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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Relationship between lutein and mycotoxin content in durum wheat a
b
a
a
b
Rosa M. Delgado , Michael Sulyok , Ondřej Jirsa , Tomáš Spitzer , Rudolf Krska & Ivana a
Polišenská a
Agrotest fyto s.r.o., Kroměříž, Czech Republic
b
Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Tulln, Austria Accepted author version posted online: 20 May 2014.Published online: 09 Jun 2014.
Click for updates To cite this article: Rosa M. Delgado, Michael Sulyok, Ondřej Jirsa, Tomáš Spitzer, Rudolf Krska & Ivana Polišenská (2014) Relationship between lutein and mycotoxin content in durum wheat, Food Additives & Contaminants: Part A, 31:7, 1274-1283, DOI: 10.1080/19440049.2014.925589 To link to this article: http://dx.doi.org/10.1080/19440049.2014.925589
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 7, 1274–1283, http://dx.doi.org/10.1080/19440049.2014.925589
Relationship between lutein and mycotoxin content in durum wheat Rosa M. Delgadoa*, Michael Sulyokb, Ondřej Jirsaa, Tomáš Spitzera, Rudolf Krskab and Ivana Polišenskáa a
Agrotest fyto s.r.o., Kroměříž, Czech Republic; bDepartment for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Tulln, Austria
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(Received 25 February 2014; accepted 12 May 2014) Levels of lutein and a number of mycotoxins were determined in seven varieties of durum wheat (Triticum durum) and two varieties of common wheat (Triticum aestivum) in order to explore possible relationships amongst these components. Durum wheat cultivars always showed both higher lutein and mycotoxin contents than common wheat cultivars. The mycotoxins detected in both common and durum wheat cultivars were produced by the genera Fusarium, Claviceps, Alternaria and Aspergillus. Fusarium was the major producer of mycotoxins (26 mycotoxins) followed by Claviceps (14 mycotoxins), which was present only in some cultivars such as Chevalier (common wheat), Lupidur and Selyemdur (both durum wheat), Alternaria (six mycotoxins) and Aspergillus (three mycotoxins). Positive correlations between the levels of lutein and mycotoxins in durum wheat cultivars were found for the following mycotoxins: deoxynivalenol (DON), its derivative DON-3-glucoside, moniliformin, culmorin and its derivatives (5-hydroxyculmorin and 15-hydroxyculmorin). Keywords: durum wheat; lutein; mycotoxin; Fusarium; LC-MS/MS
Introduction Wheat is amongst the most widely grown food crop with a global production of about 600 million tonnes annually. Wheat varieties have traditionally been selected for functionality, e.g. making bread or biscuits resulting in the selection of common bread wheat (Triticum aestivum L.) due to its high level of strong gluten proteins, or durum wheat (Triticum turgidum L. subsp. durum Desf. [Hus.]) used mainly for yellowcoloured pasta products. Reductions in crop yield can be caused by Fusarium infection of cereal grains which causes Fusarium head blight (FHB). Furthermore, various species of this genus produce mycotoxins that are hazardous both to humans and animals. However, apart from Fusarium, toxic secondary fungal metabolites from other genera such as Aspergillus, Penicillium, Claviceps and Alternaria can also be found in wheat grain. In general, the most important target mycotoxins analytes in small grain cereals by these genera includes trichothecenes, zearalenone and its derivatives, fumonisins, enniatins, ergot alkaloids, ochratoxins, aflatoxins, moniliformin and patulin. Field surveys indicate that DON, a mycotoxin belonging to the trichothecenes group, as well as its derivatives, are the most frequently encountered mycotoxins associated with FHB disease of wheat throughout European countries, mainly produced by F. graminearum and F. culmorum (Bottalico & Perrone 2002). Although only a very limited number of mycotoxins is subject to legal guidance and regular monitoring (aflatoxins, fumonisins, DON, *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
zearalenone and ochratoxin A), the concern about the determination of the whole complex of mycotoxins and their metabolites rises. This initiated the development of multi-mycotoxin LC-MS/MS-based methods (Sulyok et al. 2006) allowing the presence of a large range of mycotoxins to be analysed and metabolites in the same sample, including masked mycotoxins (e.g., DON-3-glucoside or ZEN-4-glucoside). Fusarium infection constitutes not only an important food safety issue, it also reduces grain quality such as semolina yield, colour, and gluten strength in durum wheat. The quality of durum wheat grain depends on the genetic potential of variety, environmental conditions and their mutual interactions. It may be evaluated from different points of view: agronomical quality (potentiality and stability of grain yield), milling quality (semolina yield, ash content, humidity, impurity of grains), technological quality (protein and gluten content), and hygienic and sanitary quality (phytopathological microorganisms or their secondary metabolites). Nevertheless, from the point of view of the quality criteria of customers, the pigmentation of durum wheat grain and as a consequence of the end-products is one of the most relevant points used to evaluate the quality of the durum wheat. Lutein is considered as the major carotenoid component in wheat and the main responsible for its pigmentation. Lanchman et al. (2013) indicated that lutein is the most abundant carotenoid in wheat cultivars (on average ≈83%) with zeaxanthin (≈10%) and β-carotene (≈7%) being minor constituents. Its content in einkorn
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Food Additives & Contaminants: Part A (T. monococcum L., subsp. monococcum) wheat can reach 91% of the total carotenoids (Hidalgo et al. 2006) and 71% and 83% in spring and winter wheat (T. aestivum), respectively (Konopka et al. 2006). Durum wheat contains in general higher amount of carotenoids (1.5–4.0 mg kg–1 dry matter, DM) in comparison with common wheat (0.1–2.4 mg kg–1 DM) (Panfili et al. 2004). This pigmentation can be modified by processing conditions and enzymes activities (Trono et al. 1999). The yellow colour of durum wheat semolina and pasta products is attributed to its carotenoid content. This content may have beneficial health effects as the consumption of wheat with high a carotenoid level is linked to a reduction in oxidative damage by the ageing processes (Granado et al. 2003). In addition, it was previously established that an increase in the antioxidative potential of plants has a positive effect on pathogen resistance (Lorenc-Kukuła et al. 2005). The aim of this study was to determine the lutein content which is responsible for the much appreciated yellow colour as well as the level of a broad spectrum of mycotoxins in different varieties of durum wheat. Furthermore, relationships between lutein and mycotoxin content were investigated in the different cultivars to establish if varieties with higher lutein content, and therefore more appreciated due to their yellowness, also have a higher or lower mycotoxin content.
