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Food Control 34 (2013) 268e273

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Food Control

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

Isabel Giménez, Marta Herrera, Jacqueline Escobar, Elena Ferruz, Susana Lorán,Antonio Herrera, Agustín Ariño*

Veterinary Faculty, University of Zaragoza, c/Miguel Servet 177, E-50013 Zaragoza, Spain

a r t i c l e i n f o

Article history:Received 1 December 2012Received in revised form15 April 2013Accepted 27 April 2013

Keywords:DeoxynivalenolZearalenoneMillingWheat germWheat germ oil

* Corresponding author. Tel.: þ34 876 554131; fax:E-mail address: aarino@unizar.es (A. Ariño).

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.04.033

a b s t r a c t

A method consisting of solvent extraction using hexane for defatting, multifunctional cleanup column,and HPLC determination was validated for the analysis of deoxynivalenol (DON) and zearalenone (ZEA)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 50samples of germ-based dietary supplements revealed that 60% of wheat germ and 40% of wheat germoils 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 and0.03 mg ZEA, representing 1.9% and 0.23% of their respective tolerable daily intakes (TDI).

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1. Introduction

Most of the wheat harvested in the world is subjected tomilling,the procedure by which wheat grains are ground and their com-ponents separated into milled fractions based on particle size. Thegerm 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, &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 thetissue structures within the wheat kernel acted as an effective bar-rier to fungal invasion and the subsequent synthesis of mycotoxins.

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

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milling redistributes DON, with the highest amounts appearing inthe bran and the lowest in the flour. Similarly, Trigo-Stockly, Deyoe,Satumbaga, & Pedersen (1996) revealed that higher concentrationsof ZEA are found in the bran fraction than in the originalwheat grainwith lower concentrations in white flour. However, little is knownon the effects ofmilling on the distribution of DONand ZEA inmilledwheat germ. On the other hand, information on the occurrence ofthese Fusarium toxins in products such as wheat germ and wheatgerm oil for direct human consumption is very scarce (Kappensteinet al., 2005; Schollenberger et al., 2005; Schollenberger, Müller,Rüfle, & Drochner, 2008). Nevertheless, notably high levels of ZEAhave been found in corn germ oil (up to 823 mg/kg) and wheat germoil (up to 150 mg/kg), and these products could make an importantcontribution to the ZEA exposure (EFSA, 2011).

Determination of DON and ZEA in foodstuffs is required for boththe control of current legislative compliance and the assessment ofhuman 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, &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 & Molyneux, 2010).

Therefore, the objectives of the present work were: (i) todevelop a method for the determination of DON and ZEA in fatty

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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.

2. Materials and methods

2.1. Sample collection

The milling study was carried out in an industrial milling plantlocated in Aragón (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 aMahlkönig EG-43mill (Hamburg, Germany), thoroughlymixed,and kept at �21 �C until analysis.

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-oughly mixed before analysis.

Fig. 1. LC-DAD chromatograms of a DON standard solution (500 mg/L)

2.2. Reagents and apparatus for mycotoxin analysis

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.

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 fluorescence 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 flow 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 flow 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.

2.3. Analysis of mycotoxins in wheat, wheat germ and wheat germoil

For the determination of DON and ZEA in wheat grain, a vali-datedmethod based on Mycosep columns and HPLC determinationwas used (Sugita-Konsihi et al. 2006). Briefly, 5 g of homogenized

and a wheat germ sample contaminated with DON (294 mg/kg).

Fig. 2. LC-FLD chromatograms of a ZEA standard solution (50 mg/L) and a wheat germ oil sample contaminated with ZEA (44 mg/kg).

I. Giménez et al. / Food Control 34 (2013) 268e273270

wheat was extracted with 20 mL acetonitrile and water (84:16, v/v)using an Ultraturrax homogenizer for 3 min, filtered with What-man #4 filter 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 filtered with Whatman #4 filter paper and collected forcleanup.

The resulting filtered 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 flange end) into one culture tube containing theextract. For ZEA analysis, the extract was acidified with 50 mL glacialacetic acid and pushed all through a Mycosep #224multifunctionalcleanup column. Two mL purified 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 filterupwards through the packing material of the column. The in-terferences adhere to the chemical packing in the column and thepurified extract, containing the analytes of interest, passes throughthe column.

