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Distribution of deoxynivalenol and zearalenone in milled germ during wheat milling and analysis of toxin levels in wheat germ and wheat germ oil
Food Control 34 (2013) 268e273
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Distribution of deoxynivalenol and zearalenone in milled germ during wheat milling and analysis of toxin levels in wheat germ and wheat germ 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
a b s t r a c t
Article history: Received 1 December 2012 Received in revised form 15 April 2013 Accepted 27 April 2013
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 industrial milling and the distribution factors in milled germ were 47% for DON and 71% for ZEA. A survey of 50 samples of germ-based dietary supplements revealed that 60% of wheat germ and 40% of wheat germ oils contained DON at mean values of 111 and 41 mg/kg, respectively, while none of germ samples and 16% oils contained ZEA (mean 6 mg/kg). Contamination levels 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 intakes (TDI). Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Deoxynivalenol Zearalenone Milling Wheat germ Wheat germ oil
1. Introduction Most of the wheat harvested in the world is subjected to milling, the procedure by which wheat grains are ground and their components separated into milled fractions based on particle size. The germ is a high fat by-product of milling with great nutritional value for the content of a-tocopherol (vitamin E). Both wheat germ and wheat germ oil can be marketed for direct human consumption as dietary supplements, and they are attractive and promising sources of vegetable functional compounds (Rizzello, Cassone, Coda, & Gobbetti, 2011). It is well documented that wheat grains and milling products may contain mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA) (FAO/WHO, 2000, 2011; EFSA, 2004, 2011; SCOOP, 2003). Pinson-Gadais et al. (2007) detected the occurrence of toxigenic Fusarium spp. by PCR assay in all wheat tissues (germ, pericarp, aleurone layer, and albumen), concluding that none of the tissue structures within the wheat kernel acted as an effective barrier to fungal invasion and the subsequent synthesis of mycotoxins. Several studies have been carried out to determine the stability and partitioning of DON and ZEA during wheat milling. Kushiro (2008) reviewed 19 published papers and concluded that wheat
* Corresponding author. Tel.: þ34 876 554131; fax: þ34 976 761612. E-mail address: [email protected] (A. Ariño). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.04.033
milling redistributes DON, with the highest amounts appearing in the bran and the lowest in the flour. Similarly, Trigo-Stockly, Deyoe, Satumbaga, & Pedersen (1996) revealed that higher concentrations of ZEA are found in the bran fraction than in the original wheat grain with lower concentrations in white flour. However, little is known on the effects of milling on the distribution of DON and ZEA in milled wheat germ. On the other hand, information on the occurrence of these Fusarium toxins in products such as wheat germ and wheat germ oil for direct human consumption is very scarce (Kappenstein et al., 2005; Schollenberger et al., 2005; Schollenberger, Müller, Rüfle, & Drochner, 2008). Nevertheless, notably high levels of ZEA have been found in corn germ oil (up to 823 mg/kg) and wheat germ oil (up to 150 mg/kg), and these products could make an important contribution to the ZEA exposure (EFSA, 2011). Determination of DON and ZEA in foodstuffs is required for both the control of current legislative compliance and the assessment of human exposure. Most analytical methods proposed in recent years for the determination of DON and ZEA in foods have been developed, primarily for solid samples (Bao, Oles, White, Sapp, & Trucksess, 2011; Shephard et al. 2010). Analysis of wheat germ and derived oil presents entirely different commodities that could potentially complicate extraction and cleanup prior to determination because of the presence of fat (Mahoney & Molyneux, 2010). Therefore, the objectives of the present work were: (i) to develop 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 dry milling on the distribution of DON and ZEA in milled wheat germ, and (iii) to examine the occurrence and concentration levels of DON and ZEA in wheat germ-based dietary supplements for direct human 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 plant located in Aragón (NE Spain) able to process 140 tons/day of wheat grain. For this study, 36 different batches of wheat grain of harvest years 2009 and 2010, coming from Spain, France, Germany, and the USA were milled in different weeks. The wheat grains were cleaned and tempered as usual practice, and the germ fraction was separated during the milling process by rolling and sieving with a total germ yield of 0.5%. Five incremental samples of 400 g were collected from different places of each lot of cleaned wheat resulting in an aggregate sample of 2 kg, together with the corresponding samples of germ fractions taken and aggregated in the same manner. For mycotoxin analysis, these samples were ground in a Mahlkönig EG-43 mill (Hamburg, Germany), thoroughly mixed, and kept at 21 C until analysis. Commercial samples of dietary supplements for direct human consumption (wheat germ and wheat germ oil) were all purchased from a number of local retail outlets including grocery stores, drug stores, general merchandise retailers, natural food stores and specialty health and nutrition stores. Wheat germ was packaged in bags of 250e500 g and wheat germ oil was supplied in bottles of 250 mL. Samples of wheat germ and wheat germ oil were thoroughly mixed before analysis.
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2.2. Reagents and apparatus for mycotoxin analysis HPLC grade acetonitrile, methanol and n-hexane were purchased from Lab-Scan (Dublin, Ireland) and HPLC grade glacial acetic and formic acids from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Plus apparatus from Millipore (Milford, MA). The multifunctional cleanup columns Mycosep #224 and #225 were supplied by Romer Labs (Union, MO). DON and ZEA standard solutions at 100 mg/mL in acetonitrile were 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 an Agilent diode-array detector (DAD) at 220 nm for the determination of DON, and an Agilent fluorescence detector (FLD) at 274 mm (excitation)/440 nm (emission) for the determination of ZEA. The LC column was Ace 5 C18, 250 4.6 mm, 5 mm particle size (Advanced Chromatography Technologies, Aberdeen, United Kingdom). For DON analysis the mobile phase was water/acetonitrile/methanol (90:5:5, v/v/v) at a flow rate of 1.0 mL/min with an injection volume of 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 of 0.8 mL/min, and the injection volume was 20 mL. Figs. 1 and 2 show chromatograms of standards and contaminated samples of wheat germ and wheat germ oil.
2.3. Analysis of mycotoxins in wheat, wheat germ and wheat germ oil For the determination of DON and ZEA in wheat grain, a validated method based on Mycosep columns and HPLC determination was used (Sugita-Konsihi et al. 2006). Briefly, 5 g of homogenized
Fig. 1. LC-DAD chromatograms of a DON standard solution (500 mg/L) and a wheat germ sample contaminated with DON (294 mg/kg).
