1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Objective 1. T-2 toxin is produced by certain fungi that infest agricultural commodities and foods, particularly grains such as wheat, barley, rye, maize, and rice. Poultry in particular are sensitive to this toxin, which causes a variety of symptoms ranging from simple weight loss to death. To keep T-2 toxin out of the human food and animal feed supplies commodities are often tested. A new technology, imaging surface plasmon resonance (iSPR) was used to develop a novel method for detecting T-2 toxin in wheat. The method was able to detect very low levels (4 ppb) of the toxin. To ensure the method could meet the needs for testing grain for export, the method was validated against a regulatory level established by the European Commission. This research completed the first step in the development of a method capable of screening for multiple toxins simultaneously, which is needed to reduce the costs associated with monitoring for such toxins in grains. Another mycotoxin that is related to T-2 toxin is deoxynivalenol (DON, aka “vomitoxin”). DON is a toxin produced by certain fungi that cause Fusarium head blight, a disease of cereal crops that results in substantial economic losses worldwide. DON is both a mycotoxin, capable of causing disease in animals, and a compound that facilitates fungal infection of the host cereal crop (a virulence factor). Tests that measure DON generally use proteins (antibodies) that bind the toxin. The efficacy of a novel antibody fragment was evaluated in a novel sensor format. The sensor format, iSPR, indicated that this particular antibody lost activity in the process of modification. Objective 2. Mass spectrometry (MS) is a commonly used tool for detecting chemical contaminants. Typically, MS methods require extracting toxins from commodities or foods and then testing the extracts. However, mass spectrometers can be modified to detect contaminants on surfaces. One such modification is known as Direct Analysis in Real Time (DART). In this work a DART-MS instrument was modified to link it with an infrared (IR) laser. An “off-the-shelf” IR laser was used to remove material from the surface, which was then detected using the DART-MS instrument. Because it uses computer control over the position and intensity of the laser, this new apparatus may allow convenient probing of two dimensional surfaces. The device was put through initial studies aimed at characterizing the efficiency of the production of sample analyte ions and the spatial resolution of the technique. After the preliminary characterization, the instrument was used to analyze solutions of aflatoxin B1, T-2 toxin, and maize extracts containing these toxins. Objective 3. As a self-protection mechanism, plants can take fungal toxins and convert them to forms that are less toxic to the plant. Such forms are generally not detected with most established screening methods and are therefore termed “masked”. When animals ingest the plant (commodity) they may digest the modified form and “unmask” the toxin. Fumonisins are a group of naturally-occurring toxins commonly found in maize. They cause a variety of diseases in domestic animals and have been implicated in several diseases in humans, in particular esophageal cancer, and neural tube defects. Plants such as maize can mask the fumonisins thereby preventing their accurate measurement. A technique known as liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of certain fumonisins and their sugar derivatives (fructosides). The developed method was applied to naturally contaminated maize. Furthermore, a sugar derivative of one of the fumonisins was produced, isolated, and used as an analytical standard. In order to characterize the production of modified fumonisins in maize, preparative chromatography was used to prepare quantities of the modified toxins sufficient for chemical characterization by other means, such as nuclear magnetic resonance (NMR) spectroscopy. Comparison to these standards should allow confident identification of the modified toxins found in naturally contaminated maize. Objective 4. Improve toxin detection methods and reduce exposure through the development and application of synthetic materials. Completed a comprehensive experimental and computational modeling study on the detection properties of zearalenone, ochratoxin, and citrinin with collaborators from Bradley University (Peoria, Illinois) and National Taiwan University (Taipei). The research identified specific spectroscopic properties that improve the accuracy and selectivity of popular methods to measure the levels of toxins in contaminated commodities. The results were applied within our laboratory for the determination of zearalenone levels, a contaminant of corn. Completed the characterization of mycotoxin binding materials using spectroscopy methods, including infrared, Raman, and surface area analysis. This knowledge is needed for the development of materials in FY2018. Significant progress has been made in design, preparation, and characterization of synthetic receptor materials to improve detection of ochratoxin and citrinin.
