Food Control. Beauvericin degradation during bread and beer making. G. Meca a, *, T. Zhou b, X.-Z. Li b, J. Mañes a. abstract

Food Control 34 (2013) 1e8 Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Beauvericin degradation during bread and beer making G. Meca a, *, T. Zhou b, X.-Z. Li b, J. Mañes a a Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain b Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Rd. W, Guelph, ON N1G 5C9, Canada article info abstract Article history: Received 14 January 2013 Accepted 23 March 2013 Keywords: Beauvericin Beer production Bread production Mycotoxin reduction LC-MS/MS LC-MS-LIT Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium species and known to have various biological activities. This study investigated the degradation of the minor Fusarium mycotoxin BEA present at the concentration of 5 mg/kg in barley and wheat flour during beer and bread making. The influence of the making processes and of the formation of degradation products of BEA were evaluated during the beer and bread making. The concentration of BEA and its evolution during the production processes were determined with the technique of the liquid chromatography tandem mass spectrometry in tandem (LC-MS/MS), whereas the formation of the BEA degradation products was determined with the technique of the LC-MS coupled to a linear ion trap (LC-MS- LIT). The degradation of BEA during beer making ranged from 23 to 82%. During bread making, BEA reduction ranged from 75 to 95%. The highest degradation activity of BEA for both beer and bread was evidenced during the heat and fermentation processes. Also two degradation products formed during these processes were identified. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Beauvericin (BEA) (Fig. 1) is a depsipeptide with antibiotic and insecticidal effects belonging to the enniatin family (Hamill, Higgens, Boaz, & Gorman, 1969). It was initially isolated from the fungus Beauveria bassiana, but it can also be produced by other fungi, including several Fusarium species (Logrieco et al., 1998); it may therefore occur in grains (such as corn, wheat, and barley) contaminated with these fungi (Jestoi et al., 2004; Logrieco et al., 1998). BEA is active against gram-positive bacteria and mycobacteria, and it is also capable of inducing programmed cell death in mammals (Meca, Font, & Ruiz, 2011). BEA has been found as a natural contaminant of maize from Poland, Italy, USA, South Africa, Switzerland and Slovakia; feed samples from USA; rye from Finland; and oats, wheat and barley from Norway and Finland (Jestoi, 2008). Logrieco et al. (1993) reported high levels of BEA up to 60 mg/kg in maize from Poland; Ritieni et al. (1997) reported high levels of BEA up to 520 mg/kg in maize from Italy. Recently, Meca, Zinedine, Blesa, Font, and Manes (2010) have reported the contamination of cereals available in the Spanish market with BEA and the levels ranged from 0.51 to 11.78 mg/kg. * Corresponding author. Tel.: þ34963544959; fax: þ3496354954. E-mail addresses: giuseppe.meca@uv.es, g.meca@virgilio.it (G. Meca). Related to the methodologies employed for BEA reduction in food, only a US patent (Duvick & Rod, 1998) and a scientific article (Meca, Ritieni, & Mañes, 2012) are available in the scientific literature. In particular Duvick & Rod (1998), employing as detoxification agent, a strain of Norocardia glubera, reduced the BEA contamination in wheat kernels by 50% with an initial contamination of the mycotoxin at1000 mg/kg; Meca, Ritieni et al. (2012) published a study on the degradation of the mycotoxin BEA during heat treatments related to crispy bread production. The degradation of several Fusarium mycotoxins by a UDPglucosyltransferase isolated from plant Arabidopsis thaliana was studied by Poppenberger et al. (2003). The authors demonstrated that the enzymes purified from the plant detoxified completely the Fusarium mycotoxins deoxynivalenol (DON) and 15-acetyl-deoxynivalenol (15-Ac-DON). The degradation of fumonisin B 1 (FB 1 ) by microbial enzymes was studied by Heinl et al. (2010). The authors isolated two enzymes from liquid culture of Sphingopyxis sp. MTA144, the bacterial strain capable of detoxifying FB 1. The enzymatic degradation of FB 1 by the bacterial strain was also studied by Hartinger et al. (2011). Cramer, Keonigs, and Humpf (2008) evaluated the degradation of mycotoxin ochratoxin A (OTA) during the coffee roasting, evidencing a reduction variable from 69 to 96%, correlating the degradation with the roasting time employed. The degradation of Fusarium toxin nivalenol (NIV) during the baking and cooking processes was studied by Bretz, Knecht, Geockler, and Humpf (2005). It was found that under all the 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.03.032

