Occurrence of deoxynivalenol and deoxynivalenol-3-glucoside in durum wheat from Argentina
Introduction
Fusarium head blight (FBH) is one of the most destructive and economically important fungal diseases of wheat and other small grain cereals worldwide. FHB is caused primarily by species within the Fusarium graminearum species complex (FGSC), mainly F. graminearum. Other species which may appear as major pathogens depending on the geographic region are Fusarium culmorum, Fusarium avenaceum and Fusarium poae (Becher et al., 2013, Parry et al., 1995). The disease can severely reduce grain yield and quality, and under favorable conditions (high humidity and warm temperatures) yield losses can reach up to 50% (Parry et al., 1995). Moreover, the infected grains could be contaminated with mycotoxins such as trichothecenes which results in difficulties for wheat trading, besides the threat to human and animal health. Trichothecenes have been classified into four groups: types A–D, according to their chemical structure (WHO, 1990), the most important in cereals are types A and B (Desjardins, 2006). The B-trichothecenes include the mycotoxins deoxynivalenol (DON), its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON), and nivalenol (NIV). DON can be found worldwide and it is the most frequent type-B trichothecene. SCOOP (2003) reported trichothecene incidence in samples from 11 European countries. DON incidence was 57% while NIV, fusarenon X (FUS-X), 15ADON and 3ADON were found in the 16, 10, 20 and 8% of the samples analyzed. DON inhibits protein synthesis by interference with peptidyl transferase function on the ribosome, and it has been associated with intoxication of humans and animals through consumption of contaminated food and feed (Pestka, 2010). In Argentina, there are not maximum permitted levels set for DON, however, being its toxicity well characterized, the maximum permitted levels set by the European Commission (EC) for DON in wheat and derived products are often taken as reference. In particular, in durum wheat for human consumption the EC maximum permitted level is 1750 μg/kg (Commission Regulation, EC N° 1881/2006). Durum wheat (Triticum turgidum L. var. durum) is an important small grain cereal, used for human consumption. In Argentina durum wheat is mainly used for pasta production. In the last few years the national production of pasta has increased to 40% and in 2013 it reached 352,062 tons. The pasta consumption per capita was estimated in 8.27 kg/year (UIFRA, 2014). Although previous studies showed that the retention level of DON from unprocessed wheat grains to cooked pasta in the plate can be of 25% or less (Visconti, Haidukowski, Pascale, & Silvestri, 2004), according to the Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluations, the incidence of DON in pasta (30% of positive samples) can be considered of concern (JECFA, 2001). Several studies have reported DON contamination of pasta but at levels below the maximum permitted level established by the EC of 750 μg/kg for adults (Commission Regulation, EC N° 1881/2006, Brera et al., 2013, EFSA, 2013, González-Osnaya et al., 2011). However, Raiola, Meca, Mañes, and Ritieni (2012) analyzed twenty-seven pasta samples intended for young children consumption and reported that seven of them exceeded the maximum tolerable limit of 200 μg/kg for DON fixed for processed cereal-based baby foods by the EC (Commission Regulation, EC N° 1881/2006).Therefore, DON contamination in wheat, mainly in those varieties intended for pasta production, is of concern for the Argentinean consumers.
Moreover, DON may acts as a virulence factor of the fungus, affecting the protein synthesis in plant cells. As a consequence, plants have developed detoxification processes which include DON glucosylation to a less toxic compound, DON-3-β-d-glucoside (D3G). This product is known as a modified mycotoxin and it is less active as a protein synthesis inhibitor than DON in vitro (Poppenberger et al., 2003). So far, D3G has been reported in naturally contaminated samples of bread and durum wheat, maize, oats, barley, malt, triticale and also derived products such as flour, breakfast cereals, snacks and beer (Berthiller et al., 2013, Dall’Asta et al., 2013, Lancova et al., 2008, Malachova et al., 2011, Rasmussen et al., 2012).
Currently, there are not regulatory levels for the most frequently occurring modified forms of DON, i.e. 3 and 15ADON and D3G, in cereals and cereal-based foods. However, the JECFA considered that D3G and the acetylated derivatives, might contribute to exposure to DON. Based on the available toxicity data, the Committee considered the toxicity of the acetylated derivatives equal to that of DON (JECFA, 2011). Some studies indicate that D3G can be released during food processing as a consequence of enzymatic degradation of polysaccharides and also it could be cleaved during mammalian digestion, thus contributing to the overall toxicity of DON (Franz Berthiller et al., 2013, Nagl et al., 2014). Furthermore, the JECFA experts pointed out that additional data on the occurrence of and the effects of processing on 3ADON, 15ADON and D3G are needed, as well as their co-occurrence with DON (JECFA, 2011).
Updated information on the occurrence of DON and its modified forms are reported in the recent report issued by the European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (EFSA, 2013). Results from the EFSA survey showed that the DON derivatives (3ADON, 15ADON) were far less frequently found and at lower levels than DON. The Panel highlighted that further data should be collected on D3G, 3ADON and 15ADON in order to better characterize their potential contribution to the total exposure to DON (EFSA, 2013).
Up to now, in Argentina only DON has been reported as grain contaminant in durum wheat, there are not data available on D3G and the acetylated DON derivatives contamination in this cereal. The aim of this study was, therefore, to measure the contamination by deoxynivalenol, 3- and 15-deoxynivalenol and deoxynivalenol-3-glucoside in different durum wheat commercial cultivars collected in the Argentinean main growing area. Furthermore, in order to get insights into the possible correlation between the glucosylation ability and the resistance to FHB, the level of glucosylation in the field was assessed.
Section snippets
Chemicals and reagents
Methanol, and acetonitrile (both HPLC grade) were purchased from Mallinckrodt Baker (Milan, Italy). Ultrapure water was produced by a Millipore Milli-Q system (Millipore, Bedford, MA, USA). Ammonium acetate (for mass spectrometry) was from Sigma-Aldrich (Milan, Italy). Oasis® HLB (3 ml, 60 mg) columns were purchased from Waters (Milan, Italy). Filter papers (Whatman no. 4) were obtained from Whatman International Ltd (Maidstone, UK). Standard mycotoxins (DON, 3ADON, 15ADON, and D3G) were
Results and discussion
Occurrence of DON, its acetylated derivatives and D3G were assessed in samples of durum wheat (n = 84) produced in three different geographic locations of the main durum wheat growing area during 2012/13 and 2013/14 harvest seasons. All samples were found positive for DON at concentrations varying between <LOQ and 9480 μg/kg, with an average of 1750 μg/kg for quantified samples, there was only one sample below the LOQ. D3G was detected in 94% of the analyzed samples at concentrations ranging from
Conclusion
In the present study, the contamination by deoxynivalenol, 3- and 15-deoxynivalenol and deoxynivalenol-3-glucoside in different durum wheat commercial cultivars from the Argentinean main growing area was investigated. All analyzed samples were found to be contaminated with DON, and 30% of them exceeded the maximum permitted level established by the EC, presenting a potential risk for Argentinean consumers. It was also observed that DON contamination was dependent on the harvest year and the
Acknowledgements
This work was supported by grant PIP 11220120100525 from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). We thank Ing. Agr. Carlos Jensen for assistance in wheat sampling.
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