Based on European Commission Regulation (EC) No. 401/2006laying down the methods of sampling and analysis for the officialcontrol of the levels of mycotoxins in foodstuffs, the analytical

procedures 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 quantification (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 proficiency testing Progetto Trieste 2010 for myco-toxins between 13 laboratories from different countries.

2.4. Confirmation of mycotoxins

Confirmatory 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 flow rate of0.3 mL/min, with injection volume of 20 mL. The mass spectrometerwas operated in the positive electrospray ionizationmode (ESIþ) 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 3.5�10�3mbar. The

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operating conditions are described in Table 1. MassLynx softwareversion 4.1 was used for data acquisition and processing.

2.5. Statistical analyses

The results from mycotoxin analyses were subjected todescriptive and comparative statistics according to Sachs (1978).The 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 þ Graphics (Abacus Concepts, Berkeley, CA)for Macintosh personal computers.

3. Results and discussion

3.1. Method validation

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 &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 immunoaffinitycleanup 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.

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 efficiency 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 optimalefficiency 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,

Table 1Optimized MS/MS conditions for mycotoxin confirmation.

Mycotoxin Precursorion

Cone (V) Collision energy(eV)

Product ion(m/z)

Deoxynivalenol 297.3 24 13 231.1Deoxynivalenol 297.3 24 10 249.1Zearalenone 317.2 60 30 131.1Zearalenone 317.2 60 15 175.1

LODs for DON and ZEA in wheat germ and wheat germ oil were 22and 8 mg/kg, respectively, as compared to 33 mg/kg and 10 mg/kgobtained with the matrix wheat.

Bao et al. (2011), using HPLC-DAD for the determination of DONinwheat germ reported a LOD about 100 mg/kg, while Schollenbergeret al. (2008) achieved a LOD of 7 mg/kg using GCeMS with derivati-zation. A LOD for ZEA of 10 mg/kg in maize germ oil was reported byMajerus, Graft, & Krämer (2009) using HPLC-FLD. A deffating stepwith hexane has been also recently described for the simultaneousdetermination of DON, ZEA, T-2-toxin and some masked metabolitesin maize, wheat, oats, cornflakes and bread with very good perfor-mance (De Boevre et al., 2012).

Finally, our study of intra-day precision in terms of repeatability,obtained RSDr values lower than 15% for both mycotoxins in alltested matrixes, in accordance with the validation criteria.Accordingly, the present study has led to the successful develop-ment of a simple, reliable method for the determination of DON andZEA inwheat germ and derived edible oil. Furthermore, the hexanedefatting step allowed the sensitivity to be increased, resulting inrelatively cleaner extracts. The method, which was validated ac-cording to European Commission criteria, could provide a basis foran intra- and interlaboratory-validated analytical method foranalysis of mycotoxins in dietary supplements and other productsformulated with germ and germ-derived oils.

3.2. DON and ZEA in the germ fraction after milling of wheat grains

DON was detected in all 36 batches of cleaned wheat grainbeforemilling (100% positives), with amean concentration (�SD) of251�144 mg/kg and amaximum of 820 mg/kg. In the correspondingmilled germ fractions, DON was detected in 34 out of 36 samples(94.4%), with a mean concentration of 117 � 61 mg/kg and amaximum of 290 mg/kg. All samples were below their respectivemaximum permitted levels for DON of 1250 mg/kg for wheat and750 mg/kg for germ (Commission Regulation (EC) no 1881/2006). Asregards of ZEA, only one batch of cleaned wheat grain (2.8% posi-tives) contained ZEA at detectable levels of 14 mg/kg, and the ZEAconcentration in the corresponding milled germ was 10 mg/kg,down their respective maximum permitted levels of 100 mg/kg forwheat and 75 mg/kg for germ. However, it is rather difficult tocompletely guarantee that starting material and milled productsare totally representative when sampling big batches in industrialmilling processes, and therefore, percents of distribution reportedshould be considered with caution. The distribution factor isdefined as the ratio between the mycotoxin content in the milledgerm fraction and the mycotoxin content in the wholegrain � 100.Then, the distribution factor for DON after commercial milling was47%, indicating that the DON concentration in milled germ is nearlyone half of the original toxin concentration in the wholegrain.Therefore, the germ is not a significant source of DON and the usualmilling practice is enough to deliver a commercially safe product.The distribution factor for ZEAwas 71%, indicating that ZEA showedsomewhat higher affinity for germ than DON during the commer-cial milling of wheat. However, there were too few data and too lowlevels at which ZEA occurred to allow confirmation of the situationwith the germ fraction.