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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).
wheat was extracted with 20 mL acetonitrile and water (84:16, v/v) using an Ultraturrax homogenizer for 3 min, filtered with Whatman #4 filter paper, and the extract collected for further cleanup as described below. Our contribution to the method involved a defatting step for the determination of the toxins in wheat germ and wheat germ oil. For this, 5 g was extracted with a mixture of 20 mL acetonitrile and water (84:16, v/v) combined with 12.5 mL nhexane 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 extract was filtered with Whatman #4 filter paper and collected for cleanup. The resulting filtered extracts for all test commodities were split into two culture tubes by pipetting 5e7 mL each. For DON analysis, a Mycosep #225 multifunctional cleanup column was slowly pushed (rubber flange end) into one culture tube containing the extract. For ZEA analysis, the extract was acidified with 50 mL glacial acetic acid and pushed all through a Mycosep #224 multifunctional cleanup column. Two mL purified extracts were transferred into 4 mL vials, evaporated to dryness at 50 C in a heating block under a gentle stream of nitrogen, and redissolved in 400 mL mobile phase. Mycosep columns allowed a one-step cleanup within 30 s without the use of any solvent for elution: the column was pushed into a test tube containing the sample extract, forcing the extract to filter upwards through the packing material of the column. The interferences adhere to the chemical packing in the column and the purified extract, containing the analytes of interest, passes through the column. Based on European Commission Regulation (EC) No. 401/2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs, the analytical
procedures of the present work were validated in-house in terms of recovery, precision, and sensitivity. Spiking procedure was done in sextuplicate by adding appropriate amounts of standards to the sample matrices: DON at 1250 mg/kg (wheat), 750 mg/kg (wheat germ), 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 in Commission Regulation (EC) No. 1881/2006. The limits of detection (LOD) and quantification (LOQ) were based on minimum amount of target analytes that produced a chromatogram peak with a signalto-noise ratio of 3 and 10 times the background chromatographic noise, respectively. The quality of results was assured by participating in the proficiency testing Progetto Trieste 2010 for mycotoxins between 13 laboratories from different countries. 2.4. Confirmation of mycotoxins Confirmatory analysis was performed using an Acquity UPLC system coupled to a Quattro Premier XE triple quadrupole mass spectrometer (Waters, Milford, MA). The LC separation was performed 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 of 0.3 mL/min, with injection volume of 20 mL. The mass spectrometer was operated in the positive electrospray ionization mode (ESIþ) for DON, while for ZEA negative electrospray ionization mode (ESI-) was used. The MS/MS parameters included the following settings: ESI source block temperature 120 C, desolvation temperature 400 C, capillary voltage 3.5 kV, and argon collision gas 3.5 103 mbar. The
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operating conditions are described in Table 1. MassLynx software version 4.1 was used for data acquisition and processing. 2.5. Statistical analyses The results from mycotoxin analyses were subjected to descriptive 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 containing levels above the corresponding limit of detection (LOD). For each mycotoxin, the mean and standard deviation (SD) were calculated using LOD/2 for results lower than LOD. Calculations were performed on StatView SE þ Graphics (Abacus Concepts, Berkeley, CA) for Macintosh personal computers. 3. Results and discussion 3.1. Method validation Numerous methods for analysis of mycotoxins in foodstuffs have been developed, primarily for solid samples. Analysis in fatty commodities and oils presents entirely different matrixes that could potentially complicate extraction and cleanup of samples prior to determination due to their fat content (Mahoney & Molyneux, 2010). Thus, the crucial step in the analytical procedure for germ and oil was anticipated to be extraction and cleanup to retain mycotoxins but eliminate as much fat as possible prior to HPLC separation. In the method applied, the fat is separated during extraction with a nonpolar solvent such as hexane. For further cleanup, liquid partitioning was discarded because it is complicated and time and solvent consuming, and so were immunoaffinity cleanup columns (IAC) because they are expensive, though IAC can be preferable in most situations for the cleanup of mycotoxins in agricultural products. We therefore sought commercial available products such as multifunctional Mycosep columns, which are simple, fast, and consistent in quality and performance for the cleanup step in the analysis of mycotoxins (Bao et al., 2011). Based on our laboratory experience, the Mycosep #224 and #225 (Romer Labs, Union, MO) were tested as these columns are designed to retain interferences and elute mycotoxins in a very simple manner with no wash or elution solvents, as described in subheading 2.3. The analytical method for wheat grain provided good recoveries for DON and ZEA of 98 9% and 99 16%, respectively, and the limit of detection (LOD) for DON was 33 mg/kg and for ZEA was 10 mg/kg. For the analysis of DON and ZEA in fatty wheat germ and wheat germ oil, the extraction efficiency was tested with a mixture of acetonitrile: water (84:16, v/v) as extraction solvent combined with the addition of a hexane defatting step, and the most optimal efficiency was calculated by applying 20 mL extraction solvent and 12.5 mL of n-hexane. The mean recoveries for DON in wheat germ and wheat germ oil were 92 and 106%, respectively, and recoveries for ZEA amounted to 98 and 104%, respectively. Therefore, though ZEA is slightly soluble in hexane, there were no noticeable losses of this mycotoxin in the hexane layer. An additional advantage of the defatting step was the obtaining of cleaner extracts that allowed increased sensitivity and lower limits of detection (LODs). Thus, Table 1 Optimized MS/MS conditions for mycotoxin confirmation. Mycotoxin
Precursor ion
Cone (V)
Collision energy (eV)
Product ion (m/z)
Deoxynivalenol Deoxynivalenol Zearalenone Zearalenone
297.3 297.3 317.2 317.2
24 24 60 60
13 10 30 15
231.1 249.1 131.1 175.1