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Materials and methods Materials Plant material Field experiments were carried out at the location in Kroměříž (Czech Republic) during the growing season 2012–13. Kroměříž (49°16–17°N/17°21–22°E. l.) is located in the sugar beet agricultural production area with the altitude of 235 m in a plain terrain of the River Morava. The region is characterised as warm and moderately wet. The soil type is classified as luvisol chernozem, soil type clayey, medium heavy. The average annual temperature measured at the meteorological station in Kroměříž in the period 1971–2010 is 9.2°C, the average total annual precipitation is 576 mm (long-term normal). Actual weather conditions in the growing season 2012–13 in comparison with long-term normal are illustrated in the Figure 1. The field experiments were established as orthogonal randomised blocks with three replications and the size of individual plot was 10 m2. Individual variants were harvested separately, the grain from appropriate replications was thoroughly mixed and analysed as one sample in triplicate. In the varietal trial, seven varieties of T. durum (IS Pentadur, Logidur, Auradur, Lunadur, Lupidur, Cliodur and Selyemdur) and two varieties of T. aestivum (Akteur and Chevalier) were involved. The trial was sown
Figure 1. Weather conditions in the growing season 2012–13, data given for pentad periods and compared with long-term normal (1971–2010) for Kroměříž, Czech Republic.
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on 11 October 2012; the preceding crop was lucerne. Before sowing, 35 kg ha–1 of P2O5 (superphosphate 43%) and 125 kg ha–1 of K2O (potassium salt 60%) were applied. Sowing rate was 4 million of germinating seeds per 1 ha. Nitrogen was added in the form of potassium nitrate (27%) in the dose equal to 30 kg N ha–1 (8 March 2013) and 50 kg N ha–1 (15 April 2013) and then in the form of DAM (ammonium nitrate 42.2% and 32.7% of urea) in the dose equal to 40 kg N ha–1. The trials was treated with growth regulators Retacel extra R 68 (chlormequat – chloride 720 g l–1) at a dose of 1.5 l ha–1 (23 April 2013) and Moddus (250 g l–1 trinexapac-ethyl) at a dose of 0.3 l ha–1 (10 May 2013). Fungicides were applied twice: 16 May 2013 – Hutton (spiroxamine 250 g l–1, prothioconazole 100 g l–1, tebuconazole 100 g l–1) at a dose of 0.8 l ha–1 and 07 June 2013 – Prosaro (prothioconazole 125 g l–1, tebuconazole 125 g l–1) at a dose of 0.75 l ha–1. Crop treatment and protection was the same as would be in a good farming practice appropriate to the area in order to achieve good grain quality. All trial plots were harvested by a small plot combine (1 August 2013 with the exception of Chevalier and Lupidur, which were not rape enough and were harvested on 8 August 2013); from harvested grain representative subsamples (approximately 1 kg each) for laboratory analyses were taken. Grains samples were milled using sieve of 0.5 mm. They were divided into plastic bags and kept at –20°C until mycotoxin analysis. Chemicals LC gradient-grade methanol, acetonitrile, isopropyl alcohol, hexane and MS-grade glacial acetic acid were purchased from Sigma Aldrich (Vienna, Austria; and Schnelldorf, Germany). A Purelab Ultra system (ELGA LabWater, Celle, Germany) was used for further purification of reverse osmosis water. All other reagents were of analytical or HPLC grade and were from Lach:ner (Neratovice, Czech Republic). Standards of lutein and trans-β-apo-8-carotenal were purchased from Sigma-Aldrich (Schnelldorf, Germany). Standards solutions of lutein and the internal standard (trans-β-apo-8-carotenal) were prepared in n-butanol (0.5 and 1 mg ml–1, respectively) and stored at –20°C. The working standards solutions were freshly prepared prior to the calibration by dilution. The internal standard spiking solution (25 ng µl) was stored inter-day analysis at –20°C. Standards of fungal and bacterial metabolites were obtained either as gifts from various research groups or from the following commercial sources: Romer Labs (Tulln, Austria), Sigma-Aldrich (Vienna, Austria), Iris Biotech GmbH (Marktredwitz, Germany), Axxora Europe (Lausanne, Switzerland), BioAustralis (Smithfiled, Australia), BiovioticaNaturstoffe GmbH (Dransfeld, Germany), Fermentek (Jerusalem, Israel) and
LGC Promochem GmbH (Wesel, Germany). Stock solutions of each analyte were prepared by dissolving the solid substance in acetonitrile (preferably), acetonitrile/water 1:1 (v/v), methanol, methanol/water 1:1 (v/v) or water. Thirty-four combined working solutions were prepared by mixing the stock solutions of the corresponding analytes for easier handling and were stored at –20°C. The final working solution was freshly prepared prior to spiking experiments by mixing of the combined working solutions.