Previous research has mainly focused on the effect of wheatmilling on the fate of DON and ZEA in flour and bran fractions, whilevery little information is available in the scientific literature on thefractionation of these toxins in milled wheat germ. As reported byAbbas, Mirocha, Pawlosky, & Pusch (1985) and Trigo-Stockly et al.(1996), when present in milled fractions of wheat, the levels ofDON and ZEA were generally highest in the bran and lowest in themilled flour. Similarly, Herrera, Juan, Estopañan, & Ariño (2009)indicated a significant effect of type of fraction on the distribution

Table 2Occurrence of DON and ZEA in samples of wheat germ and wheat germ oil. Resultsexpressed in mg/kg.

Sample number Wheat germ (n ¼ 25) Wheat germ oil (n ¼ 25)

DON ZEA DON ZEA

1 150 <LOD <LOD <LOD2 93 <LOD <LOD <LOD3 188 <LOD <LOD <LOD4 <LODa <LOD <LOD <LOD5 <LOD <LOD <LOD <LOD6 <LOD <LOD <LOD <LOD7 191 <LOD <LOD 98 <LOD <LOD <LOD <LOD9 179 <LOD 28 <LOD10 <LOD <LOD 56 <LOD11 186 <LOD <LOD <LOD12 193 <LOD <LOD <LOD13 <LOD <LOD 45 <LOD14 <LOD <LOD <LOD <LOD15 <LOD <LOD <LOD <LOD16 <LOD <LOD <LOD <LOD17 151 <LOD <LOD <LOD18 150 <LOD 99 4419 <LOD <LOD 130 <LOD20 200 <LOD 58 <LOD21 238 <LOD 96 822 150 <LOD 115 1223 153 <LOD <LOD <LOD24 240 <LOD 79 <LOD25 191 <LOD 163 <LOD% Positives 60% e 40% 16%Total mean 111 mg/kg e 41 mg/kg 6 mg/kgStd. deviation 88 mg/kg e 46 mg/kg 8 mg/kg

a Limit of detection of 22 mg/kg for DON and 8 mg/kg for ZEA in both wheat germand wheat germ oil.

I. Giménez et al. / Food Control 34 (2013) 268e273272

of DON in milled products of durum wheat, as the distributionfactors for DON after milling were 153% for bran, 87% for durumsemolina, and 108% for flour. Pascale et al. (2011) reported that themilling process of durum wheat led to an increase of T-2 and HT-2toxin contents up to 5-fold in bran as compared with the uncleanedwheat, while observed an overall reduction by 54% in cleanedwheat and by 89% in semolina, respectively. Recently, Edwards et al.(2011) reported the effect of commercial milling in 35 consign-ments of wheat, indicating that the distribution pattern between allconsignments was variable, probably as a consequence of sampling.DONwas lower in thewhite flour by an average of 30% compared tothe level in the original cleaned wheat; bran was higher by 282%,while concentration in the germ was approximately equivalent tothe cleaned wheat. ZEA was quantifiable in six consignments, fromwhich it appeared that distribution factors were 44%, 360%, and170% for white flour, bran and germ, respectively.

For comparison, there are several studies of the fate of myco-toxins in maize germ during wet- and dry-milling. Schaafsma,Frégeau-Reid, & Phibbs (2004) conducted a mass balance of DONduring wet milling of maize and concluded that the endospermfraction contained 20% of the original DON, 25% of the DON wasfound in the germ portion of the kernel and 55% was retained in thepericarp. ZEA in contaminated maize entering the wet-milling fa-cility were more concentrated in the gluten and fiber fractions,which are derived from the endosperm and pericarp, respectively,than the germ fraction (from which edible oil is obtained) (Bennet& Anderson, 1978). In contrast, for dry-milled maize the highestlevels of DON and ZEA are found in the germ and bran fractionswith lower levels in maize flour and grits (products of endosperm)(Brera et al., 2006; Scudamore & Patel, 2009).