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LODs for DON and ZEA in wheat germ and wheat germ oil were 22 and 8 mg/kg, respectively, as compared to 33 mg/kg and 10 mg/kg obtained with the matrix wheat. Bao et al. (2011), using HPLC-DAD for the determination of DON in wheat germ reported a LOD about 100 mg/kg, while Schollenberger et al. (2008) achieved a LOD of 7 mg/kg using GCeMS with derivatization. A LOD for ZEA of 10 mg/kg in maize germ oil was reported by Majerus, Graft, & Krämer (2009) using HPLC-FLD. A deffating step with hexane has been also recently described for the simultaneous determination of DON, ZEA, T-2-toxin and some masked metabolites in maize, wheat, oats, cornflakes and bread with very good performance (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 all tested matrixes, in accordance with the validation criteria. Accordingly, the present study has led to the successful development of a simple, reliable method for the determination of DON and ZEA in wheat germ and derived edible oil. Furthermore, the hexane defatting step allowed the sensitivity to be increased, resulting in relatively cleaner extracts. The method, which was validated according to European Commission criteria, could provide a basis for an intra- and interlaboratory-validated analytical method for analysis of mycotoxins in dietary supplements and other products formulated 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 grain before milling (100% positives), with a mean concentration (SD) of 251 144 mg/kg and a maximum of 820 mg/kg. In the corresponding milled germ fractions, DON was detected in 34 out of 36 samples (94.4%), with a mean concentration of 117 61 mg/kg and a maximum of 290 mg/kg. All samples were below their respective maximum permitted levels for DON of 1250 mg/kg for wheat and 750 mg/kg for germ (Commission Regulation (EC) no 1881/2006). As regards of ZEA, only one batch of cleaned wheat grain (2.8% positives) contained ZEA at detectable levels of 14 mg/kg, and the ZEA concentration in the corresponding milled germ was 10 mg/kg, down their respective maximum permitted levels of 100 mg/kg for wheat and 75 mg/kg for germ. However, it is rather difficult to completely guarantee that starting material and milled products are totally representative when sampling big batches in industrial milling processes, and therefore, percents of distribution reported should be considered with caution. The distribution factor is defined as the ratio between the mycotoxin content in the milled germ fraction and the mycotoxin content in the wholegrain 100. Then, the distribution factor for DON after commercial milling was 47%, indicating that the DON concentration in milled germ is nearly one half of the original toxin concentration in the wholegrain. Therefore, the germ is not a significant source of DON and the usual milling practice is enough to deliver a commercially safe product. The distribution factor for ZEA was 71%, indicating that ZEA showed somewhat higher affinity for germ than DON during the commercial milling of wheat. However, there were too few data and too low levels at which ZEA occurred to allow confirmation of the situation with the germ fraction. Previous research has mainly focused on the effect of wheat milling on the fate of DON and ZEA in flour and bran fractions, while very little information is available in the scientific literature on the fractionation of these toxins in milled wheat germ. As reported by Abbas, Mirocha, Pawlosky, & Pusch (1985) and Trigo-Stockly et al. (1996), when present in milled fractions of wheat, the levels of DON and ZEA were generally highest in the bran and lowest in the milled flour. Similarly, Herrera, Juan, Estopañan, & Ariño (2009) indicated a significant effect of type of fraction on the distribution
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of DON in milled products of durum wheat, as the distribution factors for DON after milling were 153% for bran, 87% for durum semolina, and 108% for flour. Pascale et al. (2011) reported that the milling process of durum wheat led to an increase of T-2 and HT-2 toxin contents up to 5-fold in bran as compared with the uncleaned wheat, while observed an overall reduction by 54% in cleaned wheat and by 89% in semolina, respectively. Recently, Edwards et al. (2011) reported the effect of commercial milling in 35 consignments of wheat, indicating that the distribution pattern between all consignments was variable, probably as a consequence of sampling. DON was lower in the white flour by an average of 30% compared to the level in the original cleaned wheat; bran was higher by 282%, while concentration in the germ was approximately equivalent to the cleaned wheat. ZEA was quantifiable in six consignments, from which it appeared that distribution factors were 44%, 360%, and 170% for white flour, bran and germ, respectively. For comparison, there are several studies of the fate of mycotoxins in maize germ during wet- and dry-milling. Schaafsma, Frégeau-Reid, & Phibbs (2004) conducted a mass balance of DON during wet milling of maize and concluded that the endosperm fraction contained 20% of the original DON, 25% of the DON was found in the germ portion of the kernel and 55% was retained in the pericarp. ZEA in contaminated maize entering the wet-milling facility 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 highest levels of DON and ZEA are found in the germ and bran fractions with 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: wheat germ and wheat germ oil Results of the natural occurrence of DON and ZEA in analyzed samples of wheat germ and wheat germ oil are summarized in Table 2. The incidence and levels of DON were higher than for ZEA in both dietary supplements. Thus, sixty percent of wheat germ samples and 40% of wheat germ oils contained detectable amounts of DON, while none of germ samples and only 4 out of 25 oils (16%) contained ZEA. The mean level of DON in germ was 111 mg/kg, with a maximum value up to 240 mg/kg, while lower DON amounts were found 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. The co-occurrence of DON and ZEA was only verified in 3 samples of wheat germ oil. None of the wheat germ samples exceeded the maximum content for DON (750 mg/kg) or ZEA (75 mg/kg) fixed by current Commission Regulation (EC) No. 1881/2006. There are no maximum contents established specifically for wheat germ oil, but if the maximum levels for processed cereal-based foods are considered (200 mg/kg for DON and 20 mg/kg for ZEA), only one sample of oil containing ZEA at 44 mg/kg exceeded the tolerance. For the exposure assessment, the level of consumption of wheat germ and wheat germ oil was combined with the mean concentrations of DON and ZEA. A consumption survey was conducted during the years 2007e2011 including 277 consumers, from which 25.6% had consumed dietary supplements in the last 12 months. For consumers only, the calculated daily consumption of wheat germ 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 intakes (TDI), indicating a low to moderate risk for consumers. Our results are similar to those reported in the few available surveys on the occurrence of DON and ZEA in wheat germ and
Table 2 Occurrence of DON and ZEA in samples of wheat germ and wheat germ oil. Results expressed in mg/kg. Sample number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 % Positives Total mean Std. deviation
Wheat germ (n ¼ 25)
Wheat germ oil (n ¼ 25)
DON
ZEA
DON
ZEA
150 93 188
a Limit of detection of 22 mg/kg for DON and 8 mg/kg for ZEA in both wheat germ and wheat germ oil.
