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.

Screening of Mycotoxigenic Fungi in Barley and Barley Malt (Hordeum vulgare L.) Using Real-Time PCR-A Comparison between Molecular Diagnostic and Culture Technique.

Filamentous fungi have a crucial impact on the food safety and technological quality of malting barley. Commonly used techniques for the detection of seed-borne fungi are based on cultivation and identification by morphological criteria. In contrast, this study established a quantitative real-time polymerase chain reaction (PCR) assay based on SYBR green technology for the detection and quantification of black fungal species (Alternaria spp., Epicoccum nigrum, Cladosporium cladosporioides, Penicillium verrucosum and Aspergillus niger) on brewing barley and compares it with the traditional cultivation technique and visual assessment. To screen the fungal spectrum over different barley varieties and harvest years, naturally infected samples of malting barley and corresponding malts (Hordeum vulgare L.) were analyzed over four consecutive years (2018-2021), grown under different climatic conditions in Germany. Alternaria and Cladosporium spp. DNA were present in all examined barley samples, even without visible contamination. In contrast, detection via culture-based methods does not reliably cover all species. Molecular analysis showed that there was less fungal biomass after malting, by 58.57% in the case of A. alternata, by 28.27% for Cladosporium spp. and by 12.79% for Epicoccum nigrum. Correlation analysis showed no causal relationship between fungal DNA and the number of black kernels. The qPCR provides a highly sensitive and time-saving screening method for detecting latent fungal infections in brewing grains to identify batches that are potentially highly contaminated with toxigenic fungi.