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

Location: Mycotoxin Prevention and Applied Microbiology Research

2021 Annual Report

Objectives
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.

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. 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
Significant progress was made on all four objectives. Rapid methods were developed for detecting economically important mycotoxins in commodities using biosensors and new component materials (Objective 1). A biosensor was developed for the simultaneous detection of multiple Fusarium mycotoxins in wheat. The method used a novel biosensor technology. By allowing for multiple toxins to be monitored simultaneously, the method reduced the time required for testing. A water-based sample preparation for screening for deoxynivalenol in wheat and wheat dust was developed in collaboration with researchers at Dublin City University and at Ghent University. The test was fast and could be performed on-site. During this project, researchers in Europe discovered a new group of trichothecene mycotoxins (“NX” toxins). We assisted researchers from the University of Natural Resources and Life Sciences in determining whether commercial mycotoxin detection test kits were able to detect these toxins. None of them could. However, an assay developed by project researchers was capable of detecting some forms of the NX toxins and therefore was useful in assessing their potential risk. The best-known group of mycotoxins, the aflatoxins, are produced by certain species of molds (Aspergilli). Many of the strains that produce aflatoxins also produce a neurotoxin, cyclopiazonic acid. A sensitive and portable antibody-based screening test was developed for measuring cyclopiazonic acid in maize. The screening assay will be useful for determining the potential risk that cyclopiazonic acid poses to human and animal health. A novel biosensor was also developed to measure cyclopiazonic acid in maize and in camembert cheese. Furthermore, in the course of this project it was discovered that cyclopiazonic acid could be rendered fluorescent through the interaction with certain rare-earth metals (terbium, europium), without the need for antibodies. This allowed for detection of cyclopiazonic acid using low cost, field-portable instrumentation. This helped to reveal how the toxin interacts with its target enzyme. The same effect was seen with the mycotoxin tenuazonic acid and an assay was used to demonstrate that this toxin binds copper and aluminum very effectively. This provided insight into how this toxin exerts its effects on plants. A novel system was developed for detecting the mycotoxin fumonisin B1. Most screening tests for the fumonisins incorporates selective antibodies. In collaboration with researchers at the Henan Academy of Agricultural Sciences a novel method based upon aptamers, rather than antibodies, was developed to detect this toxin in maize. A portable test was developed that was easy to operate and used relatively inexpensive instrumentation. Reducing the cost and improving the ease-of-use may encourage more testing and greater accuracy in the diversion of contaminated grain. Significant progress was made in the development of novel methods for improving mass-spectrometry based approaches for toxin detection (Objective 2). A variety of formats for laser desorption, coupled with ambient ionization mass spectrometry (MS), were used to achieve spatially resolved analysis of mycotoxins on surfaces. Further, the sensitive analysis of fumonisins by paper-spray ionization-MS was attained. Paper-spray ionization permitted analyses to be made directly from porous surfaces. The paper-spray approach was used to simultaneously analyze mixtures of fumonisins and their fructoside analogues in maize extracts. Significant progress was also made in the ability to detect and measure the masked mycotoxins and biomarkers of mycotoxin exposure in commodities and foods (Objective 3). T-2 toxin is produced by certain fungi that infest cereal grains. Plants protect themselves from this toxin by metabolizing it, which also ‘masks’ it from common detection systems. A biosensor for detecting this toxin and its masked forms was developed. The sensor could detect very low levels of the toxin and the method was validated to meet the criteria for testing of grain for export. In collaboration with the Institute of Sciences of Food Production we developed a new method to detect T-2 and related toxins in wheat. The method was rapid and portable. These methods detected the toxins at levels that will ensure food safety while supporting the export of U.S. commodities. The fumonisins can also exist in masked forms. An antibody-based screening assay was developed to detect masked forms of the fumonisins. To confirm the presence of masked fumonisins in corn, a liquid chromatography high resolution mass spectrometry method was developed. This facilitated the production of analytical standards, allowing confident identification in maize. These two tools will be useful for determining the extent to which the masked fumonisins represent a hazard. The potential for masked mycotoxins to regenerate the parent toxins was studied in collaboration with researchers at the University of Aberdeen. Microbes found in the human gut were able to convert certain masked mycotoxins back into their parent compounds. This suggested that grain contaminated with masked toxins may result in exposure to the parent toxins. This work provided a tool for regulators to conduct effective risk assessments and provided insights into the responses of humans to contaminated foods. Citrinin is a potential liver toxin and carcinogen that is regulated in the European Union and Japan. Common methods for detecting citrinin in corn were improved by identifying the most reliable conditions for accurate evaluation. Project researchers described changes in citrinin that facilitate selective and accurate fluorescence detection. This knowledge is useful for the development of more reliable, accurate, and economical methods to detect this toxin. Alternariol and related compounds are toxins produced by fungi that contaminate cereal grains and fruits. Researchers at the National Taiwan University and project researchers applied computational methods to characterize the detection properties of alternariol and related compounds. Modifications to certain chemical groups of the toxins were identified as being able to influence properties related to detection. By providing insights into the properties influencing detection, this research facilitated the design of improved analytical methods for these toxins. Citreoviridin is a toxin found in rice, maize, pecan nuts, and wheat products. In collaboration with researchers at Azabu University an antibody-based screening assay was developed to detect citreoviridin in white rice. This test provided a tool for diverting contaminated rice from human food and animal feed supplies. Toxin detection methods can be improved, and exposures reduced, through the development and application of synthetic materials that interact with mycotoxins (Objective 4). Zearalenone and related molecules are environmental estrogens that can have adverse effects on a variety of animals, in particular swine. Project researchers, in collaboration with researchers at Bradley University applied experimental methods and computational chemistry to identify the parameters that improve zearalenone detection. The results were improvements to the quantitative analysis and reliability of zearalenone tests. A method for detecting ochratoxin A in beverages was also improved. This toxin contaminates a wide variety of foods and beverages. Using computational chemistry and quantitative structure activity relationship techniques, project researchers developed a material to isolate ochratoxin A from beverages, which improved detection accuracy. One means to reduce spoilage of commodities and exposure to mycotoxins is by controlling fungal contamination with antifungal agents. Safer, targeted, antifungal agents are in demand. Certain phenolic compounds that have consumer-friendly properties are potential anti-fungal agents. To facilitate the selection of better antifungal compounds, project researchers applied computational artificial intelligence and machine learning methods to develop mathematical models that identified chemical properties of phenolic and heterocyclic compounds that reduce contamination by mycotoxin-producing fungi. Two of the antifungal compounds evaluated, thymol and carvacrol, are components of essential oils of many plants, including thyme. These models will help toxicologists, microbiologists, and chemists discover better antifungal agents for the benefit of the food industry. Ferulic acid, a natural antioxidant found in many plants, is usually bound to other plant components (sugars, oils). These ferulic acid compounds are of special interest due to their health benefits, antimicrobial properties, and their use in cosmetics. However, methods to determine their structures were lacking. To assist in the identification of these compounds, project researchers developed a method to rapidly generate their structural fingerprints. This method was able to easily differentiate between different ferulic acid-containing compounds that are produced industrially with vegetable oils. In order to support expanded, value-added markets for agricultural products, a cooperative agreement was used to transfer the method to industry. This project is being replaced by project 5010-42000-052-00D. The new project has an emphasis on development of materials and methods to reduce exposure to mycotoxins, determination of the occurrence of mycotoxins and fungi in the U.S. oat supply, the identification of oat cultivars associated with reduced mycotoxin levels or disease, and the development of predictive tools for the evaluation of the presence of mycotoxins in grain commodities and foods.