Methods Sample preparation for carotenoids and mycotoxins determination ● Carotenoid determination. Carotenoids were extracted according to Panfili et al. (2003) with the modifications of Peterson et al. (2007). Thus, 0.5 g of wheat samples with 100 µl of internal standard (trans-β-apo-8-carotenal, 25 ng µl–1) were combined with 0.5 ml potassium hydroxide (600 g l–1), 0.5 ml of 95% ethanol, 0.5 ml of sodium chloride (10 g l–1) and 1.25 ml of pyrogallol (60 g l–1 ethanol) in screw dark tubes. They were incubated at 70ºC under nitrogen atmosphere for 25 min, then cooled in an ice water bath. After adding 3.75 ml of sodium chloride (10 g l–1), the samples were extracted with 3.75 ml of hexane/ethyl acetate (9:1, v/v) until colourless. The organic extracts were combined and evaporated to dryness under nitrogen. The residues were dissolved in 1 ml of the mobile phase (methanol/acetonitrile/isopropyl alcohol, 38:60:2). Then, 10 µl of the samples were analysed by an HPLC-DAD system. ● Mycotoxin determination. Ground wheat (5 g) was extracted with 20 ml of acetonitrile/water/acetic acid (79/20/1) for 90 min using a GFL 3017 rotary shaker (GFL, Burgwedel, Germany). Aliquots of 100 µl were transferred into glass vials and diluted with the same amount of a mixture containing acetonitrile/water/acetic acid (20/79/1). After mixing, 5 µl of the diluted extract were injected into the LC-MS/MS system.
HPLC analysis ● Carotenoids analysis (HPLC-DAD). Chromatography was performed using a Shimadzu HPLC 20AD (Kyoto, Japan) Prominence analytical system equipped with an automatic sampler. Separation was done on a Kinetex Phenomenex C18 column
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Figure 2. Chromatogram of wheat extracts with the added internal standard (trans-β-apo-8-carotenal) at 445 nm. Retention times of lutein and trans-β-apo-8-carotenal were 3.346 and 4.851 min, respectively.
(250 × 4.6 mm i.d., particle size = 5 µm) (Torrance, CA, USA). Spectrophotometric detection was achieved by means of a diode array detector SPDM20A (λ = 445 nm). The mobile phase was methanol/acetonitrile/isopropyl alcohol (38/60/2) at a flow rate of 1.5 ml min–1. Lutein was identified through comparison of its retention time with those of known available standard solutions and quantified on the basis of calibration curves of standard solutions (r2 > 0.99) (Figure 2). Data were processed by LC Solution version 1.21 SP1 (Torrance, CA, USA). ● Mycotoxins analysis (LC-MS/MS). Detection and quantification was performed with a QTrap5500 LC-MS/MS System (Applied Biosystems, Foster City, CA) equipped with a TurboIonSpray electrospray ionisation (ESI) source and an 1290 Series UHPLC System (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25°C on a Gemini® C18-column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a C18 security guard cartridge, 4 × 3 mm i.d. (all Phenomenex). Elution was carried out in binary gradient mode. Both mobile phases contained 5 mM ammonium acetate and were composed of methanol/water/acetic acid 10:89:1 (v/v/v; eluent A) and 97:2:1 (v/v/v; eluent B), respectively. After an initial time of 2 min at 100% A, the proportion of B was increased linearly to 50% within 3 min. Further linear increase of B to 100% within 9 min was followed by a hold time of 4 min at 100% B and 2.5 min column re-equilibration at 100% A. The flow rate was 1000 μl min–1. ESI-MS/MS was performed in the scheduled multiple reaction monitoring (sMRM) mode in both positive and negative polarities in two separate chromatographic runs. The sMRM detection window of each analyte was
set to the respective retention time ±27 s and ±42 s in positive and in negative mode, respectively. The target scan time was set to 1 s. Confirmation of positive analyte identification is obtained by the acquisition of two sMRMs per analyte (with the exception of moniliformin and 3-nitropropionic acid, which exhibited only one fragment ion). This yielded 4.0 identification points according to Commission Decision 2002/657/EC (EU 2002). LOD was defined as three times the signal/noise ratio (Table 1). Analyst® software version 1.5.1 (AB Sciex, Foster City, CA, USA) was used to control the LC-MS/MS instrument, as well as for automatic and manual integration of the peak.
