologists insight into the potential variability in human responses to mycotoxin contaminated food products.

Review Publications
Maragos, C.M., Uchiyama, Y., Kobayashi, N., Kominato, F., Sugita-Konishi, Y. 2019. Development and characterization of monoclonal antibodies for the mycotoxin citreoviridin. Toxins. 11(11):630.
Tittlemier, S.A., Cramer, B., Dall'Asta, C., Iha, M.H., Lattanzio, V.M.T., Maragos, C., Solfrizzo, M., Stranska, M., Stroka, J., Sumarah, M. 2020. Developments in mycotoxin analysis: an update for 2018-19. World Mycotoxin Journal. 13(1):3-24.
Lippolis, V., Porricelli, A.C.R., Mancini, E., Ciasca, B., Lattanzio, V.M.T., De Girolamo, A., Maragos, C.M., McCormick, S., Li, P., Logrieco, A.F., Pascale, M. 2019. Fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins and their glucosides in wheat. Toxins. 11(7):380.
Compton, D.L., Appell, M. 2020. Rapid Raman spectroscopic determination of 1-feruloyl-sn-glycerol and 1,3-diferuloyl-sn-glycerol. Spectrochimica Acta. 229:118020.
Maragos, C.M. 2020. Commercial immunoassays for detecting aflatoxins. Chapter 4, In: Kintzios, S., Mavrikou, S. editors. Aflatoxins: Biochemistry, Toxicology, Public Health, Policies and Modern Methods of Analysis., Hauppauge, NY: Nova Science Publishers, Inc. p. 91-115.
Appell, M.D., Tu, Y., Compton, D.L., Evans, K.O., Wang, L.C. 2020. Quantitative structure-activity relationship study for prediction of antifungal properties of phenolic compounds. Structural Chemistry. 31:1621-1630.
Cowger, C., Ward, T.J., Nilsson, K., Arellano, C., McCormick, S.P., Busman, M. 2020. Regional and field-specific differences in Fusarium species and mycotoxins associated with blighted North Carolina wheat. International Journal of Food Microbiology.
Laraba, I., Kim, H.-S., Proctor, R.H., Busman, M., O'Donnell, K., Felker, F.C., Aime, M.C., Koch, R.A., Wurdack, K.J. 2019. Fusarium xyrophilum, sp. nov., a member of the Fusarium fujikuroi species complex recovered from pseudoflowers on yellow-eyed grass (Xyris spp.) from Guyana. Mycologia. 112(1):39-51.
Daud, N., Currie, V., Duncan, G., Busman, M., Gratz, S.W. 2019. Intestinal hydrolysis and microbial biotransformation of diacetoxyscirpenol-a-glucoside, HT-2-ß-glucoside and N-(1-deoxyd-d-fructos-1-yl) fumonisin B1 by human gut microbiota in vitro. International Journal of Food Sciences and Nutrition. 71(5):540-548.