3.3. Occurrence of DON and ZEA in dietary supplements: wheatgerm and wheat germ oil

Results of the natural occurrence of DON and ZEA in analyzedsamples of wheat germ and wheat germ oil are summarized inTable 2. The incidence and levels of DON were higher than for ZEAin both dietary supplements. Thus, sixty percent of wheat germsamples and 40% of wheat germ oils contained detectable amountsof DON, while none of germ samples and only 4 out of 25 oils (16%)contained ZEA. The mean level of DON in germwas 111 mg/kg, witha maximum value up to 240 mg/kg, while lower DON amounts werefound in oils with a mean of 41 mg/kg and a maximum of 163 mg/kg.The total mean of ZEA in wheat germ oil amounted to 6 mg/kg,showing a maximum concentration of 44 mg/kg in one sample. Theco-occurrence of DON and ZEA was only verified in 3 samples ofwheat germ oil. None of the wheat germ samples exceeded themaximum content for DON (750 mg/kg) or ZEA (75 mg/kg) fixed bycurrent Commission Regulation (EC) No. 1881/2006. There are nomaximum contents established specifically for wheat germ oil, butif the maximum levels for processed cereal-based foods areconsidered (200 mg/kg for DON and 20 mg/kg for ZEA), only onesample of oil containing ZEA at 44 mg/kg exceeded the tolerance.

For the exposure assessment, the level of consumption of wheatgerm and wheat germ oil was combined with the mean concen-trations of DON and ZEA. A consumption survey was conductedduring the years 2007e2011 including 277 consumers, fromwhich25.6% had consumed dietary supplements in the last 12 months.For consumers only, the calculated daily consumption of wheatgerm was 10 g, and amounted to 5 g for wheat germ oil (Giménez,2012). Our data lead to a daily intake of 1.3 mg DON and 0.03 mg ZEA,representing 1.9% and 0.23% of their respective tolerable daily in-takes (TDI), indicating a low to moderate risk for consumers.

Our results are similar to those reported in the few availablesurveys on the occurrence of DON and ZEA in wheat germ and

wheat germ oil. In Germany, Kappenstein et al. (2005) detected ZEAin 10 out of 11 samples of wheat germ oil, with a mean value of13 mg/kg and a maximum value of 46 mg/kg Schollenberger et al.(2005) conducted a survey on 219 samples of foodstuffs of plantorigin in which DON was detected in all 5 samples of wheat germanalyzed, ranging from 31 to 95 mg/kg, while ZEAwas only found inone sample at 3 mg/kg. Subsequently, a total of 110 samples of edibleoil marketed in Germany were analyzed for 13 trichothecene toxins(including DON) and for ZEA and its derivatives by Schollenbergeret al. (2008). None of the toxins analysed was detected in wheatgerm oil, but several Fusarium toxins were found in oil from soy-bean, sunflower and maize germ, with 14 positive samples.Trichothecene concentrations did not exceed 116 mg/kg, whereaslevels up to 1730 mg/kg ZEAwere found in maize germ oil. The EFSA(2011) also reported very high levels of ZEA (823 mg/kg) in corngerm oil, and other studies on the redistribution of ZEA during dryand wet milling of maize had revealed high concentrations of ZEA(up to 4.6mg/kg) in the oil fraction (Lauren & Ringrose,1997). Theseresults indicate that ZEA is predominant in maize germ and it canbe easily carried over to the edible corn germ oil.

As final conclusions, the developed method allowed the deter-mination of the target mycotoxins at low ppb levels in fatty ma-trixes such as wheat germ andwheat germ oil. Themilling of wheatreduced DON and ZEA levels by 53% and 29% in milled germ,respectively, as compared to the starting cleaned wholegrain.Therefore, wheat germwas not a significant source of DON and ZEAand the usual dry milling practice was enough to deliver acommercially safe product. The survey of 50 samples of dietarysupplements revealed a moderate incidence of DON in wheat germoil (40% positives) and wheat germ (60% positives) with averagelevels ranging from 41 to 111 mg/kg, respectively. ZEA was notdetected inwheat germ and appeared in 16% wheat germ oil with amaximum of 44 mg/kg.

I. Giménez et al. / Food Control 34 (2013) 268e273 273

Acknowledgments

This research was supported by the Spanish MICINN (ProjectsAGL2008-03555 and AGL2011-26808), the Government of Aragón(Grupo de Investigación Consolidado A01), and the European SocialFund. Author E. Ferruz acknowledges a grant from FundaciónCuenca Villoro. We are very grateful to the milling industry thatprovided the batch samples of wheat and germ.

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