Germination induces the glucosylation of the Fusarium mycotoxin deoxynivalenol in various grains

Food Chemistry 131 (2012) 274–279

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Germination induces the glucosylation of the Fusarium mycotoxin deoxynivalenol in various grains Ronald Maul a,⇑, Christian Müller b, Stephanie Rieß a, Matthias Koch a, Frank-Jürgen Methner b, Nehls Irene a a b

BAM Federal Institute for Materials Research and Testing, Division Organic Chemical Analysis, Reference Materials, Richard-Willstätter-Straße 11, 12489 Berlin, Germany Berlin Institute of Technology, Department of Biotechnology, Chair of Brewing Science, Seestraße 13, 13353 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 13 May 2011 Received in revised form 30 June 2011 Accepted 25 August 2011 Available online 1 September 2011 Keywords: Mycotoxins Masked mycotoxins Deoxynivalenol (DON) DON-3-glucoside Acetyl-DON Germination Malting

a b s t r a c t In food, the mycotoxin deoxynivalenol (DON) often occurs in conjunction with its 3-b-D-glucopyranoside (D3G). The transformation of DON to D3G through glucosylation is catalysed by plant enzymes, however, the exact circumstances are not well understood. In order to investigate the role of enzymatic glucosylation in germinating grains, DON treated kernels were steeped and germinated under laboratory conditions. Furthermore, the effect of malting on the DON content of the contaminated barley was investigated. In all cases, DON and its derivatives were quantified by HPLC-MS/MS before, during and after the experiments. Amongst the six tested cereals; wheat, rye, barley, spelt, and millet transformed DON to D3G during germination whilst the oats were inactive. For wheat, barley, and spelt the initial DON content was reduced by 50%, with the loss being almost entirely accounted for by D3G formation. As D3G might be cleaved during digestion, the elevated D3G concentration may obscure the toxicologically relevant DON content in processed food and beer. The germination process has a major influence on the ‘‘masking’’ of DON, leading to high quantities of D3G that may be missed in common mycotoxin analyses. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Mycotoxins are natural contaminants produced by a range of fungal species. The Food and Agriculture Organisation (FAO) estimates that 25% of the Worlds food crops, including many basic foods, are affected by mycotoxin producing fungi (Köppen et al., 2010). Therefore, mycotoxin analysis represents a major challenge in the control and inspection of foodstuffs as a high proportion of cereal based foods is affected. Also, the toxins may occur associated to the matrix or as conjugates (so called ‘‘masked mycotoxins’’) (Berthiller, Schuhmacher, Adam, & Krska, 2009). The mycotoxin deoxynivalenol (DON) predominantly occurs in wheat and barley cultivars infected with Fusarium graminearum or Fusarium culmorum, which cause Fusarium head blight (Kokkonen, Ojala, Parikka, & Jestoi, 2010). In addition to free DON, masked forms like DONb-D-glucopyranoside (D3G) or the acetylated forms 3- and 15acetyldeoxynivalenol (3-AcDON and 15-AcDON) are of particular

Abbreviations: 15-AcDON, 15-acetyldeoxynivalenol; 3-AcDON, 3-acetyldeoxynivalenol; D3G, DON-3-b-D-glucopyranoside; DON, deoxynivalenol; ESI, electrospray ionisation; HPLC, high-performance liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MS, mass spectrometry; MS/MS, tandem mass spectrometry. ⇑ Corresponding author. Tel.: +49 30 8104 5960; fax: +49 30 8104 1127. E-mail addresses: [email protected], [email protected] (R. Maul). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.08.077

