Distribution of deoxynivalenol and zearalenone in milled germ during wheat milling and analysis of toxin levels in wheat germ and wheat germ oil

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<ul><li><p>lels</p><p>E</p><p>n</p><p>lvenas</p><p>oils contained DON at mean values of 111 and 41 mg/kg, respectively, while none of germ samples and16% oils contained ZEA (mean 6 mg/kg). Contamination levels lead to a daily intake of 1.3 mg DON and</p><p>e world</p><p>tissue structures within the wheat kernel acted as an effective bar-rier to fungal invasion and the subsequent synthesis of mycotoxins.</p><p>Several studies have been carried out to determine the stabilityand partitioning of DON and ZEA during wheat milling. Kushiro(2008) reviewed 19 published papers and concluded that wheat</p><p>and ZEA inmilledthe occurrence oft germ and wheatarce (Kappensteinllenberger, Mller,high levels of ZEAg) and wheat germake an important</p><p>s required for boththe assessment of</p><p>human exposure. Most analytical methods proposed in recent yearsfor the determination of DON and ZEA in foods have been devel-oped, primarily for solid samples (Bao, Oles, White, Sapp, &amp;Trucksess, 2011; Shephard et al. 2010). Analysis of wheat germand derived oil presents entirely different commodities that couldpotentially complicate extraction and cleanup prior to determina-tion because of the presence of fat (Mahoney &amp; Molyneux, 2010).</p><p>Therefore, the objectives of the present work were: (i) todevelop a method for the determination of DON and ZEA in fatty</p><p>* Corresponding author. Tel.: 34 876 554131; fax: 34 976 761612.</p><p>Contents lists available at</p><p>Food Co</p><p>lse</p><p>Food Control 34 (2013) 268e273E-mail address: aarino@unizar.es (A. Ario).germ is a high fat by-product of milling with great nutritional valuefor the content of a-tocopherol (vitamin E). Both wheat germ andwheat germ oil can be marketed for direct human consumption asdietary supplements, and they are attractive and promising sourcesof vegetable functional compounds (Rizzello, Cassone, Coda, &amp;Gobbetti, 2011). It is well documented that wheat grains and mill-ing productsmaycontainmycotoxins such as deoxynivalenol (DON)and zearalenone (ZEA) (FAO/WHO, 2000, 2011; EFSA, 2004, 2011;SCOOP, 2003). Pinson-Gadais et al. (2007) detected the occurrenceof toxigenic Fusarium spp. by PCR assay in all wheat tissues (germ,pericarp, aleurone layer, and albumen), concluding that none of the</p><p>on the effects ofmilling on the distribution of DONwheat germ. On the other hand, information onthese Fusarium toxins in products such as wheagerm oil for direct human consumption is very scet al., 2005; Schollenberger et al., 2005; SchoRe, &amp; Drochner, 2008). Nevertheless, notablyhave been found in corn germ oil (up to 823 mg/koil (up to 150 mg/kg), and these products could mcontribution to the ZEA exposure (EFSA, 2011).</p><p>Determination of DON and ZEA in foodstuffs ithe control of current legislative compliance andthe procedure by which wheat grains are ground and their com-ponents separated into milled fractions based on particle size. The</p><p>of ZEA are found in the bran fraction than in the originalwheat grainwith lower concentrations in white our. However, little is knownKeywords:DeoxynivalenolZearalenoneMillingWheat germWheat germ oil</p><p>1. Introduction</p><p>Most of the wheat harvested in th0956-7135/$ e see front matter 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.04.0330.03 mg ZEA, representing 1.9% and 0.23% of their respective tolerable daily intakes (TDI). 2013 Elsevier Ltd. All rights reserved.