2 G. Meca et al. / Food Control 34 (2013) 1e8 2.3. Beer making Fig. 1. BEA chemical structure. conditions employed the degradation of NIV was accelerated with the increase of the temperatures used during the heat treatments. Bretz, Beyer, Cramer, Knecht, and Humpf (2006) studied the degradation products of DON using model heating experiments with compounds that mimicked the typical food constituents. The model solutions were heated at different temperatures (150e 200 C) for different time periods. The results showed that the degradation of DON was correlated to the increase of the temperature. The aims of this study were to evaluate: a) the degradation of mycotoxin BEA during bread and beer production and b) the formation of BEA degradation products during the heat and fermentation processes in the bread and beer production. Beer making was carried out with a homemade process based on the following steps: 10 g of malt were contaminated with 25 mg/ kg of BEA. The malt was toasted (T) at two different toasting conditions to simulate the low (40 C for 2 days) and medium (100 C for 1 day) toasting processes normally used in the brewery industry. The malt was finely grounded to produce malt flour that was boiled (B) with 30 ml of water for 30 min to permit the macronutrient dissolution necessary for the yeast fermentation. After that, a-amylase was added to promote starch hydrolysis (E) at 63 C for 30 min. The mixture was centrifuged at 4000 rpm for 5 min. The supernatant was boiled at 100 C for 5 min, and the hoops were added (H). The fermentation (F) of the malt extract was carried out with the three yeast strains, A34, LO9 and QRI individually for 4 days at 23 C under anaerobic conditions. After the fermentation, the mixtures were centrifuged and the resulting beers were analyzed for BEA levels as described below. The BEA level was also determined at the end of each step during the beer making (Fig. 2). Barley 10g Toasting: Low: 40ºC during 2 days Medium: 100ºC for 1 day Grounding of the toasted barley to produce barley flour T 2. Materials and methods 2.1. Materials Boiling with 30 ml of water for 30 min. B Sodium chloride (NaCl), sucrose (C 12 H 22 O 11 ) and formic acid (HCOOH) were obtained from SigmaeAldrich (Madrid, Spain). Acetonitrile, methanol and ethyl acetate were purchased from Fisher Scientific (Madrid, Spain). Deionized water (<18 MU cm resistivity) was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Chromatographic solvents and water were degassed for 20 min using a Branson 5200 (Branson Ultrasonic Corp., CT, USA) ultrasonic bath. The BEA, utilized in this study was purchased from SigmaeAldrich (Madrid, Spain). Enzymatic starch hydrolysis with α-amylase at 63ºC for 30 min. Centrifugation at 4000 rpm for 5 min. E 2.2. Yeast strains and methodology The study was carried out using three strains of Saccharomyces cerevisiae named LO9 (for Bavarian Weizen beer), A34 (for scotch Ale beer), and QRI (commercial bread instant yeast, Fleischmann s Quick-Rise Instant Yeast, www.breadworld.com). The strains were obtained by the personal collection of Dr. Ting Zhou of the Guelph Food Research Centre, Agriculture and Agri-Food Canada in PDA slant at 4 C. For longer survival and higher quantitative retrieval of the cultures, they were stored in sterile 18% glycerol at 80 C. When needed, recovery of strains was undertaken by two consecutive subcultures in appropriate media prior to use. The microorganisms were cultured in 50 ml sterile plastic centrifuge tubes with 20 ml Potato Dextrose Broth (PDB) as growth medium. They were incubated at 25 C in aerobic conditions with 150 rpm shaking for 48 h. Hops adding 100ºC for 15 min. Fermentation 4 days at 23ºC Centrifugation at 4000 rpm for 5 min. Beer H F Fig. 2. Experimental plan used for the production of beer.