importance in contaminated food. In cereals, they may reach concentration levels of about 50% of the DON content and the toxicological fate of D3G after digestion by animals is still largely unknown (Berthiller et al., 2009). Whilst D3G is a product of the plant metabolism (Sewald, Vongleissenthall, Schuster, Muller, & Aplin, 1992) 3- and 15-AcDON are intermediates of fungal DON biosynthesis (Bretz, Beyer, Cramer, & Humpf, 2005). Whether or not plant enzymes also catalyse the acetylation of DON was never reported. However, it is known that D3G may be converted to DON during food processing and vice versa (Voss & Snook, 2010). Also, the presence of D3G and acetylated DON in food has been previously reported (Berthiller et al., 2005; Vendl, Crews, MacDonald, Krska, & Berthiller, 2010). In various studies, concentration levels of the acetylated DON derivatives were typically less than 10% of those reported for DON. The highest mean levels reported for wheat and barley for 3-AcDON were 193 lg/kg and 19 lg/kg, respectively; for 15-AcDON, the corresponding levels were 365 lg/kg and 0.3 lg/kg (JECFA, 2011). Generally, the extent of DON acetylation depends on the fungal strain with 3-AcDON appearing to be the prevalent acetylated derivative (Kokkonen et al., 2010). However, in other studies considerable amounts of 15-AcDON have been reported as well (Schollenberger et al., 2006). For D3G only very limited data about the occurrence in cereals or food is available. In a recent study investigating the DON and D3G content in numerous wheat and maize samples the mean

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concentration levels for D3G were 393 lg/kg and 141 lg/kg, respectively, representing about 17% and 14% of the measured DON content (Berthiller et al., 2009). For malt, only few investigations concerning the content of masked mycotoxins have been carried out so far: Lancova et al. (2008) reported that a significant increase of D3G in malt as compared to the original barley could be seen. In beer, the concentration of masked DON even exceeded the one of free DON (Lancova et al., 2008). In a large study of Kostelanska and co-workers the occurrence of DON and its conjugates in 176 beer samples was investigated. Here, it was shown that traces of Fusarium toxins can be detected in almost all beers worldwide (Kostelanska et al., 2009). The level of contamination varied between 1.0–35.9 lg/l DON and 1.4–37.0 lg/l D3G. For brewing barley, the mycotoxin contamination had been monitored over 10 years showing mean annual DON levels ranging from 0.4 to 10.3 lg/g with 32–81% of the barley exceeding 0.5 lg/g (Schwarz, Horsley, Steffenson, Salas, & Barr, 2006). A more recent study investigating DON in a small set of 20 samples reported levels ranging from 0.02 to 0.64 lg/g DON in barley (Dohnal et al., 2010). Since 3-AcDON is converted to DON in the mammal metabolism and therefore contributes to the total DON-induced toxicity, the Joint FAO/WHO (World Health Organisation) Expert Committee on Food Additives decided to convert the provisional maximum tolerable daily intake (PMTDI) for DON to a group PTMDI of 1 lg/ kg bodyweight for DON and its acetylated derivatives (JECFA, 2011). A similar assessment for D3G was not made due to a lack of toxicological data, however, the JECFA experts considered it possible that this compound would be hydrolysed in the body and the DON would become bioavailable. They noted, however, that toxicokinetic studies would be necessary to confirm this (JECFA, 2011). Still, for the corresponding glycoside of the mycotoxin zearalenone a cleavage during mammalian digestion has already been shown (Gareis et al., 1990). Whilst an analytical surveillance of conjugates in foodstuff thus appears advisable, the lacks of reference standards have obstructed reliable quantification to date. In addition, existing analytical techniques respond differently to masked DON: whilst immunological methods in many cases also detect the DON derivatives because of cross-reactivities of the employed antibodies, common HPLC-MS techniques miss the conjugates because the relevant mass transitions are not implemented (Köppen et al., 2010). In any case, understanding the formation process of masked mycotoxins is a key issue when dealing with the associated analytical and technological problems. It is known that plant enzymes from the class of the uridine diphosphate glycosyltransferases have the capability to transform mycotoxins into their glucosides (Poppenberger et al., 2006). During the first days of germination high quantities of glucosidase enzymes (and therefore also elevated amounts of free glucose) are present in the kernels (Frandsen, Lok, Mirgorodskaya, Roepstorff, & Svensson, 2000). Raised amounts of glucose may be transformed to the activated uridine diphosphate glucose and consecutively attached to the mycotoxin by various plant glycosyltransferases. Likewise, it was reported that a- or b-glucosidases are unable to cleave the DON-glucoside, (Sewald et al., 1992), thus, avoiding a possible back reaction. Hence, it is reasonable to expect the formation of mycotoxin glucosides during the first days of germination. Germinated grains can be the basis for regular food, for example in beer brewing or in the production of gluten free dough for bread making (Loponen et al., 2009). It can be assumed that the mycotoxins are, at least partially, transformed into their conjugated form during food processing, entering the food chain as masked derivatives. Hence, we investigated the capability of various germinating grains to transform DON into D3G, 3- and 15-AcDON. Possible grain type dependency as well as the time course of the transformation was monitored using DON spiked, Fusarium free grains. A special focus