</p><p>is subjected tomilling,</p><p>milling redistributes DON, with the highest amounts appearing inthe bran and the lowest in the our. Similarly, Trigo-Stockly, Deyoe,Satumbaga, &amp; Pedersen (1996) revealed that higher concentrations15 April 2013Accepted 27 April 2013samples of germ-based dietary supplements revealed that 60% of wheat germ and 40% of wheat germReceived in revised form in wheat germ and wheat germ oil. A total of 36 batches of grain wheat were subjected to industrialmilling and the distribution factors in milled germ were 47% for DON and 71% for ZEA. A survey of 50Distribution of deoxynivalenol and zearawheat milling and analysis of toxin levegerm oil</p><p>Isabel Gimnez, Marta Herrera, Jacqueline Escobar,Antonio Herrera, Agustn Ario*</p><p>Veterinary Faculty, University of Zaragoza, c/Miguel Servet 177, E-50013 Zaragoza, Spai</p><p>a r t i c l e i n f o</p><p>Article history:Received 1 December 2012</p><p>a b s t r a c t</p><p>A method consisting of soand HPLC determination w</p><p>journal homepage: www.eAll rights reserved.none in milled germ duringin wheat germ and wheat</p><p>lena Ferruz, Susana Lorn,</p><p>t extraction using hexane for defatting, multifunctional cleanup column,validated for the analysis of deoxynivalenol (DON) and zearalenone (ZEA)</p><p>SciVerse ScienceDirect</p><p>ntrol</p><p>vier .com/locate/ foodcont</p></li><li><p>wheat germ and wheat germ oil, (ii) to study the effect of drymilling on the distribution of DON and ZEA in milled wheat germ,and (iii) to examine the occurrence and concentration levels of DONand ZEA in wheat germ-based dietary supplements for direct hu-man consumption such as wheat germ and wheat germ oil.</p><p>2. Materials and methods</p><p>2.1. Sample collection</p><p>The milling study was carried out in an industrial milling plantlocated in Aragn (NE Spain) able to process 140 tons/day of wheatgrain. For this study, 36 different batches of wheat grain of harvestyears 2009 and 2010, coming from Spain, France, Germany, and theUSAwere milled in different weeks. The wheat grains were cleanedand tempered as usual practice, and the germ fraction was sepa-rated during the milling process by rolling and sieving with a totalgerm yield of 0.5%. Five incremental samples of 400 g werecollected from different places of each lot of cleaned wheatresulting in an aggregate sample of 2 kg, together with the corre-sponding samples of germ fractions taken and aggregated in thesame manner. For mycotoxin analysis, these samples were groundin aMahlknig EG-43mill (Hamburg, Germany), thoroughlymixed,and kept at 21 C until analysis.</p><p>Commercial samples of dietary supplements for direct humanconsumption (wheat germ and wheat germ oil) were all purchasedfrom a number of local retail outlets including grocery stores, drugstores, general merchandise retailers, natural food stores and spe-cialty health and nutrition stores. Wheat germ was packaged inbags of 250e500 g and wheat germ oil was supplied in bottles of250 mL. Samples of wheat germ and wheat germ oil were thor-</p><p>2.2. Reagents and apparatus for mycotoxin analysis</p><p>HPLC grade acetonitrile, methanol and n-hexane were pur-chased from Lab-Scan (Dublin, Ireland) and HPLC grade glacialacetic and formic acids from Merck (Darmstadt, Germany). Ultra-pure water was obtained from a Milli-Q Plus apparatus from Mil-lipore (Milford, MA). The multifunctional cleanup columnsMycosep #224 and #225 were supplied by Romer Labs (Union,MO). DON and ZEA standard solutions at 100 mg/mL in acetonitrilewere provided by Sigma (St. Louis, MO) and stored at 21 C.</p><p>The LC system consisted of an Agilent Technologies (Santa Clara,CA) 1100 high performance liquid chromatograph coupled to anAgilent diode-array detector (DAD) at 220 nm for the determinationof DON, and an Agilent uorescence detector (FLD) at 274 mm(excitation)/440 nm (emission) for the determination of ZEA. The LCcolumnwas Ace 5 C18, 250 4.6 mm, 5 mm particle size (AdvancedChromatography Technologies, Aberdeen, United Kingdom). ForDON analysis the mobile phase was water/acetonitrile/methanol(90:5:5, v/v/v) at a ow rate of 1.0 mL/minwith an injection volumeof 100 mL. For the analysis of ZEA the mobile phase was water/acetonitrile/methanol (46:46:8, v/v/v) pumped at a ow rate of0.8 mL/min, and the injection volume was 20 mL. Figs. 1 and 2 showchromatograms of standards and contaminated samples of wheatgerm and wheat germ oil.