G. Meca et al. / Food Control 34 (2013) 1e8 3 2.4. Bread making Bread making was carried out with a homemade process. Wheat flour was pre-contaminated with 25 mg/kg of BEA. The dough composed by wheat flour, water, sucrose, and sodium chloride (NaCl), and was fermented at 27 C for 2 h using the three yeast strains individually. The raised dough was cooked at 200 C for 20 min. BEA levels were determined at each step of the bread making (Fig. 3). 2.5. Mycotoxin extraction procedure from cereals and bread The method used for BEA extraction was described by Jestoi (2008). Briefly, 3 g of sample was extracted with 20 ml of a mixture of water/acetonitrile (85/15, v/v) using an Ultra Ika T18 basic Ultraturrax (Staufen, Germany) for 3 min. The extract was centrifuged at 4500 g for 5 min and the supernatant was evaporated to dryness with a Büchi Rotavapor R-200 (Postfach, Switzerland) before it was re-dissolved in 2 ml of extraction solvent. This final solution was filtered through 0.22 mm nylon filter purchased from Análisis Vínicos (Tomelloso, Spain) and injected into the LC-MS system for analysis. 2.6. Mycotoxin extraction from beer BEA contained in beer and also in the intermediates of beer making was extracted as follows: Five milliliters of each mixture were placed in a 20 ml test tube, and extracted three times with 5 ml of ethyl acetate using a vortex VWR international (Barcelona, Spain) for 1 min. Then, the mixtures were centrifuged (Centrifuge 5810R, Eppendorf, Germany) at 4000 rpm and at 4 C for 10 min. The organic phases were completely evaporated by a rotary evaporator (Buchi, Switzerland) operating at 30 C and 30 mbar pressure, resuspended in 1 ml of methanol and filtered with a 0.22 mm filter (Phenomenex, Madrid, Spain) before being analyzed by LC- MS/MS (Meca, Fernandez-Franzon et al., 2010). 2.7. LC-MS/MS and LC-MS-LIT The separation of BEA was achieved by LC Agilent 1100 (Agilent Technologies, Santa Clara, California, USA) coupled to a mass Ingredients: Wheat flour (460g), water (255mL), sugar (7g), salt (3.5 g) Dough production Fermentation 27ºC for 2h Cooking 200ºC for 20 min Bread Fig. 3. Experimental plan used for the production of bread. F C spectrometer Applied Biosystems/MDS SCIEX Q TRAP TM linear ion trap mass spectrometer (Concord, Ontario, Canada). A Gemini (150 2.0 mm, 5 mm) Phenomenex (Torrance, California, USA) column was used. LC conditions were set up using a constant flow at 0.2 ml/min and acetonitrile/water (70:30, v/v with 0.1% of HCOOH) as mobile phases in isocratic condition. The instrument was set in the positive ion electrospray mode using the following parameters: cone voltage 40 V, capillary voltage 3.80 kv, source temperature 350 C, desolvation temperature 270 C and collision gas energy 5 ev. Multiple reaction monitoring (MRM) technique was used for identification and quantification, in which protonated molecule [M þ H]þ of the BEA m/z 784.50, was fragmented in the collision cell to the product-ion m/z 244.20. For the quantification, we used the product-ions m/z 244.20 (Jestoi, 2008). The samples corresponding to the model solution experiments and to the crispy bread production were also injected in the modality full scan with the Enhanced Resolution (ER) scan procedure: utilizing the mass range from 200 to 1500 Da, to obtain the general spectra of the molecule and the principally fragments related. The utilization of the mass spectrometry associated at the detection with the linear ion trap, utilized in this modality permitted us to obtain a total characterization of the compound isolated (Meca, Luciano, Zhou, Tsao, & Mañes, 2012). 2.8. Calculation and statistical analysis All experiments were performed three times. Statistical analysis was carried out using analysis of variance (ANOVA), followed by Dunnet s multiple comparison tests. Differences were considered significant if p < 0.05. 