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was put on the enzymatic DON glucosylation during the malting of brewing barley. Samples were analysed by means of HPLC-MS/MS using 13C-labelled DON as an internal standard. 2. Materials and methods 2.1. Chemicals D3G (OEKANALÒ, certified purity: 96.0%) was purchased from Sigma–Aldrich (Steinheim, Germany). DON (certified purity: 99.4%), 3AcDON (certified purity: 99.4%), 15AcDON (certified purity: 98.8%), and 13C15-DON solution (certified purity: 99.5%) were purchased from Biopure (Tulln, Austria). Stock solutions were prepared in pure acetonitrile and kept at 20 °C in the dark. Working solutions were prepared in acetonitrile/water 20:80 (v/v) or water for the treatment of the grains. Acetonitrile (HPLC grade) was purchased from Labscan. Deionised water (used for sample dilution, extraction and as a HPLC eluent) was sourced from a Seralpur Pro 90 CN system (Seral Erich Alhäuser, Ransbach-Baumbach, Germany). All grains used for the experiments were obtained from a local supermarket, except barley which was supplied by the Chair of Brewing Science, Berlin Institute of Technology. 2.2. HPLC–MS equipment HPLC–MS/MS analysis was performed on an 1100 Series HPLC system from Agilent Technologies (Waldbronn, Germany) used in connection with an API 4000 triple-quadrupole MS/MS system from Applied Biosystems (Foster City, CA, USA). MS/MS measurements were exclusively carried out in selected-reaction-monitoring mode using the mass transitions and parameters given in Table 1. 2.3. Chromatographic conditions HPLC separation of DON and its metabolites was carried out on a Prontosil (120 mm  3.0 mm i.d., particle size 3 lm, pore size 120 Å) reversed-phase column (Bischoff, Leonberg, Germany). The column temperature was set to 30 °C .The solvent system consisted of water (solvent A) and acetonitrile (solvent B). The following gradient was applied with a flow rate of 0.7 ml/min: 0–2 min isocratic with 10% B; then a linear increase from 10% to 30% B from 2–5.5 min; a linear increase from 30% to 60% for 5.5–7 min; a linear increase from 60% to 65% B for 7–12 min; a linear increase from 65% to 100% for 12–13.5 min, followed by an isocratic washout step for 2 min and after a reconditioning step was used for shifting back to acetonitrile–water (10:90; v/v) for 3.5 min. 20 ll was used as the standard injection volume. 2.4. Mass spectrometric conditions The ESI interface was used in negative-ionisation mode at 450 °C with the following settings: 20 psi curtain gas; 60 psi nebulizer gas (GS1); 50 psi auxiliary gas (GS2); 4500 V ionisation voltage, and 8 psi collision gas. Selected-reaction-monitoring (SRM) dwell time was 150 ms. Two mass transitions were recorded for each analyte by applying individually optimised MS/MS conditions (Table 1). 2.5. Sample treatment and extraction Each grain sample was milled in a Retsch Mixer Mill MM 400 (Retsch GmbH, Haan, Germany) for 5 min at a vibrational frequency of 30 s 1. One gramme of each milled sample was extracted with 4 ml ACN/H2O (50:50; v/v) containing ISTD (13C15-DON,

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Table 1 Precursor ions, declustering potentials (V), quantifier ions, qualifier ions, and collision energies (eV) for DON and derivatives. Analyte

Precursor ion (m/z)

Declustering potential (V)

Quantifier ion, m/z (collision energy (eV))

D3G

457.1 [M–H]

65 55

247.1 (25)

DON

295.1 [M–H] 355.1 [M–H + CH3COO ]

55 55

264.9 (16)

15AcDON

337.1 [M–H]

45 50

219.1 (15)

3-AcDON

337.1 [M–H]

45

307.2 (17)

19 mM) for 60 min under constant shaking of a horizontal shaker. Subsequently, 500 ll of the extract was dried under a stream of nitrogen and redissolved in 300 ll ACN/H2O (10:90; v/v). The samples were passed through a regenerated cellulose syringe filter (pore size of 0.45 lm) prior to HPLC-MS/MS analysis. Additionally a blank sample of the untreated grains was extracted analogously.