Statistical analysis The extraction was carried out in triplicate and data were reported as mean ± SD. Analysis of variance (ANOVA) was carried out and a Fisher least significant difference (LSD) test was used to describe means with 95% confidence. The Spearman correlation coefficients were calculated by STATISTICA Cz 10 statistical software (StatSoft CR, Prague, Czech Republic) at a probability level of p < 0.05.
Results and discussion Lutein content in common and durum wheat cultivars Figure 3 shows the lutein content determined by HPLCDAD for seven durum and two common wheat cultivars from our trial. Significant differences were found in lutein content between all cultivars both for durum and common
6.0 0.1 1.0 6.9 9.1 18.2 0.3 0.0
± ± ± ± ± ± ± ± ± 3.3 ± 1.2
0.1 13.9 1.4 0.9
± ± ± ±
± 19.2 ± 2.9
Akteur
Claviceps Elymoclavine 0.5 Ergine 0.08 Ergocornine 0.8 Ergocorninine 0.2 Ergocristine 1.0 Ergocristinine 0.1 Ergocryptine 0.4 Ergocryptinine 0.06 Ergometrinine 0.03 Ergosin 0.80 Ergotamine 0.3 Ergovalin 0.5 Chanoclavine 0.008 Secalonic acid D 4.0 Fusarium 15-Hydroxyculmorin 3 136.9e 15-Hydroxyculmoron n.d. 26.2c 5-Hydroxyculmorin n.d. Apicidin 0.1 1.0ef Aurofusarin 1.0 144.1cd Beauvericin 0.002 6.1bc Culmorin 0.8 34.2h Diacetoxyscirpenol 0.15 Don 0.5 116.4h Don glucoside n.d. 2.4f Enniatin A 0.01 3.9f Enniatin A1 0.02 37.8e Enniatin B 0.01 157.8f Enniatin B1 0.02 133.5f Enniatin B2 0.06 8.6f Enniatin B3 0.001 0.1g Equisetin 0.20 HT2 toxin 16 Moniliformin 0.5 127.5e Monoacetoxyscirpenol 1.5 13.2b
LOD
68.0f ± 2.1 16.7a ± 0.6
53.1i ± 1.9 1.6g ± 0.1 2.8f ± 0.2 27.5e ± 4.5 102.1g ± 3.8 85.1g ± 1.3 5.1g ± 0.1 0.1g ± 0.0 ± ± ± ± ± ± ± ±
27.9 158.6e ± 13.3 6.4 34.9c ± 1.7 9.3 61.3e ± 12.3 0.1 4.5c ± 0.8 63.0 203.3c ± 29.4 0.5 4.2d ± 1.0 8.4 202.4f ± 10.0
Logidur
± ± ± ± ± ± ± ±
23.8 0.8 0.8 3.3 22.1 14.8 0.8 0.0
9.2 5.5 3.3 0.2 43.7 0.8 9.4
283.4c ± 12.0 9.5cd ± 1.1
582.7e 6.5d 23.1c 147.4c 454.5c 392.7d 25.8c 0.2e
± ± ± ± ± ± ±
Lunadur
464.2a ± 28.6 167.4e 87.8a ± 12.0 25.4c a 242.2 ± 13.7 158.0bc 2.2d ± 0.2 1.6de a 603.9 ± 24.2 305.1b 4.5d ± 0.4 4.7cd a 667.6 ± 10.8 240.2e
Auradur
13.0 460.1f ± 16.1 1275.7a ± 61.1 0.2 4.1e ± 0.2 12.4a ± 0.6 f 2.3 2.4 ± 0.4 12.8de ± 1.2 33.3 31.7e ± 5.0 163.7c ± 31.2 11.5 239.1e ± 23.9 619.5a ± 5.0 19.2 154.0f ± 15.7 564.1b ± 14.9 1.5 13.8e ± 1.5 41.2a ± 0.7 0.0 0.1f ± 0.0 0.4b ± 0.0 c 1.6c ± 0.2 4.6 ± 1.1 39.9a ± 4.3 19.5b ± 1.3 262.9c ± 5.7 192.3d ± 5.3 417.2a ± 51.4 8.8cd ± 0.1 7.3ef ± 0.4 10.1c ± 1.3
309.8g 4.2e 26.9b 197.2b 525.8b 529.6c 31.1b 0.3c
0.6f ± 0.1 22.2e ± 5.2 4.9cd ± 0.9 46.1h ± 5.7
± ± ± ± ± ± ±
0.9c ± 0.1
1.4d ± 0.1 1.2c ± 0.2
Pentadur
216.0d 24.5c 92.7d 1.5de 360.6b 6.9b 167.5g
0.8a ± 0.2 0.8a ± 0.1 462.7b ± 22.8 365.4a ± 16.5 276.9a ± 49.1 88.4a ± 12.3 260.1a ± 28.6 67.6a ± 2.8 253.0a ± 14.4 126.6a ± 5.6 65.4a ± 7.3 31.6a ± 3.3 14.2a ± 2.2 1792.0a ± 61.4
Chevalier
Mycotoxins level (ppb) in common and durum wheat classified by mycotoxin producer.