wheat germ oil. In Germany, Kappenstein et al. (2005) detected ZEA in 10 out of 11 samples of wheat germ oil, with a mean value of 13 mg/kg and a maximum value of 46 mg/kg Schollenberger et al. (2005) conducted a survey on 219 samples of foodstuffs of plant origin in which DON was detected in all 5 samples of wheat germ analyzed, ranging from 31 to 95 mg/kg, while ZEA was only found in one sample at 3 mg/kg. Subsequently, a total of 110 samples of edible oil marketed in Germany were analyzed for 13 trichothecene toxins (including DON) and for ZEA and its derivatives by Schollenberger et al. (2008). None of the toxins analysed was detected in wheat germ oil, but several Fusarium toxins were found in oil from soybean, sunflower and maize germ, with 14 positive samples. Trichothecene concentrations did not exceed 116 mg/kg, whereas levels up to 1730 mg/kg ZEA were found in maize germ oil. The EFSA (2011) also reported very high levels of ZEA (823 mg/kg) in corn germ oil, and other studies on the redistribution of ZEA during dry and wet milling of maize had revealed high concentrations of ZEA (up to 4.6 mg/kg) in the oil fraction (Lauren & Ringrose, 1997). These results indicate that ZEA is predominant in maize germ and it can be easily carried over to the edible corn germ oil. As final conclusions, the developed method allowed the determination of the target mycotoxins at low ppb levels in fatty matrixes such as wheat germ and wheat germ oil. The milling of wheat reduced DON and ZEA levels by 53% and 29% in milled germ, respectively, as compared to the starting cleaned wholegrain. Therefore, wheat germ was not a significant source of DON and ZEA and the usual dry milling practice was enough to deliver a commercially safe product. The survey of 50 samples of dietary supplements revealed a moderate incidence of DON in wheat germ oil (40% positives) and wheat germ (60% positives) with average levels ranging from 41 to 111 mg/kg, respectively. ZEA was not detected in wheat germ and appeared in 16% wheat germ oil with a maximum of 44 mg/kg.
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Acknowledgments This research was supported by the Spanish MICINN (Projects AGL2008-03555 and AGL2011-26808), the Government of Aragón (Grupo de Investigación Consolidado A01), and the European Social Fund. Author E. Ferruz acknowledges a grant from Fundación Cuenca Villoro. We are very grateful to the milling industry that provided the batch samples of wheat and germ. References Abbas, H. K., Mirocha, C. J., Pawlosky, R. J., & Pusch, D. J. (1985). Effect of cleaning, milling, and baking on deoxynivalenol in wheat. Applied and Environmental Microbiology, 50, 482e486. Bao, L., Oles, C. J., White, K. D., Sapp, C., & Trucksess, M. W. (2011). Use of multifunctional column for the determination of deoxynivalenol in grains, grain products, and processed foods. Journal of AOAC International, 94, 1506e1512. Bennett, G. A., & Anderson, R. A. (1978). Distribution of aflatoxin and/or zearalenone in wet-milled corn products: a review. Journal of Agricultural and Food Chemistry, 26, 1055e1960. Brera, C., Catano, C., De Santis, B., Debegnach, F., Giacomo, M., Pannunzi, E., et al. (2006). Effect of industrial processing on the distribution of aflatoxins and zearalenone in corn-milling fractions. Journal of Agricultural and Food Chemistry, 54, 5014e5019. Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. (2006). Official Journal of the European Union, L364, 5e24. Commission Regulation (EC) No. 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. (2006). Official Journal of the European Union, L70, 12e34. De Boevre, M., Di Mavungu, J. D., Maene, P., Audenaert, K., Deforce, D., Haesaert, G., et al. (2012). Development and validation of an LC-MS/MS method for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin and some masked metabolites in different cereals and cereal-derived food. Food Additives and Contaminants Part A, 29, 819e835. Edwards, S. G., Dickin, E. T., MacDonald, S., Buttler, D., Hazel, C. M., Patel, S., et al. (2011). Distribution of Fusarium mycotoxins in UK wheat mill fractions. Food Additives and Contaminants, 28, 1694e1704. EFSA (European Food Safety Authority). (2004). Opinion of the scientific panel of contaminants in the food chain on a request from the commission related to deoxynivalenol as undesirable substance in animal feed. The EFSA Journal, 73, 1e41. EFSA (European Food Safety Authority). (2011). Scientific opinion on the risks for public health related to the presence of zearalenone in food. EFSA panel on contaminants in the food Chain. The EFSA Journal, 9(6), 2197. FAO/WHO. (2000). Position paper on zearalenone. CX/FAC 00/19, Rome, Italy. FAO/WHO. (2011). Proposed draft maximum levels for deoxynivalenol (DON) and its acetylated derivatives in cereals and cereal-based products. CX/CF 11/5/6, Rome, Italy. Giménez, I. (2012). Fusarium toxins in wheat germ: Effect of milling and contamination in derived products. Doctoral thesis. Spain: University of Zaragoza. Herrera, M., Juan, T., Estopañan, G., & Ariño, A. (2009). Comparison of deoxynivalenol, ochratoxin A and aflatoxin B1 levels in conventional and organic
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durum semolina and the effect of milling. Journal of Food and Nutrition Research, 48, 92e99. Kappenstein, O., St. Klaffke, H., Mehlitz, I., Tiebach, R., Weber, R., Lepschy, J., et al. (2005). Determination of zearalenone in edible oils with SEC and LC-ESI-MS/ MS. Mycotoxin Research, 21, 3e6. Kushiro, M. (2008). Effects of milling and cooking processes on the deoxynivalenol content in wheat. International Journal of Molecular Science, 9, 2127e2145. Lauren, D. R., & Ringrose, M. A. (1997). Determination of the fate of three Fusarium mycotoxins through wet-milling of maize using an improved HPLC analytical technique. Food Additives and Contaminants, 14, 435e443. Mahoney, N., & Molyneux, R. J. (2010). Rapid analytical method for the determination of aflatoxins in plant-derived dietary supplement and cosmetic oils. Journal of Agricultural and Food Chemistry, 58, 4065e4070. Majerus, P., Graft, N., & Krämer, M. (2009). Rapid determination of zearalenone in edible oils by HPLC with fluorescence detection. Mycotoxin Research, 25, 117e121. Pascale, M., Haidukowski, M., Lattanzio, V. M. T., Silvestri, M., Ranieri, R., & Visconti, A. (2011). Distribution of T-2 and HT-2 toxins in milling fractions of durum wheat. Journal of Food Protection, 74, 1700e1707. Pinson-Gadais, L., Barreau, C., Chaurand, M., Gregoire, S., Normanson, M., & RichardForget, F. (2007). Distribution of toxigenic Fusarium spp. and mycotoxin production in milling fractions of durum wheat. Food Additives and Contaminants, 24, 53e62. Rizzello, C. G., Cassone, A., Coda, R., & Gobbetti, M. (2011). Antifungal activity of sourdough fermented wheat germ used as an ingredient for bread making. Food Chemistry, 127, 952e959. Sachs, L. (Ed.). (1978). Estadística aplicada (1st ed.) Barcelona, Spain: Labor. Schaafsma, A. W., Frégeau-Reid, J., & Phibbs, T. (2004). Distribution of deoxynivalenol in Gibberella-infected food-grade corn kernels. Canadian Journal of Plant Science, 84, 909e913. Schollenberger, M., Müller, H.