Qualifier ion, m/z (collision energy (eV)) 427.1 (16) 295.1 (14) 150.0 (25) 172.9 (12)

tion and dry the malt a kilning step followed in which the malt was dried for 15 h at 50 °C and then for 4 h at 80 °C. Samples were taken after every processing day and kept frozen at 20 °C until analysis. Subsequently, all samples were dried at 60 °C and treated further as described above (see Section 2.5). 3. Results and discussion

2.6. Steeping and germination procedure 3.1. Grain type dependency Six types of grains (barley (Hordeum vulgare), millet (Panicum miliaceum), oats (Avena sativa), rye (Secale cereale), spelt (Triticum spelta), and wheat (Triticum aestivum)) were tested for their general ability to transform DON to D3G and AcDON during germination. Two grammes of each type had been steeped overnight with 2 ml of either distilled water or an aqueous DON solution (810 nmol/kg, equivalent to 241 ng/g DON in the grain after complete resorption) in closed petri-dishes at room temperature in the dark. After total imbibition of the liquid by the grains, wetted filter paper was added to maintain a constant moisture level throughout the experiment. After five days of germination at room temperature the samples were transferred to an oven and dried at 60 °C overnight. In order to investigate the time course of the DON conjugation, multiple wheat samples were prepared and the germination process was stopped at various time points. Further analysis was then carried out as described before. The use of grains that were not contaminated with Fusarium before was chosen in order to reduce the risk of a DON formation on the grains caused by newly outgrowing Fusarium, as it was assumed by Lancova et al. (2008). Only healthy looking grains were used for the experiments and more than 90% of them had germinated under the applied conditions. 2.7. Model malting experiment For the investigation of the DON conjugation under malting conditions two portions of 800 g barley, either untreated or pretreated with DON solution, were subjected to the steeping and malting process in a pilot malting plant (Type A1-2008, SchmidtSeeger GmbH, Beilngries, Germany). In the first steeping step, the barley grains were adjusted from initial moisture of 13.6% to a moisture content of 41.5%.This step was carried out in an aerated laboratory beaker for 5 h at 13.5 °C using an aqueous DON solution (6.9 lM) or water for the control sample. After the wet steep, the barley was aerated and kept at 16 °C without water for 16 h of so called air rest. A second steeping step of 3 h in the pilot plant followed in order to further increase the moisture content. The second steeping was done with water for both, the DON treated as well as the control sample. Then, the barely was germinated for 5 days in the plant at a constant humidity of 100% and a temperature of 16 °C. After the second air rest and at the first germination day the moisture content was adjusted by spraying water onto the malt to realise the desired moisture of 46%. To stop the germina-

Initially, six types of grains were tested for their ability to germinate in the presence of DON supplemented water. Wheat and barley both developed rootlets within three days, whilst oats, millet, spelt, and rye required five days to develop shoots of at least 1 mm length. All experiments were terminated after five days and the samples were treated as described in Section 2.6. The HPLC-MS/MS results show that all tested grains, except oats, were able to generate D3G in the presence of DON, whilst in the absence of DON no DON or D3G could be detected in amounts higher than in the control experiments (Fig. 1). Although the grains used for this study were carefully selected, low amounts of background contamination could not be excluded in all cases. However, it could be shown for the control samples (germinated with water) that all grain samples initially contained merely insignificant DON or D3G concentrations, i.e. levels lower than 58 nmol/kg (in wheat, barley, and spelt; whilst all others had
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Fig. 1. Grain specificity for the glucosylation of deoxynivalenol (DON) to the b-3-glucoside (D3G). Each bar represents the mean value of two independent experiments. The first bar gives the theoretical maximum content resulting from a complete resorption of the added DON solution.