Mycotoxins
Table 1.
882.9c 7.4bc 13.5d 142.9c 614.1a 592.2a 40.9a 0.4a 123.6a 23.5b 320.2b 8.6de
361.8b 61.2b 181.6b 8.9b 312.7b 8.5a 562.1c
± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ±
3.6 0.5 0.6 10.0 20.3 19.4 1.0 0.0 4.5 1.8 15.3 0.1
9.0 9.6 11.3 0.8 42.7 1.0 10.5
0.0 11.7 9.9 1.4 1.6 8.2 1.6 2.5 3.1 0.8 0.2 0.2 70.7
Lupidur
0.1c 111.5c 90.0b 83.8b 25.2b 139.7b 22.4c 27.3c 45.8c 10.8b 1.5b 2.3c 442.4c
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± ± ± ± ± ± ±
22.4 6.5 16.0 0.1 26.5 0.6 5.1
± ± ± ± ± ± ± ±
297.9a 55.1b 179.3b 99.4b 4.5b 5.2b 7.3b 671.9b
33.8 5.9 23.8 0.5 17.9 0.2 15.4 0.1 42.1 0.3 1.9 10.0 10.4 3.0 1.5 0.0 0.7 1.3 5.8 0.9
24.7 2.1 33.4 11.9 0.5 0.6 1.4 79.3
0.2 0.0 55.3 64.5
(continued )
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ±
1.0a 0.5b 535.8a 419.3a
Selyemdur
292.8c 66.2b 152.0c 41.2a 85.5de 5.1cd 373.7d 1.5 974.4b ± 23.2 766.2d 7.7b ± 0.3 7.0cd e 11.4 ± 0.6 39.8a 91.8d ± 0.9 254.9a 425.0d ± 12.7 427.8d 345.6e ± 8.8 576.6ab 22.2d ± 0.8 25.0c 0.3d ± 0.0 0.2e c 1.9 ± 0.2 22.3b 14.4c bc 288.4 ± 9.7 221.5d 7.1f ± 0.7 13.6b
279.1c 61.5b 232.9a 0.3f 193.2c 5.3cd 623.1b
0.6c ± 0.0
Cliodur
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0.03 0.5 3.0 0.1 0.03 0.3
Monocerin Nivalenol T2 tetraol T2 toxin Zea-4-sulfate Zearalenon Alternaria Altenariol Altenariolmethylether Altertoxin I Infectopyron Macrosporin Tentoxin Aspergillus 3-Nitropropionic acid Asperloxin A Emodin Unspecific Tryptophol ± ± ± ± ±
0.0 0.1 9.8 0.4 0.2
37.4b ± 1.0
n.d.
1.0de ± 0.1
0.5de ± 0.3 1.6c ± 0.1 142.0c ± 6.8 9.5f ± 0.7 10.8c ± 0.2
49.6d ± 3.1 10.1b ± 1.5
Chevalier
± ± ± ± ±
0.1 0.3 6.8 7.7 0.0
4.1c ± 0.3 10.7b ± 0.4 0.9fg ± 0.1
0.5de 1.7c 245.3a 36.4e 5.7fg
1.5a ± 0.0 94.7a ± 5.4 30.7a ± 3.9
0.5c ± 0.1 67.3c ± 3.3
± ± ± ± ± ±
2.6 0.6 0.1 17.7 1.5 1.5
3.3 ± 0.2 0.8g ± 0.1
e
12.0a 4.9a 2.2b 225.4b 25.0ef 12.6b
0.5a ± 0.1
Logidur
Pentadur
11.7c ± 3.0 7.3d ± 0.4 0.9ef ± 0.0
0.2e ± 0.0 2.6a ± 0.2 74.2e ± 8.4 60.4d ± 4.7 4.5g ± 0.1
1.1b ± 0.3 45.5d ± 1.7 16.2b ± 2.8 7.6ab ± 1.1 1.1a ± 0.2 0.7a ± 0.4
Auradur
± ± ± ± ± ±
2.2 0.1 0.2 13.9 12.6 0.8 28.4b ± 6.5 16.3a ± 1.1 1.2ab ± 0.1
± ± ± ± ± ±
0.5a ± 0.1
31.6e ± 1.6 26.7a ± 3.7 8.1a ± 1.2
Lupidur
0.2 7.6b 0.2 1.6b 0.1 2.8a 8.6 221.6b 15.8 149.5bc 0.5 13.0b 10.3 ± 0.0 1.1bc ± 0.0
b
1.5c 1.3bc 2.1b 110.6d 346.1a 17.2a
0.4a ± 0.2
4.6d ± 0.3
1.5a ± 0.2 53.9d ± 5.9
Lunadur
Notes: Values are means ± SD. Means in the same row with different superscripts are significantly different (p < 0.05). LOD = limit of detection by LC-MS/MS.