-M., Rüfle, M., Suchy, S., Planck, S., & Drochner, W. (2005). Survey of Fusarium toxins in foodstuffs of plant origin marketed in Germany. International Journal of Food Microbiology, 97, 317e326. Schollenberger, M., Müller, H.-M., Rüfle, M., & Drochner, W. (2008). Natural occurrence of 16 Fusarium toxins in edible oil marketed in Germany. Food Control, 19, 475e482. SCOOP (Scientific Cooperation Task 3.2.10 of the European Commission). (2003). Collection of ocurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states. Final report, 2003. Brussels, Belgium: DirectorateeGeneral Health and Consumer Protection. European Commission. Scudamore, K. A., & Patel, S. (2009). Fusarium mycotoxins in milling streams from the commercial milling of maize imported to the UK, and relevance to current legislation. Food Additives and Contaminants Part A, 26, 744e753. Shephard, G. S., Berthiller, F., Dorner, J., Krska, R., Lombaert, G. A., Malone, B., et al. (2010). Developments in mycotoxin analysis: an update for 2008e2009. World Mycotoxin Journal, 3, 3e23. Sugita-Konsihi, Y., Tanaka, T., Tabata, S., Nakajima, M., Nouno, M., Nakaie, Y., et al. (2006). Validation of an HPLC analytical method coupled to a multifunctional clean-up column for the determination of deoxynivalenol. Mycopathologia, 161, 239e243. Trigo-Stockli, D. M., Deyoe, C. W., Satumbaga, R. F., & Pedersen, J. R. (1996). Distribution of deoxynivalenol and zearalenone in milled fractions of wheat. Cereal Chemistry, 73, 388e391.
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Distribution of deoxynivalenol and zearalenone in milled germ during wheat milling and analysis of toxin levels in wheat germ and wheat germ 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
a b s t r a c t
Article history: Received 1 December 2012 Received in revised form 15 April 2013 Accepted 27 April 2013
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 industrial milling and the distribution factors in milled germ were 47% for DON and 71% for ZEA. A survey of 50 samples of germ-based dietary supplements revealed that 60% of wheat germ and 40% of wheat germ oils contained DON at mean values of 111 and 41 mg/kg, respectively, while none of germ samples and 16% oils contained ZEA (mean 6 mg/kg). Contamination levels 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 intakes (TDI). Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Deoxynivalenol Zearalenone Milling Wheat germ Wheat germ oil
1. Introduction Most of the wheat harvested in the world is subjected to milling, the procedure by which wheat grains are ground and their components separated into milled fractions based on particle size. The germ is a high fat by-product of milling with great nutritional value for the content of a-tocopherol (vitamin E). Both wheat germ and wheat germ oil can be marketed for direct human consumption as dietary supplements, and they are attractive and promising sources of vegetable functional compounds (Rizzello, Cassone, Coda, & Gobbetti, 2011). It is well documented that wheat grains and milling products may contain mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA) (FAO/WHO, 2000, 2011; EFSA, 2004, 2011; SCOOP, 2003). Pinson-Gadais et al. (2007) detected the occurrence of toxigenic Fusarium spp. by PCR assay in all wheat tissues (germ, pericarp, aleurone layer, and albumen), concluding that none of the tissue structures within the wheat kernel acted as an effective barrier to fungal invasion and the subsequent synthesis of mycotoxins. Several studies have been carried out to determine the stability and partitioning of DON and ZEA during wheat milling. Kushiro (2008) reviewed 19 published papers and concluded that wheat
* Corresponding author. Tel.: þ34 876 554131; fax: þ34 976 761612. E-mail address: [email protected] (A. Ariño). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.04.033
milling redistributes DON, with the highest amounts appearing in the bran and the lowest in the flour. Similarly, Trigo-Stockly, Deyoe, Satumbaga, & Pedersen (1996) revealed that higher concentrations of ZEA are found in the bran fraction than in the original wheat grain with lower concentrations in white flour. However, little is known on the effects of milling on the distribution of DON and ZEA in milled wheat germ. On the other hand, information on the occurrence of these Fusarium toxins in products such as wheat germ and wheat germ oil for direct human consumption is very scarce (Kappenstein et al., 2005; Schollenberger et al., 2005; Schollenberger, Müller, Rüfle, & Drochner, 2008). Nevertheless, notably high levels of ZEA have been found in corn germ oil (up to 823 mg/kg) and wheat germ oil (up to 150 mg/kg), and these products could make an important contribution to the ZEA exposure (EFSA, 2011). Determination of DON and ZEA in foodstuffs is required for both the control of current legislative compliance and the assessment of human exposure. Most analytical methods proposed in recent years for the determination of DON and ZEA in foods have been developed, primarily for solid samples (Bao, Oles, White, Sapp, & Trucksess, 2011; Shephard et al. 2010). Analysis of wheat germ and derived oil presents entirely different commodities that could potentially complicate extraction and cleanup prior to determination because of the presence of fat (Mahoney & Molyneux, 2010). Therefore, the objectives of the present work were: (i) to develop 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 dry milling on the distribution of DON and ZEA in milled wheat germ, and (iii) to examine the occurrence and concentration levels of DON and ZEA in wheat germ-based dietary supplements for direct human 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 plant located in Aragón (NE Spain) able to process 140 tons/day of wheat grain. For this study, 36 different batches of wheat grain of harvest years 2009 and 2010, coming from Spain, France, Germany, and the USA were milled in different weeks. The wheat grains were cleaned and tempered as usual practice, and the germ fraction was separated during the milling process by rolling and sieving with a total germ yield of 0.5%. Five incremental samples of 400 g were collected from different places of each lot of cleaned wheat resulting in an aggregate sample of 2 kg, together with the corresponding samples of germ fractions taken and aggregated in the same manner. For mycotoxin analysis, these samples were ground in a Mahlkönig EG-43 mill (Hamburg, Germany), thoroughly mixed, and kept at 21 C until analysis. Commercial samples of dietary supplements for direct human consumption (wheat germ and wheat germ oil) were all purchased from a number of local retail outlets including grocery stores, drug stores, general merchandise retailers, natural food stores and specialty health and nutrition stores. Wheat germ was packaged in bags of 250e500 g and wheat germ oil was supplied in bottles of 250 mL. Samples of wheat germ and wheat germ oil were thoroughly mixed before analysis.