1. Rapid detection of cyclopiazonic acid in maize. Cyclopiazonic acid (CPA) is a naturally occurring neurotoxin produced by certain fungi, in particular the species Aspergillus flavus. Many strains of A. flavus also produce the more widely known aflatoxins, and these two groups of toxins are known to occur together in commodities. Despite this, techniques for measuring CPA in commodities and foods have lagged behind efforts to detect other toxins. To address this issue, antibodies against CPA were developed by an ARS scientist in Peoria, Illinois. The antibodies developed and the resulting screening assay will be useful tools for further evaluation of the prevalence of this mycotoxin in maize. Evaluating the prevalence of this toxin is important for determining the potential risk it poses to human and animal health.
2. Improvements in detection of zearalenone. Agricultural commodities, including corn, can be contaminated with fungi capable of producing the toxin zearalenone. Zearalenone and several similar molecules are well known environmental estrogens that can have adverse effects on a variety of species, in particular swine. Detection of zearalenone facilitates diversion of contaminated commodities from human food or swine feed into the feed of more resistant domestic animals or alternative uses. ARS scientists in Peoria, Illinois, and collaborators at Bradley University applied experimental methods and computational chemistry to identify the parameters that improve zearalenone detection for the commonly used fluorescence method. The findings of this study improved popular quantitative analysis tests for more reliable determination of zearalenone levels. This research benefits the efforts of scientists and regulators to keep food safe by improving the accuracy of popular methods to detect zearalenone contamination.
3. A novel system for detecting fumonisins. Fumonisin B1 (FB1) is a mycotoxin commonly found in maize and maize products throughout the world, it is linked with several diseases in humans and domestic animals. Most rapid detection methods for fumonisins rely upon antibodies to bind the toxin for detection. Scientists at the Henan Academy of Agricultural Sciences (Zhengzhou, China), in collaboration with ARS scientists in Peoria, Illinois, developed a novel method for detecting FB1 in maize. Rather than using toxin specific antibodies, the method relies upon the binding of the toxin to an oligonucleotide (aptamer). A technique called fluorescence polarization was used to indirectly measure the presence of FB1. The technique is easy to operate and uses relatively low cost instrumentation and it may find application for the detection of this toxin in maize in place of the more traditional antibody-based screening assays. Reducing the cost and improving the ease-of-use of existing methods may encourage more testing and greater accuracy in the diversion of contaminated grain.
4. Enzymatic detoxification of the mycotoxin deoxynivalenol. Deoxynivalenol (DON) is produced by fungi on a variety of commodities including maize, wheat, and barley. While it is toxic to many types of animals, it also helps the fungus to overcome the defenses of the host plant. Infestation with this fungus and the associated toxins is a significant and recurring problem known as Fusarium Head Blight. Plants can render DON less toxic by attaching a sugar to it, forming a glucoside. This is accomplished by an enzyme that may provide the plant with some resistance. In this study, ARS scientists in Peoria, Illinois, used X-ray crystallography to identify structural features of such an enzyme in rice that can reduce the toxicity of DON. This information will provide a guide for various approaches designed to increase resistance to the harmful effects of DON in cereals.
Wang, H., Wang, J., Hong, H., Yin, H., Maragos, C.M., Zhang, L., Liu, J. 2017. Selection and application of strand displacement probes for a fumonisin B1 aptamer. Quality and Safety of Agro-Products. 1:44-48.
Maragos, C.M., Sieve, K.K., Bobell, J. 2017. Detection of cyclopiazonic acid (CPA) in maize by immunoassay. Mycotoxin Research. 33(2):157-165.
Appell, M., Wang, L.C., Bosma, W.B. 2017. Analysis of the photophysical properties of zearalenone using density functional theory. Luminescence. 188:551-557.
Wetterhorn, K.M., Newmister, S.A., Caniza, R.K., Busman, M., McCormick, S.P., Berthiller, F., Adam, G., Rayment, I. 2016. Crystal structure of Os79 (Os04g0206600) from Oryza sativa: a UDP-glucosyltransferase involved in the detoxification of deoxynivalenol. Journal of Biochemistry. 55(44):6175-6186.
Stroka, J., Maragos, C.M. 2016. Challenges in the analysis of multiple mycotoxins. World Mycotoxin Journal. 9(5):847-861.
Berthiller, F., Brera, C., Iha, M.H., Krska, R., Lattanzio, V.M.T., MacDonald, S., Malone, R.J., Maragos, C., Solfrizzo, M., Stranska-Zachariasov, M., Stroka, J., Tittlemier, S.A. 2017. Developments in mycotoxin analysis: an update for 2015-2016. World Mycotoxin Journal. 10(1):5-29.