European food safety research: An explorative study with funding experts' consultation.

The European research area exhibits considerable opacity and fragmentation in food safety research funding and organizational structures, impeding the exploitation of existing research potential across European countries. Given that food safety is inherently linked to the societal challenges of our time, identifying and removing existing barriers to research funding in this area is crucial. Towards investigating this matter, interviews were conducted with funding bodies from six European countries to assess key issues related to research funding in general and food safety in particular. Funding experts were then invited to a workshop to jointly discuss the challenges identified and explore strategies to address them. Evaluation of the food safety research funding situation in selected European countries revealed both convergences and significant differences among national funding bodies. Engaging with funding experts provided invaluable insights into the issues encountered with research funding, such as inadequate call management staff or insufficient research funds, culminating in a set of recommendations for action to remedy the situation.

Root uptake and metabolization of Alternaria toxins by winter wheat plants using a hydroponic system.

Fungi of the genus Alternaria are ubiquitous in the environment. Their mycotoxins can leach out of contaminated plants or crop debris into the soil entering the plant via the roots. We aim to evaluate the importance of this entry pathway and its contribution to the overall content of Alternaria toxins (ATs) in wheat plants to better understand the soil-plant-phytopathogen system. A hydroponic cultivation system was established and wheat plants were cultivated for up to two weeks under optimal climate conditions. One half of the plants was treated with a nutrient solution spiked with alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA), whereas the other half of the plants was cultivated without mycotoxins. Plants were harvested after 1 and 2 weeks and analyzed using a QuEChERS-based extraction and an in-house validated LC-MS/MS method for quantification of the ATs in roots, crowns, and leaves separately. ATs were taken up by the roots and transported throughout the plant up to the leaves after 1 as well as 2 weeks of cultivation with the roots showing the highest ATs levels followed by the crowns and the leaves. In addition, numerous AOH and AME conjugates like glucosides, malonyl glucosides, sulfates, and di/trihexosides were detected in different plant compartments and identified by high-resolution mass spectrometry. This is the first study demonstrating the uptake of ATs in vivo using a hydroponic system and whole wheat plants examining both the distribution of ATs within the plant compartments and the modification of ATs by the wheat plants.

Development of analytical methods to study the effect of malting on levels of free and modified forms of Alternaria mycotoxins in barley.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) multi-mycotoxin method was developed for the analysis of the Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), altertoxin I (ATX I), altertoxin II (ATX II), alterperylenol (ALTP), and altenuene (ALT), as well as the modified toxins AOH-3-glucoside (AOH-3-G), AOH-9-glucoside (AOH-9-G), AME-3-glucoside (AME-3-G), AOH-3-sulfate (AOH-3-S), and AME-3-sulfate (AME-3-S) in barley and malt. The toxin tenuazonic acid (TeA) was analyzed separately as it could not be included into the multi-mycotoxin method. Quantitation was conducted by using a combination of stable isotope dilution analysis (SIDA) for AOH, AME, and TeA, and matrix-matched calibration for all other toxins. Limits of detection were between 0.05 µg/kg (AME) and 2.45 µg/kg (ALT), whereas limits of quantitation ranged from 0.16 µg/kg (AME) to 8.75 µg/kg (ALT). Recoveries between 96 and 107% were obtained for the analytes when SIDA was applied, while recoveries between 84 and 112% were found for analytes quantified by matrix-matched calibration. The method was applied for the analysis of 50 barley samples and their respective malts from the harvest years 2016-2020 for their mycotoxin content, showing the overall potential of toxin formation during the malting process. The toxins ALTP and ATX I were mainly found in the malt samples, but not in barley.

Analysis of 13 Alternaria mycotoxins including modified forms in beer.

A multi-mycotoxin LC-MS/MS method was developed to quantify 13 free and modified Alternaria toxins in different beer types by applying a combination of stable-isotope dilution assays (SIDAs) and matrix-matched calibration. With limits of detection (LODs) between 0.03 µg/L (alternariol monomethyl ether, AME) and 5.48 µg/L (altenuene, ALT), limits of quantitation (LOQs) between 0.09 µg/L (AME) and 16.24 µg/L (ALT), and recoveries between 72 and 113%, we obtained a sensitive and reliable method, which also covers the emerging toxins alternariol-3-glucoside (AOH-3-G), alternariol-9-glucoside (AOH-9-G), alternariol monomethyl ether-3-glucoside (AME-3-G) and alternariol-3-sulfate (AOH-3-S) and alternariol monomethylether-3-sulfate (AME-3-S). Furthermore, 50 different beer samples were analyzed, showing no contamination with Alternaria toxins apart from tenuazonic acid (TeA) in concentrations between 0.69 µg/L and 16.5 µg/L. According to this study, the exposure towards TeA through beer consumption can be considered as relatively low, as the threshold of toxicological concern (TTC) value of 1500 ng/kg body weight per day might not be reached when consuming reasonable amounts of beer.

Enzymatic Synthesis of Modified Alternaria Mycotoxins Using a Whole-Cell Biotransformation System.

Reference standards for Alternaria mycotoxins are rarely available, especially the modified mycotoxins alternariol-3-glucoside (AOH-3-G), alternariol-9-glucoside (AOH-9-G), and alternariol monomethylether-3-glucoside (AME-3-G). To obtain these three glucosides as analytical standards for method development and method validation, alternariol and alternariol monomethylether were enzymatically glycosylated in a whole-cell biotransformation system using a glycosyltransferase from strawberry (Fragaria x ananassa), namely UGT71A44, expressed in Escherichia coli (E. coli). The formed glucosides were isolated, purified, and structurally characterized. The exact amount of the isolated compounds was determined using high-performance liquid chromatography with UV-detection (HPLC-UV) and quantitative nuclear resonance spectroscopy (qNMR). This method has proved to be highly effective with biotransformation rates of 58% for AOH-3-G, 5% for AOH-9-G, and 24% for AME-3-G.