Table 2 Molar recovery of the initial DON content in the germinated grains calculated as the sum of DON and D3G. Grain type

Recovery [%]

Oats Millet Barley Rye Spelt Wheat

91.7 99.5 111.2 91.1 56.0 73.9

the sum of DON and D3G after germination, is almost complete, for spelt a significant fraction of DON is missing. Here, it seems likely that other metabolites were formed, being responsible for the ‘‘loss’’ of 44% of the initially doped toxin amount. Also wheat, the phylogenetically most closely related grain, shows a combined recovery of only 74%. The HPLC-MS/MS analysis of the wheat and spelt did not indicate di-glucose or sulphate adducts. Hence, currently no information on the nature of the unknown metabolite/ adducts can be given. 3.2. Time course of the D3G formation In order to investigate the time dependency of D3G formation, five wheat samples were prepared and were allowed to germinate for 17, 21, 41, 46, and 113 h. Fig. 2 shows the time dependent decrease of DON, as well as the increase of D3G. The 0 h data point was calculated theoretically by adding the DON content of untreated wheat (44 nmol/kg) to the spiked DON amount. The data indicate a weak DON transformation within the first 17 h, as can be seen from a decrease in the DON content of only 6.6% and the low D3G amount of 15.2 nmol/kg. The same accounts for the last 60 h of the experiment. Between 17 and 46 h after the start of the experiment an intense D3G formation of 4.8 nmol kg 1 hour 1 on average was observed representing the most relevant time slot for the D3G formation. This may be explained by the 20 h phase that is needed for the activation of the enzymes responsible for the glucose transfer in the grain (Frandsen et al., 2000). After about 50 h a concentration equilibrium of DON and D3G is reached in the germinated grains. Experiments with a germination time of more than five days did not lead to more D3G formation (data not shown), thus it can be assumed that after 50 h the transformation

Fig. 2. Time course of the measured deoxynivalenol (DON) and DON-3-glucoside (D3G) content in germinated wheat samples. Each data point represents the mean value measured for two independent sample preparations. The conversion of DON to D3G can be tentatively fitted according to a sigmoidal model.

process is finished under the chosen conditions. These findings are in line with general plant physiological data obtained for other grains. Particularly, between day three and five after the initiation of the germination process a-glucosidase is highly expressed in barley, causing elevated amounts of free glucose in the kernel (Frandsen et al., 2000). The determination of which specific enzyme is responsible for the conversion of DON into D3G can only be clarified by further molecularbiological investigations. However, during germination, conditions favourable for trans-glucosylation prevail because of a high circulation of glucose and an increased activity of glucose metabolism related enzymes.

3.3. DON glucosylation during steeping and malting In order to investigate the relevance of the transformation of DON into D3G during brewing, an experiment with barley was conducted in a model malting plant. The first steeping step using an aqueous DON solution was done in a beaker and hereafter the artificially contaminated barley was transferred into the model plant. In this experiment the DON solution was not resorbed entirely. Hence, after the first steeping, considerable amounts of