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2.2. Reagents and apparatus for mycotoxin analysis HPLC grade acetonitrile, methanol and n-hexane were purchased from Lab-Scan (Dublin, Ireland) and HPLC grade glacial acetic and formic acids from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Plus apparatus from Millipore (Milford, MA). The multifunctional cleanup columns Mycosep #224 and #225 were supplied by Romer Labs (Union, MO). DON and ZEA standard solutions at 100 mg/mL in acetonitrile were 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 an Agilent diode-array detector (DAD) at 220 nm for the determination of DON, and an Agilent fluorescence detector (FLD) at 274 mm (excitation)/440 nm (emission) for the determination of ZEA. The LC column was Ace 5 C18, 250 4.6 mm, 5 mm particle size (Advanced Chromatography Technologies, Aberdeen, United Kingdom). For DON analysis the mobile phase was water/acetonitrile/methanol (90:5:5, v/v/v) at a flow rate of 1.0 mL/min with an injection volume of 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 of 0.8 mL/min, and the injection volume was 20 mL. Figs. 1 and 2 show chromatograms of standards and contaminated samples of wheat germ and wheat germ oil.
2.3. Analysis of mycotoxins in wheat, wheat germ and wheat germ oil For the determination of DON and ZEA in wheat grain, a validated method based on Mycosep columns and HPLC determination was used (Sugita-Konsihi et al. 2006). Briefly, 5 g of homogenized
Fig. 1. LC-DAD chromatograms of a DON standard solution (500 mg/L) and a wheat germ sample contaminated with DON (294 mg/kg).
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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).
wheat was extracted with 20 mL acetonitrile and water (84:16, v/v) using an Ultraturrax homogenizer for 3 min, filtered with Whatman #4 filter paper, and the extract collected for further cleanup as described below. Our contribution to the method involved a defatting step for the determination of the toxins in wheat germ and wheat germ oil. For this, 5 g was extracted with a mixture of 20 mL acetonitrile and water (84:16, v/v) combined with 12.5 mL nhexane 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 extract was filtered with Whatman #4 filter paper and collected for cleanup. The resulting filtered extracts for all test commodities were split into two culture tubes by pipetting 5e7 mL each. For DON analysis, a Mycosep #225 multifunctional cleanup column was slowly pushed (rubber flange end) into one culture tube containing the extract. For ZEA analysis, the extract was acidified with 50 mL glacial acetic acid and pushed all through a Mycosep #224 multifunctional cleanup column. Two mL purified extracts were transferred into 4 mL vials, evaporated to dryness at 50 C in a heating block under a gentle stream of nitrogen, and redissolved in 400 mL mobile phase. Mycosep columns allowed a one-step cleanup within 30 s without the use of any solvent for elution: the column was pushed into a test tube containing the sample extract, forcing the extract to filter upwards through the packing material of the column. The interferences adhere to the chemical packing in the column and the purified extract, containing the analytes of interest, passes through the column. Based on European Commission Regulation (EC) No. 401/2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs, the analytical
procedures of the present work were validated in-house in terms of recovery, precision, and sensitivity. Spiking procedure was done in sextuplicate by adding appropriate amounts of standards to the sample matrices: DON at 1250 mg/kg (wheat), 750 mg/kg (wheat germ), 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 in Commission Regulation (EC) No. 1881/2006. The limits of detection (LOD) and quantification (LOQ) were based on minimum amount of target analytes that produced a chromatogram peak with a signalto-noise ratio of 3 and 10 times the background chromatographic noise, respectively. The quality of results was assured by participating in the proficiency testing Progetto Trieste 2010 for mycotoxins between 13 laboratories from different countries. 2.4. Confirmation of mycotoxins Confirmatory analysis was performed using an Acquity UPLC system coupled to a Quattro Premier XE triple quadrupole mass spectrometer (Waters, Milford, MA). The LC separation was performed 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 of 0.3 mL/min, with injection volume of 20 mL. The mass spectrometer was operated in the positive electrospray ionization mode (ESIþ) for DON, while for ZEA negative electrospray ionization mode (ESI-) was used. The MS/MS parameters included the following settings: ESI source block temperature 120 C, desolvation temperature 400 C, capillary voltage 3.5 kV, and argon collision gas 3.5 103 mbar. The
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operating conditions are described in Table 1. MassLynx software version 4.1 was used for data acquisition and processing. 2.5. Statistical analyses The results from mycotoxin analyses were subjected to descriptive 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 containing levels above the corresponding limit of detection (LOD). For each mycotoxin, the mean and standard deviation (SD) were calculated using LOD/2 for results lower than LOD. Calculations were performed on StatView SE þ Graphics (Abacus Concepts, Berkeley, CA) for Macintosh personal computers. 3. Results and discussion 3.1. Method validation Numerous methods for analysis of mycotoxins in foodstuffs have been developed, primarily for solid samples. Analysis in fatty commodities and oils presents entirely different matrixes that could potentially complicate extraction and cleanup of samples prior to determination due to their fat content (Mahoney & Molyneux, 2010). Thus, the crucial step in the analytical procedure for germ and oil was anticipated to be extraction and cleanup to retain mycotoxins but eliminate as much fat as possible prior to HPLC separation. In the method applied, the fat is separated during extraction with a nonpolar solvent such as hexane. For further cleanup, liquid partitioning was discarded because it is complicated and time and solvent consuming, and so were immunoaffinity cleanup columns (IAC) because they are expensive, though IAC can be preferable in most situations for the cleanup of mycotoxins in agricultural products. We therefore sought commercial available products such as multifunctional Mycosep columns, which are simple, fast, and consistent in quality and performance for the cleanup step in the analysis of mycotoxins (Bao et al., 2011). Based on our laboratory experience, the Mycosep #224 and #225 (Romer Labs, Union, MO) were tested as these columns are designed to retain interferences and elute mycotoxins in a very simple manner with no wash or elution solvents, as described in subheading 2.3. The analytical method for wheat grain provided good recoveries for DON and ZEA of 98 9% and 99 16%, respectively, and the limit of detection (LOD) for DON was 33 mg/kg and for ZEA was 10 mg/kg. For the analysis of DON and ZEA in fatty wheat germ and wheat germ oil, the extraction efficiency was tested with a mixture of acetonitrile: water (84:16, v/v) as extraction solvent combined with the addition of a hexane defatting step, and the most optimal efficiency was calculated by applying 20 mL extraction solvent and 12.5 mL of n-hexane. The mean recoveries for DON in wheat germ and wheat germ oil were 92 and 106%, respectively, and recoveries for ZEA amounted to 98 and 104%, respectively. Therefore, though ZEA is slightly soluble in hexane, there were no noticeable losses of this mycotoxin in the hexane layer. An additional advantage of the defatting step was the obtaining of cleaner extracts that allowed increased sensitivity and lower limits of detection (LODs). Thus, Table 1 Optimized MS/MS conditions for mycotoxin confirmation. Mycotoxin