3. Results 3.1. Degradation of BEA during beer making Fig. 4, shows the LC-MS chromatogram related to the presence of BEA and its relative mass spectrum beer made with low toasting process and fermentation with S. cerevisiae A34. During beer making as shown in Fig. 5, two important steps ware responsible for BEA degradation: the toasting and the fermentation. The low toasting process resulted in a mean BEA degradation ranged from 23 to 26%, whereas employing the medium toasting process the degradation ranged from 33 to 38%. The other steps in the beer making, like the boiling of the grounded malt, the enzymatic hydrolysis of the starch and the boiling related to the hops adding do not produce a significant reduction of the BEA. The step in the beer making process that influenced significantly the level of BEA was the fermentation. In particular, in the fermentation of the boiled malt carried out with S. cerevisiae A34, BEA reduced by 72 and 75%, BEA degradation ranged by, whereas using the strain of S. cerevisiae LO9 the degradation evidenced ranged from 76 to 79%. The highest BEA degradation data was evidenced in the experiment carried out with the strain of bread yeast, with a degradation of BEA ranged from 78 to 82%. The BEA degradation evidenced during the fermentation step was from 1.5 to 2 fold higher than the degradation evidenced in the other points of production. 3.2. Degradation of BEA during bread production During bread production the degradation of BEA was evidenced during the fermentation process as well and with the cumulative process of the fermentation þ cooking (Fig. 6). During the fermentation step the strain that produced the highest reduction of the BEA was the S. cerevisiae A34 with 82%, whereas for the other

4 G. Meca et al. / Food Control 34 (2013) 1e8 Fig. 4. a) LC-MS chromatogram and b) mass spectra of mycotoxin BEA presents in beer made with low toasting process after the fermentation with Saccharomyces cerevisiae A34. two strains the reduction evidenced ranged from 75 to 76%. Adding the cooking process to the fermentation process the percentage of BEA degradation in homemade breads ranged from 92 to 95%. 3.3. Identification of degradation products During the heat treatment related to the malt toasting and bread cooking and also during the fermentation of beer and bread two BEA degradation products were identified as evidenced in Figs. 7 and 8. In particular in Fig. 7a, is evidenced the LC-MS chromatogram of the BEA present in toasted malt samples treated at the temperature of 100 C during one day incubation. After the BEA peak is possible to observe a peak of another compound characterized as a BEA degradation product. Fig. 7b shows three main fragments that characterize the structure of this degradation product. In particular the fragment with a m/z of 485.4 represents the BEA with a loss of a two structural components of the mycotoxin, that are the phenylalanine (Phe) and the hydroxyvaleric acid (HyLv) (BEA-(Phe þ HyLv) þ ). The loss of these two compounds was confirmed by the fragment with a m/z of 281.5 that correspond to the Phe þ HyLv unit. Another important fragment that confirms the structure of the degradation BEA compound was the fragment with a m/z of 207.1, that represents the BEA with the loss of two (Phe þ HyLv) þ units. The thermic degradation product of the mycotoxin BEA founded and also the mass fragments detected in this study were the same evidenced by Meca, Ritieni et al. (2012). Fig. 8a, shows the LC-MS chromatogram of BEA presents in fermented dough treated with the strain of A34. After BEA peak is

G. Meca et al. / Food Control 34 (2013) 1e8 5 % BEA degradation % BEA degradation % BEA degradation 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 a) b) c) T B E H F T B E H F T B E H F Fig. 5. Degradation of mycotoxin BEA in beer making with three year strains a) Saccharomyces cerevisiae A34, b) S. cerevisiae LO9 and c) S. cerevisiae QRI. Barley was treated with medium toasting (white column) and low toasting (black column). The letters are major steps in the beer making: T, malt toasting; B, malt boiling; E, enzymatic hydrolysis; H, hoops adding; F, yeast fermentation. possible to evidence another compound characterized as a BEA degradation product of fermentative origin as demonstrated in the mass spectra evidenced in Fig. 8b. The figure shows the ER spectrum of this product, where are present several diagnostic fragments that confirm the degradation of BEA. In particular the signal at m/z 573.3 correspond to the BEA with the loss of N-Phenylalanine (N-Phe) that is an important structural amino acid of this compound more two molecules of water. The formation of this new product is confirmed by the % BEA degradation 120.00 100.00 80.00 60.00 40.00 20.00 0.00 F=fermentation F+C=ferm+cook A34 LO9 Br Yeast Fig. 6. Degradation of mycotoxin BEA during bread making with Saccharomyces cerevisiae A34, S. cerevisiae LO9 and S. cerevisiae QRI, respectively. fragment with a m/z of 471.4 that corresponds