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DON were lost together with the waste steeping water. A further washout effect occurred during in the second steeping in the model plant. Barley samples were taken after the first and second steeping (st1 and st2), on days one to four of the germination (g1–g4), as well as after kilning of the malt (Fig. 3). Control samples of untreated barley were taken only after the second steeping, on day four of the germination and after kilning (Fig. 3). After the first steeping, the DON concentration in the treated barley reached 1413 nmol/kg of dry matter. The measured D3G concentration was 101 nmol/kg. Whilst the DON content decreased significantly during the second steeping, the D3G concentration rose moderately. The DON decrease is most likely due to a washout effect of DON adhering to the grains after the first steeping without having been absorbed. D3G is formed in the interior of the kernels and therefore cannot be washed out to a great extent at this stage. Until the second day of germination, the D3G content in the barley increases continuously, whilst the DON content decreases constantly until the end of the germination. As the first steeping (including the subsequent air rest) lasted for 21 h, the occurrence of D3G after the first processing stage is in good accordance with the previous model experiments where a D3G formation had been already observed after 17 h of steeping (see Section 3.2). Furthermore, 48 h after initiation of the experiment the biggest part of the glucosylation had already passed off, corresponding to a time point between the second steeping and the first day of germination. Also, hardly any additional D3G was formed after the first day of germination (i.e. 72 h post initiation) which is in clear agreement with plant physiological observations that reported the highest enzymatic activity from 48 to 72 h after the initiation of the germination process (Frandsen et al., 2000). Hence, with the first appearance of visible sprouts, the conversion was almost complete. Whilst in the petri-dish experiments with barley the sum of DON and D3G remained constant during germination, here a decline of about 30% (from 684 nmol/kg to 497 nmol/kg) between the second steeping and the final malt was observed. In contrast to the laboratory experiments, the model malting plant is constantly aerated and water is added in order to maintain constant moisture, which might lead to oxidative processes and further washout effects. Whether or not additional conjugated forms of DON are responsible for this effect is unknown. The MS transitions that would have to be expected for di-glucose, sulphate or glutathione adducts were monitored without any observable peaks in the chromatogram. However, the ratio of D3G to DON at the end of the malting experiment (D3G/DON: 0.67) is similar to the five day experiment in the petri-dish (ratio D3G/DON: 0.81). These results show the same tendency as the findings of Kostelanska and co-workers who stated that in wort and beer samples the molar ratio of

Fig. 3. Measured deoxynivalenol (DON) and DON-3-glucoside (D3G) contents in barley throughout the process of steeping (st1; 21 h and st2; 24 h), germination (g1–g4 for day one to four of the process), and after malting. Each data point represents the mean value measured for two independent sample preparations.

D3G/DON is increased and a ratio 1 can be found in many cases (Kostelanska et al., 2010), whilst in untreated barley and other grains the free DON is the dominating form and D3G only accounts for up to 20% (i.e. ratio D3G/DON < 0.2) (Berthiller et al., 2009; Malachova et al., 2010). It is well-established that plants or plant cells have the capability to form D3G from DON. Respective reports exist for thale cress (Arabidopsis thaliana) or maize (Zea mays) (Berthiller and Schuhmacher et al., 2009). However, the extent of conjugation during the process of germination, as determined in our study, is remarkable. We could demonstrate for the first time that D3G was formed by the direct glucosylation of DON in germinating grains. This finding may explain earlier reports of an increased D3G content in malt and beer compared to raw barley, which in our opinion is not exclusively caused by an altered extractability, as assumed by the authors, but also has its origin in a formation of D3G during the processing of the barley (Lancova et al., 2008; Malachova et al., 2010). However, it remains unclear which distinct enzyme catalyses the DON glucosylation. The DON conversion was previously shown to be catalysed by the UGT73C family enzymes of Arabidopsis thaliana, as well as by the HvUGT13248 enzyme in barley (Poppenberger et al., 2003; Schweiger et al., 2010). Thus, a possible elevated expression of these enzymes during germination should be monitored in future research. In addition, it is known that certain glucosamideases possess an enhanced enzymatic activity (Vuylsteker, Cuvellier, Berger, Faugeron, & Karamanos, 2000) and that certain proteins are intensely glycosylated (Lindholm et al., 2000) during the germination of barley. Thus, a high circulation of glucose and an increased activity of glucose related enzymes such as the glycosyltransferases can be assumed in the phase of cereal germination (Frandsen et al., 2000). For the sum of these reasons, it seems likely that the combination of glucose release and presence of enzymes, possessing an activity that catalyses DON glucosylation during germination is responsible for the effects observed in this study. The impact of D3G formation is not only of relevance in brewing, but also in cereal processing and analysis. According to our results D3G is formed in most germinating grains. Wherever such processing steps occur, e.g. in bread production, for preparation of cereal based meals, or in poultry feeding, only the free DON will be detected in the products by most conventional HPLC based methods (Köppen et al., 2010). Although, the metabolic fate of D3G is still unclear, it can be assumed that the glucoside can at least partially be cleaved by intestinal microbiota, thus liberating DON. A similar mechanism was previously shown for the zearalenone glucoside (Berthiller et al., 2009; Gareis et al., 1990). In contrast to the pronounced formation of D3G in the presented germination and malting experiments no formation of acetylated DON could be observed. After all, measuring the sole DON content of such products may be misleading and therefore, a sum value for DON and its conjugates should be obtained for toxicological evaluations and considered in future legal limit fixations. A further consequence of the present findings is that analytical results, obtained during the processing of germinated grains, should be considered in a critical fashion. Whilst in HPLC-MS analysis it is possible to include D3G as an additional analyte for a detailed measurement of the mycotoxin content, immunological analysis is dependent on the DON antibodies. The latter can have highly variable cross reactivities towards D3G depending on production method and lot.