Precursor ion
Cone (V)
Collision energy (eV)
Product ion (m/z)
Deoxynivalenol Deoxynivalenol Zearalenone Zearalenone
297.3 297.3 317.2 317.2
24 24 60 60
13 10 30 15
231.1 249.1 131.1 175.1
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LODs for DON and ZEA in wheat germ and wheat germ oil were 22 and 8 mg/kg, respectively, as compared to 33 mg/kg and 10 mg/kg obtained with the matrix wheat. Bao et al. (2011), using HPLC-DAD for the determination of DON in wheat germ reported a LOD about 100 mg/kg, while Schollenberger et al. (2008) achieved a LOD of 7 mg/kg using GCeMS with derivatization. A LOD for ZEA of 10 mg/kg in maize germ oil was reported by Majerus, Graft, & Krämer (2009) using HPLC-FLD. A deffating step with hexane has been also recently described for the simultaneous determination of DON, ZEA, T-2-toxin and some masked metabolites in maize, wheat, oats, cornflakes and bread with very good performance (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 all tested matrixes, in accordance with the validation criteria. Accordingly, the present study has led to the successful development of a simple, reliable method for the determination of DON and ZEA in wheat germ and derived edible oil. Furthermore, the hexane defatting step allowed the sensitivity to be increased, resulting in relatively cleaner extracts. The method, which was validated according to European Commission criteria, could provide a basis for an intra- and interlaboratory-validated analytical method for analysis of mycotoxins in dietary supplements and other products formulated 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 grain before milling (100% positives), with a mean concentration (SD) of 251 144 mg/kg and a maximum of 820 mg/kg. In the corresponding milled germ fractions, DON was detected in 34 out of 36 samples (94.4%), with a mean concentration of 117 61 mg/kg and a maximum of 290 mg/kg. All samples were below their respective maximum permitted levels for DON of 1250 mg/kg for wheat and 750 mg/kg for germ (Commission Regulation (EC) no 1881/2006). As regards of ZEA, only one batch of cleaned wheat grain (2.8% positives) contained ZEA at detectable levels of 14 mg/kg, and the ZEA concentration in the corresponding milled germ was 10 mg/kg, down their respective maximum permitted levels of 100 mg/kg for wheat and 75 mg/kg for germ. However, it is rather difficult to completely guarantee that starting material and milled products are totally representative when sampling big batches in industrial milling processes, and therefore, percents of distribution reported should be considered with caution. The distribution factor is defined as the ratio between the mycotoxin content in the milled germ fraction and the mycotoxin content in the wholegrain 100. Then, the distribution factor for DON after commercial milling was 47%, indicating that the DON concentration in milled germ is nearly one half of the original toxin concentration in the wholegrain. Therefore, the germ is not a significant source of DON and the usual milling practice is enough to deliver a commercially safe product. The distribution factor for ZEA was 71%, indicating that ZEA showed somewhat higher affinity for germ than DON during the commercial milling of wheat. However, there were too few data and too low levels at which ZEA occurred to allow confirmation of the situation with the germ fraction. Previous research has mainly focused on the effect of wheat milling on the fate of DON and ZEA in flour and bran fractions, while very little information is available in the scientific literature on the fractionation of these toxins in milled wheat germ. As reported by Abbas, Mirocha, Pawlosky, & Pusch (1985) and Trigo-Stockly et al. (1996), when present in milled fractions of wheat, the levels of DON and ZEA were generally highest in the bran and lowest in the milled flour. Similarly, Herrera, Juan, Estopañan, & Ariño (2009) indicated a significant effect of type of fraction on the distribution
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of DON in milled products of durum wheat, as the distribution factors for DON after milling were 153% for bran, 87% for durum semolina, and 108% for flour. Pascale et al. (2011) reported that the milling process of durum wheat led to an increase of T-2 and HT-2 toxin contents up to 5-fold in bran as compared with the uncleaned wheat, while observed an overall reduction by 54% in cleaned wheat and by 89% in semolina, respectively. Recently, Edwards et al. (2011) reported the effect of commercial milling in 35 consignments of wheat, indicating that the distribution pattern between all consignments was variable, probably as a consequence of sampling. DON was lower in the white flour by an average of 30% compared to the level in the original cleaned wheat; bran was higher by 282%, while concentration in the germ was approximately equivalent to the cleaned wheat. ZEA was quantifiable in six consignments, from which it appeared that distribution factors were 44%, 360%, and 170% for white flour, bran and germ, respectively. For comparison, there are several studies of the fate of mycotoxins in maize germ during wet- and dry-milling. Schaafsma, Frégeau-Reid, & Phibbs (2004) conducted a mass balance of DON during wet milling of maize and concluded that the endosperm fraction contained 20% of the original DON, 25% of the DON was found in the germ portion of the kernel and 55% was retained in the pericarp. ZEA in contaminated maize entering the wet-milling facility 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 highest levels of DON and ZEA are found in the germ and bran fractions with 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: wheat germ and wheat germ oil Results of the natural occurrence of DON and ZEA in analyzed samples of wheat germ and wheat germ oil are summarized in Table 2. The incidence and levels of DON were higher than for ZEA in both dietary supplements. Thus, sixty percent of wheat germ samples and 40% of wheat germ oils contained detectable amounts of DON, while none of germ samples and only 4 out of 25 oils (16%) contained ZEA. The mean level of DON in germ was 111 mg/kg, with a maximum value up to 240 mg/kg, while lower DON amounts were found 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. The co-occurrence of DON and ZEA was only verified in 3 samples of wheat germ oil. None of the wheat germ samples exceeded the maximum content for DON (750 mg/kg) or ZEA (75 mg/kg) fixed by current Commission Regulation (EC) No. 1881/2006. There are no maximum contents established specifically for wheat germ oil, but if the maximum levels for processed cereal-based foods are considered (200 mg/kg for DON and 20 mg/kg for ZEA), only one sample of oil containing ZEA at 44 mg/kg exceeded the tolerance. For the exposure assessment, the level of consumption of wheat germ and wheat germ oil was combined with the mean concentrations of DON and ZEA. A consumption survey was conducted during the years 2007e2011 including 277 consumers, from which 25.6% had consumed dietary supplements in the last 12 months. For consumers only, the calculated daily consumption of wheat germ 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 intakes (TDI), indicating a low to moderate risk for consumers. Our results are similar to those reported in the few available surveys on the occurrence of DON and ZEA in wheat germ and
Table 2 Occurrence of DON and ZEA in samples of wheat germ and wheat germ oil. Results expressed in mg/kg. Sample number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 % Positives Total mean Std. deviation
Wheat germ (n ¼ 25)
Wheat germ oil (n ¼ 25)
DON
ZEA
DON
ZEA
150 93 188
a Limit of detection of 22 mg/kg for DON and 8 mg/kg for ZEA in both wheat germ and wheat germ oil.