Accomplishments
1. Models to predict antifungal and antibiotic activity. Harmful fungal and bacterial pathogens sometimes contaminate and spoil food and can negatively impact human and livestock health. One means to reduce spoilage of commodities and exposure to mycotoxins is by controlling fungal contamination through the use of antifungal agents. ARS researchers at Peoria, Illinois, developed scientific models that predict the antifungal and antibiotic activity for the triazolothiadiazine class of chemicals. The models identified the chemical properties that are important for reducing fungal and bacterial growth. and were able to reliably predict chemicals with antifungal activity. Using these models, researchers will be able to quickly and economically predict the antifungal and antimicrobial activities of other compounds and aid in the development of new antimicrobials with better properties.

2. Detection of the fungal toxin roquefortine C. Roquefortine C (ROQC), has been associated with diseases in humans, dogs, and cattle following consumption of moldy food (walnuts, beer) or feedstuffs (corn silages). ARS researchers at Peoria, Illinois, developed a rapid screening assay for ROQC in nut extracts ('milks') and in the serum of dogs. A small survey of commercial nut ‘milks’ indicated the presence of extremely low levels that are unlikely to be hazardous. The assay, however, is the first to be developed to detect ROQC in serum, and provides a rapid, sensitive, screening tool that may be useful in the diagnosis of mycotoxin-induced poisoning of dogs.

Review Publications
Appell, M.D., Compton, D.L., Evans, K.O. 2020. Predictive quantitative structure-activity relationship modeling of the antifungal and antibiotic properties of triazolothiadiazine compounds. Methods and Protocols. 4(1). Article 2. https://doi.org/10.3390/mps4010002.
Evans, K.O., Compton, D.L., Kim, S., Appell, M.D. 2019. Charged phospholipid effects on AAPH oxidation assay as determined using liposomes. Chemistry and Physics of Lipids. 220:49-56. https://doi.org/10.1016/j.chemphyslip.2019.02.004.
Maragos, C.M. 2020. Development and characterisation of a monoclonal antibody to detect the mycotoxin roquefortine C. Food Additives & Contaminants. 37(10):1777-1790. https://doi.org/10.1080/19440049.2020.1781937.
Tittlemeir, S.A., Brunkhorst, J., Cramer, B., DeRosa, M.C., Lattanzio, V.M.T., Malone, R., Maragos, C., Stranska, M., Sumarah, M.W. 2021. Developments in mycotoxin analysis: an update for 2019-2020. World Mycotoxin Journal. 14(1):3-26. https://doi.org/10.3920/WMJ2020.2664.