4. Conclusion In conclusion, we could show that an extensive ‘‘masking’’ of DON occurs during germination process, particularly in barley,

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wheat and rye grains. Hereby previous findings on an elevated D3G levels in malt and beer may well be explained. Further, D3G occurrence has to be expected in other germinated cereal food and feed. Toxicological investigation should clarify whether D3G uptake is of relevance for human health and thus, D3G may have to be taken into consideration for future legislation. Acknowledgements David Siegel is acknowledged for his support in preparing the manuscript and Ramona Pielhau is acknowledged for assistance with the germination experiments. References Berthiller, F., Dall’asta, C., Corradini, R., Marchelli, R., Sulyok, M., Krska, R., et al. (2009). Occurrence of deoxynivalenol and its 3-beta-D-glucoside in wheat and maize. Food Additives and Contaminants: Part A, Chemistry, Analysis, Control, Exposure and Risk Assessment, 26(4), 507–511. Berthiller, F., Dall’Asta, C., Schuhmacher, R., Lemmens, M., Adam, G., & Krska, R. (2005). Masked mycotoxins: Determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatographytandem mass spectrometry. Journal of Agricultural and Food Chemistry, 53(9), 3421–3425. Berthiller, F., Schuhmacher, R., Adam, G., & Krska, R. (2009). Formation, determination and significance of masked and other conjugated mycotoxins. Analytical and Bioanalytical Chemistry, 395(5), 1243–1252. Bretz, M., Beyer, M., Cramer, B., & Humpf, H. U. (2005). Synthesis of stable isotope labeled 3-acetyldeoxynivalenol. Molecular Nutrition & Food Research, 49(12), 1151–1153. Dohnal, V., Jezkova, A., Pavlikova, L., Musilek, K., Jun, D., & Kuca, K. (2010). Fluctuation in the ergosterol and deoxynivalenol content in barley and malt during malting process. Analytical and Bioanalytical Chemistry, 397(1), 109–114. Frandsen, T. P., Lok, F., Mirgorodskaya, E., Roepstorff, P., & Svensson, B. (2000). Purification, enzymatic characterization, and nucleotide sequence of a highisoelectric-point alpha-glucosidase from barley malt. Plant Physiology, 123(1), 275–286. Gareis, M., Bauer, J., Thiem, J., Plank, G., Grabley, S., & Gedek, B. (1990). Cleavage of zearalenone-glycoside, a ‘‘masked’’ mycotoxin, during digestion in swine. Zentralbl Veterinarmed B, 37(3), 236–240. JECFA (2011). Evaluation of certain contaminants in food: Seventy-second report of the Joint FAO/WHO Expert Committee on Food Additives. WHO technical report series, 959. Kokkonen, M., Ojala, L., Parikka, P., & Jestoi, M. (2010). Mycotoxin production of selected Fusarium species at different culture conditions. International Journal of Food Microbiology, 143(1–2), 17–25. Köppen, R., Koch, M., Siegel, D., Merkel, S., Maul, R., & Nehls, I. (2010). Determination of mycotoxins in foods: Current state of analytical methods and limitations. Applied Microbiology and Biotechnology, 86(6), 1595–1612. Kostelanska, M., Hajslova, J., Zachariasova, M., Malachova, A., Kalachova, K., Poustka, J., et al. (2009). Occurrence of deoxynivalenol and its major conjugate,

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