0.6h ± 0.0
7.3c ± 0.5
0.1e 1.5c 90.2e 2.2f 6.3ef
86.4ab ± 10.0 14.3b ± 3.6
Akteur
1.0 0.4 0.2
0.3 0.02 0.3 n.d. 0.5 0.15
LOD
Mycotoxins
Table 1. Continued .
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± ± ± ± ± ±
± ± ± ± ±
0.2 0.2 0.3 5.3 23.4 0.8
0.2 1.9 1.8 0.3 0.2
60.6a ± 9.0
5.5c ± 0.7 8.9c ± 0.2 1.1cd ± 0.0
1.0c 0.9cd 2.7a 146.6c 154.4b 8.9d
0.7c 49.7d 11.9b 6.5bc 1.3a
Cliodur
± ± ± ± ± ±
± ± ± ±
0.0 0.1 0.2 5.9 25.1 0.5
0.4 5.7 6.7 0.5
48.6a ± 11.9 3.4e ± 0.1 1.2a ± 0.1
0.3c 0.4e 2.9a 133.1c 130.9c 6.9e
1.2ab 80.0b 29.9a 5.9cd
Selyemdur
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Figure 3. Determination of lutein content by LC-DAD in common and durum wheat cultivars. Letters indicate significant difference (p < 0.05).
wheat. As expected, common wheat (Akteur and Chevalier) showed a lower lutein content (2.9 and 3.8 µg g–1, respectively) than most durum wheat (values in the range from 3.7 to 7.3 µg g–1 with the exception of the cultivar Auradur with the highest value, 12 µg g–1). These values are in accordance with earlier observations by Leenhardt et al. (2006) who explored the genetic variability of carotenoid concentration among cultivated diploid (einkorn, T. monococcum L.), tetraploid (durum wheat, T. turgidum L. subsp. durum (Desf.) Husn.) and hexaploid (common wheat, T. aestivum L.) wheat species. They reported a varietal variation in the lutein concentration of 5.7, 3.3 and 1.2 mg kg–1 DM for einkorn, durum and common wheat, respectively. Durum wheat is appreciated by its elevated yellow pigment which is provided by its lutein content. The findings of the current study provide information about the lutein variation based on the cultivar of durum wheat. These results are consistent with previous research which found that lutein was present in Canadian durum wheat in the amount ranging from 0.37 to 5.6 µg g–1 (Van Hung & Hatcher 2011). The most striking result to emerge from the data is that Auradur cultivar can reach lutein values of 12 µg g–1. The relevance of the lutein that contributes to the desirable yellowness of semolina and flours to make a healthy yellow alkaline noodle without artificial yellow agent (Van Hung & Hatcher 2011) provides an interest in cultivars such as Auradur with higher lutein content. Mycotoxins in common and durum wheat cultivars Wheat harvested in our trial was contaminated by toxins produced by fungi belonging to Fusarium, Alternaria, Aspergillus and Claviceps genera. In total, 50 fungal secondary metabolites were detected with most of them attributed to Fusarium (26 metabolites) were found,
followed by Claviceps (14 metabolites), Alternaria (six metabolites), Aspergillus (three metabolites), in addition to one unspecific metabolite (tryptophol) (Table 1). However, the presence of these mycotoxins depended on the cultivar under study. While Claviceps affected mainly one cultivar of common (Chevalier) and two cultivars of durum wheat (Lupidur and Selyemdur), Fusarium, Alternaria and Aspergillus mycotoxins were present in almost all the cultivars (common and durum wheat). Many durum cultivars are more susceptible to Fusarium infection and subsequent mycotoxin contamination in comparison with common wheat and other small cereal grains. This is supported by Clear et al. (2005) who found higher DON and moniliformin concentrations in durum samples grown in the same crop districts as common wheat in western Canada. Although our study only comprised two common wheat cultivars and no general statements can be made, the trend of our data is consistent with those previous results. Thus, Fusarium metabolite production was higher in all durum than in common wheat cultivars mainly in enniatins, DON, aurofusarin, culmorin and its derivates, and moniliformin. European Union legislation establishes legal limits for DON in unprocessed cereals other than durum wheat, oats and maize at the level of 1250 µg kg–1. However, the limit is higher for unprocessed durum wheat, maize and oats (1750 µg kg–1) (EU 2006). All our durum wheat samples showed a DON content between 53 and 1275 µg kg–1 which is under the appropriate limit of legislation. The highest value was found in durum cultivar Auradur (1275 µg kg–1), while the lowest was found in common wheat (Akteur (116 µg kg–1) and Chevalier (53 µg kg–1)). Besides DON, DON-3-glucoside was detected in all samples being also higher in durum than in common wheat. Recent studies have pointed out that lactic acid bacteria commonly found in the digestive tract of mammals are able to cleave a significant proportion of DON-3-glucoside