to the ion with m/z 573.3 with the loss of another structural compound of BEA that is the hydroxyvaleric acid (HyLv). Another important fragment is the signal with m/z 429.4 that represents the ion with m/z 573.3 with the loss of a structural compound of BEA represented by a N-Phe. 4. Discussion In the scientific literature only a study was published on the thermic degradation of the mycotoxin BEA, whereas the influence of the technological process on the degradation of other fusarotoxins has been studied by many researchers. In particular Meca, Ritieni et al. (2012) investigated the degradation of the minor Fusarium mycotoxin BEA present in the concentration of 5 mg/kg in a model solution and in different crispy breads produced with different flours typologies (corn, hole, wheat, durum wheat, soy and rice) during the heat treatment carried out in an oven at three different temperatures of 160, 180 and 200 C and at 3, 6, 10, 15 and 20 min incubation. The concentration of the bioactive compound studied, decreased in the experiment carried out in the model solution from 2.8 mg/kg of the assay at 160 C for 3 min until the complete degradation at 200 C during 20 min incubation. In the experiments carried out using the crispy breads prepared with different kind of flours, as system to simulate a food preparation, the percentage of BEA degradation, was variable from 20 to 90%, with no significant difference showed in the use of the different flour matrices. Wu, Lohrey, Cramer, Yuan, and Hans-Ulrich Humpf (2011), studied the optimal condition for deoxynivalenol (DON) reduction during extrusion, the effects of a range of physicochemical parameters such as temperature, moisture, compression, residence time, ph value, and protein content on the reduction of DON levels in wheat. Considering that the fate of DON during extrusion cooking was not really clear, with the data produced by the authors, the use of Good Agricultural and Manufacturing Practices (GAP/GMP) on the field to reduce DON levels is strongly recommended as the first approach to control this contaminant in cereal products. Janotová, Cízková, Pivonka, and Voldrich (2011) studied the patulin (PAT) degradation during the processing of apple puree under the conditions close to the real production process. The apple samples were spiked with PAT at four levels of concentration (539 mg/kg, 140 mg/kg, 23 mg/kg and <2 mg/kg). The PAT content changes during the processing were followed. The samples were taken after the homogenization, pulping, pasteurization and aseptic packaging. All operations of apple puree production contributed to PAT reduction. The principle operation was pulping, where the PAT levels were reduced from 29% to 80% of original content. Beyer, Ferse, Mulac, Wu, and Humpf (2009) studied the stability of T-2 toxin under the conditions of baking or cooking using heating experiments with the model substances R-D-glucose, R-D-methylglucopyranosid, N-R-acetyl-L-lysine methyl ester, and N-R-acetylcysteine methyl ester. The reaction residue was screened for degradation products using gas chromatographyemass spectrometry (GCeMS) and high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). Although T-2 toxin was degraded under all conditions, only heating of T-2 toxin with R-D-glucose produced a mixture of three degradation products, which were isolated and identified by MS and nuclear magnetic resonance (NMR) experiments. Bretz et al. (2006) studied the stability of DON under foodprocessing conditions such as cooking or baking, and also the metabolites produced during the heat treatments. The results obtained by the authors evidenced that DON and 3- acetyldeoxynivalenol (3-AcDON), were transformed in a mixture

6 G. Meca et al. / Food Control 34 (2013) 1e8 Fig. 7. LC-MS chromatogram a) of the minor Fusarium mycotoxin BEA present in toasted malt samples treated at the temperature of 100 C during one day incubation. After BEA peak is possible to evidence another compound characterized as a BEA degradation product. b) Mass spectra (m/z 200e1500) of the BEA degradation product produced during the heat treatments. In the mass spectra some diagnostic fragments confirm the identification of the degradation product analyzed. of compounds known as nordon A, nordon B, and nordon C, while four new compounds were identified and named 9- hydroxymethyl DON lactone, nordon D, nor-don E, and nordon F. DON degradation products were determined by the authors in commercially available food samples. In particular nordon A, B, and C were detected in 29 of 66 of the samples in mean concentrations ranging from 3 to 15 mg/kg. Bullerman and Bianchini (2007) evaluated that the various food processes that may have effects on mycotoxins include sorting, trimming, cleaning, milling, brewing, cooking, baking, frying, roasting, canning, flaking, alkaline cooking, nixtamalization, and extrusion. Most of the food processes have variable effects on mycotoxins, with those that utilize the highest temperatures having greatest effects. In general the processes reduce mycotoxin