</p><p>2.3. Analysis of mycotoxins in wheat, wheat germ and wheat germoil</p><p>For the determination of DON and ZEA in wheat grain, a vali-datedmethod based on Mycosep columns and HPLC determination</p><p>I. Gimnez et al. / Food Control 34 (2013) 268e273 269oughly mixed before analysis.Fig. 1. LC-DAD chromatograms of a DON standard solution (500 mg/L)was used (Sugita-Konsihi et al. 2006). Briey, 5 g of homogenizedand a wheat germ sample contaminated with DON (294 mg/kg).</p></li><li><p>ContI. Gimnez et al. / Food270wheat was extracted with 20 mL acetonitrile and water (84:16, v/v)using an Ultraturrax homogenizer for 3 min, ltered with What-man #4 lter paper, and the extract collected for further cleanup asdescribed below. Our contribution to the method involved adefatting step for the determination of the toxins in wheat germand wheat germ oil. For this, 5 g was extracted with a mixture of20mL acetonitrile andwater (84:16, v/v) combined with 12.5mL n-hexane for defatting using the Ultraturrax homogenizer for 3 min,and then centrifuged at 3500 rpm for 15 min. The supernatant(hexane layer) was discarded, and the remaining aqueous extractwas ltered with Whatman #4 lter paper and collected forcleanup.</p><p>The resulting ltered extracts for all test commodities were splitinto two culture tubes by pipetting 5e7 mL each. For DON analysis,a Mycosep #225 multifunctional cleanup column was slowlypushed (rubber ange end) into one culture tube containing theextract. For ZEA analysis, the extract was acidied with 50 mL glacialacetic acid and pushed all through a Mycosep #224multifunctionalcleanup column. Two mL puried extracts were transferred into4mL vials, evaporated to dryness at 50 C in a heating block under agentle stream of nitrogen, and redissolved in 400 mL mobile phase.Mycosep columns allowed a one-step cleanup within 30 s withoutthe use of any solvent for elution: the columnwas pushed into a testtube containing the sample extract, forcing the extract to lterupwards through the packing material of the column. The in-terferences adhere to the chemical packing in the column and thepuried extract, containing the analytes of interest, passes throughthe column.</p><p>Based on European Commission Regulation (EC) No. 401/2006laying down the methods of sampling and analysis for the ofcialcontrol of the levels of mycotoxins in foodstuffs, the analytical</p><p>Fig. 2. LC-FLD chromatograms of a ZEA standard solution (50 mg/L) arol 34 (2013) 268e273procedures of the present work were validated in-house in terms ofrecovery, precision, and sensitivity. Spiking procedure was done insextuplicate by adding appropriate amounts of standards to thesample matrices: DON at 1250 mg/kg (wheat), 750 mg/kg (wheatgerm), and 200 mg/kg (wheat germ oil), and with ZEA at 100 mg/kg(wheat), 75 mg/kg (wheat germ), and 30 mg/kg (wheat germ oil).Repeatability was carried out with blank matrices spiked at 0.5, 1.0,and 1.5 times their respective maximum levels established inCommission Regulation (EC) No. 1881/2006. The limits of detection(LOD) and quantication (LOQ) were based onminimum amount oftarget analytes that produced a chromatogram peak with a signal-to-noise ratio of 3 and 10 times the background chromatographicnoise, respectively. The quality of results was assured by partici-pating in the prociency testing Progetto Trieste 2010 for myco-toxins between 13 laboratories from different countries.