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Food Additives & Contaminants: Part A releasing DON in vitro (Berthiller et al. 2011). Thus, the total bioavailable amount of DON in contaminated cereal could be increased by the presence of DON-3-glucoside. However, DON-3-glucoside showed a considerably lower toxicological relevance than DON in rats in vivo (Nagl et al. 2012). Furthermore, even if we consider the conversion of DON-3-glucoside in DON, the total amount of bioavailable DON would not surpass the European Commission guidance value in any sample. As with DON, durum wheat showed a higher content of culmorin and hydroxyculmorin derivates than common wheat with the exception of Pentadur in 15-hydroxyculmoron content. A correlation was found between DON and culmorin (r = 1.000) as well as DON with hydroxylated analogues of culmorin such as 5-hydroxyculmorin (r = 0.929). This co-occurrence, which has also been pointed out by other authors (Uhlig et al. 2013), might be due to F. graminearum being a producer of both mycotoxins, DON and culmorin (Desjardins 2006). The content of depsipeptide compounds (the sum of enniatins A, A1, B, B1, B2 and beauvericin) showed a higher content in durum wheat as well (Lupidur (1412 µg kg–1), Auradur (1406 µg kg–1), Selyemdur (1329 µg kg–1), Pentadur (1317 µg kg–1), Lunadur (1048 µg kg–1), Cliodur (901 µg kg–1), Logidur (445 µg kg–1)) than in common wheat (Akteur (347 µg kg–1) and Chevalier (227 µg kg–1)). The results of the current study are within the range determined in previous research. Thus, mean and median concentrations of the sum of beauvericin and enniatins A, A1, B, B1 were 4150 and 215 µg kg–1 for wheat harvested in 2001 in Finland (Jestoi et al. 2004). Furthermore, Uhlig et al. (2006) showed that the content of enniatin B was the highest of the depsipeptide compounds followed by enniatin B1 in Norwegian wheat harvested in 2000–02. The same trend was found in our study where enniatin B followed by enniatin B1 was the major depsipeptide in all the varieties under study, with the exception of Selyemdur and Pentadur. According to our previous results, we found a higher content of moniliformin in durum wheat peaked at 417 µg kg–1 in Auradur than in common wheat being the minimum at 68 µg kg–1 in Chevalier. Different to other studies, while in our findings all the samples showed values of moniliformin above the LOD, only the 47% of Canada western amber durum harvested in Canada in 2000–02 contained moniliformin in the range from
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Relationship between lutein and mycotoxin content in durum wheat a
b
a
a
b
Rosa M. Delgado , Michael Sulyok , Ondřej Jirsa , Tomáš Spitzer , Rudolf Krska & Ivana a
Polišenská a
Agrotest fyto s.r.o., Kroměříž, Czech Republic
b
Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Tulln, Austria Accepted author version posted online: 20 May 2014.Published online: 09 Jun 2014.
Click for updates To cite this article: Rosa M. Delgado, Michael Sulyok, Ondřej Jirsa, Tomáš Spitzer, Rudolf Krska & Ivana Polišenská (2014) Relationship between lutein and mycotoxin content in durum wheat, Food Additives & Contaminants: Part A, 31:7, 1274-1283, DOI: 10.1080/19440049.2014.925589 To link to this article: http://dx.doi.org/10.1080/19440049.2014.925589
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 7, 1274–1283, http://dx.doi.org/10.1080/19440049.2014.925589
Relationship between lutein and mycotoxin content in durum wheat Rosa M. Delgadoa*, Michael Sulyokb, Ondřej Jirsaa, Tomáš Spitzera, Rudolf Krskab and Ivana Polišenskáa a
Agrotest fyto s.r.o., Kroměříž, Czech Republic; bDepartment for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Tulln, Austria
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(Received 25 February 2014; accepted 12 May 2014) Levels of lutein and a number of mycotoxins were determined in seven varieties of durum wheat (Triticum durum) and two varieties of common wheat (Triticum aestivum) in order to explore possible relationships amongst these components. Durum wheat cultivars always showed both higher lutein and mycotoxin contents than common wheat cultivars. The mycotoxins detected in both common and durum wheat cultivars were produced by the genera Fusarium, Claviceps, Alternaria and Aspergillus. Fusarium was the major producer of mycotoxins (26 mycotoxins) followed by Claviceps (14 mycotoxins), which was present only in some cultivars such as Chevalier (common wheat), Lupidur and Selyemdur (both durum wheat), Alternaria (six mycotoxins) and Aspergillus (three mycotoxins). Positive correlations between the levels of lutein and mycotoxins in durum wheat cultivars were found for the following mycotoxins: deoxynivalenol (DON), its derivative DON-3-glucoside, moniliformin, culmorin and its derivatives (5-hydroxyculmorin and 15-hydroxyculmorin). Keywords: durum wheat; lutein; mycotoxin; Fusarium; LC-MS/MS
Introduction Wheat is amongst the most widely grown food crop with a global production of about 600 million tonnes annually. Wheat varieties have traditionally been selected for functionality, e.g. making bread or biscuits resulting in the selection of common bread wheat (Triticum aestivum L.) due to its high level of strong gluten proteins, or durum wheat (Triticum turgidum L. subsp. durum Desf. [Hus.]) used mainly for yellowcoloured pasta products. Reductions in crop yield can be caused by Fusarium infection of cereal grains which causes Fusarium head blight (FHB). Furthermore, various species of this genus produce mycotoxins that are hazardous both to humans and animals. However, apart from Fusarium, toxic secondary fungal metabolites from other genera such as Aspergillus, Penicillium, Claviceps and Alternaria can also be found in wheat grain. In general, the most important target mycotoxins analytes in small grain cereals by these genera includes trichothecenes, zearalenone and its derivatives, fumonisins, enniatins, ergot alkaloids, ochratoxins, aflatoxins, moniliformin and patulin. Field surveys indicate that DON, a mycotoxin belonging to the trichothecenes group, as well as its derivatives, are the most frequently encountered mycotoxins associated with FHB disease of wheat throughout European countries, mainly produced by F. graminearum and F. culmorum (Bottalico & Perrone 2002). Although only a very limited number of mycotoxins is subject to legal guidance and regular monitoring (aflatoxins, fumonisins, DON, *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