G. Meca et al. / Food Control 34 (2013) 1e8 7 Fig. 8. LC-MS chromatogram a) of the minor Fusarium mycotoxin BEA present in fermented dough treated with the strain of A34. After BEA peak is possible to evidence another compound characterized as a BEA degradation product of fermentative origin and b) mass spectra (m/z 200e900) of the BEA degradation product produced during the dough fermentation. concentrations significantly, but do not eliminate them completely. However, roasting and extrusion processing show promise for lowering mycotoxin concentrations, though very high temperatures are needed to bring about much of a reduction in mycotoxin concentrations. Extrusions processing at temperatures greater than 150 C are needed to give good reduction of zearalenone (ZEA), moderate reduction of aflatoxins (AFs), variable to low reduction of DON and good reduction of fumonisins (FBs). The greatest

8 G. Meca et al. / Food Control 34 (2013) 1e8 reductions of FBs occur at extrusion temperatures of 160 Cor higher and in the presence of glucose. Extrusion of FBs contaminated corn grits with 10% added glucose resulted in 75e85% reduction in FB 1 levels. Some FBs degradation products are formed during extrusion, including small amounts of hydrolyzed FB 1 and N-(Carboxymethyl)-FB 1 and somewhat higher amounts of N-(1- deoxy-d-fructos-1-yl) FB 1 in extruded grits containing added glucose. Feeding trial toxicity tests in rats with extruded FBs contaminated corn grits show some reduction in toxicity of grits extruded with glucose. Cenkowski, Pronyk, Zmidzinska, and Muir (2007) studied the effects of superheated steam (SS) as a processing medium on grains contaminated with the Fusarium mycotoxin DON. The temperature used by the authors ranged between 110 and 185 C. Reductions in DON concentration of up to 52% were achieved at 185 C and 6 min processing time. In particular Cramer et al. (2008) determined the decrease of the ochratoxin A (OTA) in naturally contaminated green coffee, roasted under various conditions. The roasting conditions were kept within the range of commercial practice and varied from 2.5 to 10 min, and the roast color varied from light medium to dark. The authors evidenced at the end of the heat treatments used, a mean OTA degradation of 69%, with a range of variation variable from 50 to 96%. Meca, Fernandez-Franzon et al. (2010) evaluated as the FB 1 content in corn products can be transformed, during the heating process in foods containing reducing sugars, in the Maillard reaction product N-(carboxymethyl)-FB 1. In this study operated by the authors a rapid method was developed for the determination of both compounds in corn products using a high-speed blender, UltraTurrax, for solvent extraction and the liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination. The kinetics of FB 1 degradation and the formation of the Maillard adduct were studied in a model system constituted by corn bread spiked with 1 mg/kg of FB 1, heated in an oven at 160, 180, and 200 C during 3, 6, 10, 15, and 20 min. The data evidenced by the authors demonstrated that during the treatments operated, the FB 1 decreased from 0.96 to 0.30 mg/kg and N-(carboxymethyl)-FB 1 increased until to 0.1 mg/kg. 5. Conclusions This research was supported by the Ministry of Science and Innovation (AGL2010 17024), the Developing Innovative Agricultural Products Program, Agriculture & Agri Food Canada (Project ID: AGR-0479) and by the pre-phd program of University of Valencia Cinc Segles. References Beyer, M., Ferse, I., Mulac, D., Wu, E. U., & Humpf, H. U. (2009). Structural elucidation of T-2 toxin thermal degradation products and investigations toward their occurrence in retail food. Journal of Agricultural and Food Chemistry, 57, 1867e 1875. 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