</p><p>2.4. Conrmation of mycotoxins</p><p>Conrmatory analysis was performed using an Acquity UPLCsystem coupled to a Quattro Premier XE triple quadrupole massspectrometer (Waters, Milford, MA). The LC separation was per-formed using a Waters Acquity UPLC BEH C18 analytical column(2.1 50 mm, 1.7 mm particle size) kept at 40 C in a column oven.Mobile phase was a time programmed gradient using A (water,formic acid 0.1%) and B (methanol, formic acid 0.1%) at a ow rate of0.3 mL/min, with injection volume of 20 mL. The mass spectrometerwas operated in the positive electrospray ionizationmode (ESI) forDON,while for ZEAnegative electrospray ionizationmode (ESI-)wasused. The MS/MS parameters included the following settings: ESIsource block temperature 120 C, desolvation temperature 400 C,capillary voltage 3.5 kV, and argon collision gas 3.5103mbar. The</p><p>nd a wheat germ oil sample contaminated with ZEA (44 mg/kg).</p></li><li><p>operating conditions are described in Table 1. MassLynx softwareversion 4.1 was used for data acquisition and processing.</p><p>2.5. Statistical analyses</p><p>The results from mycotoxin analyses were subjected todescriptive and comparative statistics according to Sachs (1978).</p><p>I. Gimnez et al. / Food ContThe incidence of batches and samples containing DON and ZEA (%positives) were expressed as the percentage of samples containinglevels above the corresponding limit of detection (LOD). For eachmycotoxin, the mean and standard deviation (SD) were calculatedusing LOD/2 for results lower than LOD. Calculations were per-formed on StatView SE Graphics (Abacus Concepts, Berkeley, CA)for Macintosh personal computers.</p><p>3. Results and discussion</p><p>3.1. Method validation</p><p>Numerousmethods for analysis ofmycotoxins in foodstuffs havebeen developed, primarily for solid samples. Analysis in fattycommodities and oils presents entirely different matrixes thatcould potentially complicate extraction and cleanup of samplesprior to determination due to their fat content (Mahoney &amp;Molyneux, 2010). Thus, the crucial step in the analytical proce-dure for germ and oil was anticipated to be extraction and cleanupto retain mycotoxins but eliminate as much fat as possible prior toHPLC separation. In the method applied, the fat is separated duringextraction with a nonpolar solvent such as hexane. For furthercleanup, liquid partitioning was discarded because it is complicatedand time and solvent consuming, and so were immunoafnitycleanup columns (IAC) because they are expensive, though IAC canbe preferable in most situations for the cleanup of mycotoxins inagricultural products. We therefore sought commercial availableproducts such as multifunctional Mycosep columns, which aresimple, fast, and consistent in quality and performance for thecleanup step in the analysis of mycotoxins (Bao et al., 2011). Basedon our laboratory experience, the Mycosep #224 and #225 (RomerLabs, Union, MO) were tested as these columns are designed toretain interferences and elute mycotoxins in a very simple mannerwith no wash or elution solvents, as described in subheading 2.3.</p><p>The analytical method for wheat grain provided good recoveriesfor DON and ZEA of 98 9% and 99 16%, respectively, and thelimit of detection (LOD) for DON was 33 mg/kg and for ZEA was10 mg/kg. For the analysis of DON and ZEA in fatty wheat germ andwheat germ oil, the extraction efciency was tested with a mixtureof acetonitrile: water (84:16, v/v) as extraction solvent combinedwith the addition of a hexane defatting step, and the most optimalefciency was calculated by applying 20 mL extraction solvent and12.5 mL of n-hexane. The mean recoveries for DON in wheat germand wheat germ oil were 92 and 106%, respectively, and recoveriesfor ZEA amounted to 98 and 104%, respectively. Therefore, thoughZEA is slightly soluble in hexane, there were no noticeable losses ofthis mycotoxin in the hexane layer. An additional advantage of thedefatting step was the obtaining of cleaner extracts that allowedincreased sensitivity and lower limits of detection (LODs). Thus,</p><p>Table 1...</p></li></ul>

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