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

Abstract

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.

Evolution of Alternaria toxins during the brewing process and the usability of optical sorting methods to reduce mycotoxin concentrations in beer

“…As previously described, fungi of the genus Alternaria constitute a large part of the barley mycoflora [10]. Accordingly, in a recent study, various Alternaria toxins have been detected in brewing malts [11].…”

“…Until now, TeA, AOH and AME were the only Alternaria toxins to be found in the final beer, which indicates a mycotoxin transfer of these three toxins during brewing [17][18][19][20]. However, a recent study on mycotoxin concentrations in 50 representative malt samples could show the presence of other relevant Alternaria toxins such as ALTP, ATX I and, to some extent, also the modified form AOH-3-sulfate (AOH-3-S) in this raw material [11]. This finding emphasizes the need to pursue the concentrations of these toxins during the brewing process as well as to evaluate why none of these toxins have been detected in beer so far.…”

“…This value was then used to refer the mycotoxin content to the wet weight of all samples. All samples were thoroughly ground, and sample preparations were performed as previously published [11,20]. In brief, 1 g of grist, mash and spent grains, and 0.5 g of yeast sediment and hot break were weighted into a 50 mL centrifuge tube.…”

“…All samples were measured with a previously published LC-MS/MS method for the analysis of 13 free and modified Alternaria toxins [11,20].…”

“…To determine the amount of A. alternata DNA, a previously established qPCR assay based on SYBR green technology for the detection and quantification of black fungal species on malting barley raw material was used [10]. To follow up on the concentration of Alternaria toxins throughout the brewing process, we applied the recently developed multi-mycotoxin liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for cereals and beer [11,20] for the analysis of TeA, AOH, AME, TEN, altenuene (ALT), ALTP, ATX I, ATX II, AOH-3-glucoside (AOH-3-G), AOH-9-glucoside (AOH-9-G), AME-3glucoside (AME-3-G), AOH-3-S, and AME-3-sulfate (AME-3-S) (see Fig. 1) during the single processing steps, and determined the transfer rates from barley malt grist into by-products and in the final product beer.…”