wheat germ oil. In Germany, Kappenstein et al. (2005) detected ZEA in 10 out of 11 samples of wheat germ oil, with a mean value of 13 mg/kg and a maximum value of 46 mg/kg Schollenberger et al. (2005) conducted a survey on 219 samples of foodstuffs of plant origin in which DON was detected in all 5 samples of wheat germ analyzed, ranging from 31 to 95 mg/kg, while ZEA was only found in one sample at 3 mg/kg. Subsequently, a total of 110 samples of edible oil marketed in Germany were analyzed for 13 trichothecene toxins (including DON) and for ZEA and its derivatives by Schollenberger et al. (2008). None of the toxins analysed was detected in wheat germ oil, but several Fusarium toxins were found in oil from soybean, sunflower and maize germ, with 14 positive samples. Trichothecene concentrations did not exceed 116 mg/kg, whereas levels up to 1730 mg/kg ZEA were found in maize germ oil. The EFSA (2011) also reported very high levels of ZEA (823 mg/kg) in corn germ oil, and other studies on the redistribution of ZEA during dry and wet milling of maize had revealed high concentrations of ZEA (up to 4.6 mg/kg) in the oil fraction (Lauren & Ringrose, 1997). These results indicate that ZEA is predominant in maize germ and it can be easily carried over to the edible corn germ oil. As final conclusions, the developed method allowed the determination of the target mycotoxins at low ppb levels in fatty matrixes such as wheat germ and wheat germ oil. The milling of wheat reduced DON and ZEA levels by 53% and 29% in milled germ, respectively, as compared to the starting cleaned wholegrain. Therefore, wheat germ was not a significant source of DON and ZEA and the usual dry milling practice was enough to deliver a commercially safe product. The survey of 50 samples of dietary supplements revealed a moderate incidence of DON in wheat germ oil (40% positives) and wheat germ (60% positives) with average levels ranging from 41 to 111 mg/kg, respectively. ZEA was not detected in wheat germ and appeared in 16% wheat germ oil with a maximum of 44 mg/kg.
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Acknowledgments This research was supported by the Spanish MICINN (Projects AGL2008-03555 and AGL2011-26808), the Government of Aragón (Grupo de Investigación Consolidado A01), and the European Social Fund. Author E. Ferruz acknowledges a grant from Fundación Cuenca Villoro. We are very grateful to the milling industry that provided the batch samples of wheat and germ. References Abbas, H. K., Mirocha, C. J., Pawlosky, R. J., & Pusch, D. J. (1985). Effect of cleaning, milling, and baking on deoxynivalenol in wheat. Applied and Environmental Microbiology, 50, 482e486. Bao, L., Oles, C. J., White, K. D., Sapp, C., & Trucksess, M. W. (2011). Use of multifunctional column for the determination of deoxynivalenol in grains, grain products, and processed foods. Journal of AOAC International, 94, 1506e1512. Bennett, G. A., & Anderson, R. A. (1978). Distribution of aflatoxin and/or zearalenone in wet-milled corn products: a review. Journal of Agricultural and Food Chemistry, 26, 1055e1960. Brera, C., Catano, C., De Santis, B., Debegnach, F., Giacomo, M., Pannunzi, E., et al. (2006). Effect of industrial processing on the distribution of aflatoxins and zearalenone in corn-milling fractions. Journal of Agricultural and Food Chemistry, 54, 5014e5019. Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. (2006). Official Journal of the European Union, L364, 5e24. Commission Regulation (EC) No. 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. (2006). Official Journal of the European Union, L70, 12e34. De Boevre, M., Di Mavungu, J. D., Maene, P., Audenaert, K., Deforce, D., Haesaert, G., et al. (2012). Development and validation of an LC-MS/MS method for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin and some masked metabolites in different cereals and cereal-derived food. Food Additives and Contaminants Part A, 29, 819e835. Edwards, S. G., Dickin, E. T., MacDonald, S., Buttler, D., Hazel, C. M., Patel, S., et al. (2011). Distribution of Fusarium mycotoxins in UK wheat mill fractions. Food Additives and Contaminants, 28, 1694e1704. EFSA (European Food Safety Authority). (2004). Opinion of the scientific panel of contaminants in the food chain on a request from the commission related to deoxynivalenol as undesirable substance in animal feed. The EFSA Journal, 73, 1e41. EFSA (European Food Safety Authority). (2011). Scientific opinion on the risks for public health related to the presence of zearalenone in food. EFSA panel on contaminants in the food Chain. The EFSA Journal, 9(6), 2197. FAO/WHO. (2000). Position paper on zearalenone. CX/FAC 00/19, Rome, Italy. FAO/WHO. (2011). Proposed draft maximum levels for deoxynivalenol (DON) and its acetylated derivatives in cereals and cereal-based products. CX/CF 11/5/6, Rome, Italy. Giménez, I. (2012). Fusarium toxins in wheat germ: Effect of milling and contamination in derived products. Doctoral thesis. Spain: University of Zaragoza. Herrera, M., Juan, T., Estopañan, G., & Ariño, A. (2009). Comparison of deoxynivalenol, ochratoxin A and aflatoxin B1 levels in conventional and organic
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