zearalenone and ochratoxin A), the concern about the determination of the whole complex of mycotoxins and their metabolites rises. This initiated the development of multi-mycotoxin LC-MS/MS-based methods (Sulyok et al. 2006) allowing the presence of a large range of mycotoxins to be analysed and metabolites in the same sample, including masked mycotoxins (e.g., DON-3-glucoside or ZEN-4-glucoside). Fusarium infection constitutes not only an important food safety issue, it also reduces grain quality such as semolina yield, colour, and gluten strength in durum wheat. The quality of durum wheat grain depends on the genetic potential of variety, environmental conditions and their mutual interactions. It may be evaluated from different points of view: agronomical quality (potentiality and stability of grain yield), milling quality (semolina yield, ash content, humidity, impurity of grains), technological quality (protein and gluten content), and hygienic and sanitary quality (phytopathological microorganisms or their secondary metabolites). Nevertheless, from the point of view of the quality criteria of customers, the pigmentation of durum wheat grain and as a consequence of the end-products is one of the most relevant points used to evaluate the quality of the durum wheat. Lutein is considered as the major carotenoid component in wheat and the main responsible for its pigmentation. Lanchman et al. (2013) indicated that lutein is the most abundant carotenoid in wheat cultivars (on average ≈83%) with zeaxanthin (≈10%) and β-carotene (≈7%) being minor constituents. Its content in einkorn
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Food Additives & Contaminants: Part A (T. monococcum L., subsp. monococcum) wheat can reach 91% of the total carotenoids (Hidalgo et al. 2006) and 71% and 83% in spring and winter wheat (T. aestivum), respectively (Konopka et al. 2006). Durum wheat contains in general higher amount of carotenoids (1.5–4.0 mg kg–1 dry matter, DM) in comparison with common wheat (0.1–2.4 mg kg–1 DM) (Panfili et al. 2004). This pigmentation can be modified by processing conditions and enzymes activities (Trono et al. 1999). The yellow colour of durum wheat semolina and pasta products is attributed to its carotenoid content. This content may have beneficial health effects as the consumption of wheat with high a carotenoid level is linked to a reduction in oxidative damage by the ageing processes (Granado et al. 2003). In addition, it was previously established that an increase in the antioxidative potential of plants has a positive effect on pathogen resistance (Lorenc-Kukuła et al. 2005). The aim of this study was to determine the lutein content which is responsible for the much appreciated yellow colour as well as the level of a broad spectrum of mycotoxins in different varieties of durum wheat. Furthermore, relationships between lutein and mycotoxin content were investigated in the different cultivars to establish if varieties with higher lutein content, and therefore more appreciated due to their yellowness, also have a higher or lower mycotoxin content.
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Materials and methods Materials Plant material Field experiments were carried out at the location in Kroměříž (Czech Republic) during the growing season 2012–13. Kroměříž (49°16–17°N/17°21–22°E. l.) is located in the sugar beet agricultural production area with the altitude of 235 m in a plain terrain of the River Morava. The region is characterised as warm and moderately wet. The soil type is classified as luvisol chernozem, soil type clayey, medium heavy. The average annual temperature measured at the meteorological station in Kroměříž in the period 1971–2010 is 9.2°C, the average total annual precipitation is 576 mm (long-term normal). Actual weather conditions in the growing season 2012–13 in comparison with long-term normal are illustrated in the Figure 1. The field experiments were established as orthogonal randomised blocks with three replications and the size of individual plot was 10 m2. Individual variants were harvested separately, the grain from appropriate replications was thoroughly mixed and analysed as one sample in triplicate. In the varietal trial, seven varieties of T. durum (IS Pentadur, Logidur, Auradur, Lunadur, Lupidur, Cliodur and Selyemdur) and two varieties of T. aestivum (Akteur and Chevalier) were involved. The trial was sown
Figure 1. Weather conditions in the growing season 2012–13, data given for pentad periods and compared with long-term normal (1971–2010) for Kroměříž, Czech Republic.
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on 11 October 2012; the preceding crop was lucerne. Before sowing, 35 kg ha–1 of P2O5 (superphosphate 43%) and 125 kg ha–1 of K2O (potassium salt 60%) were applied. Sowing rate was 4 million of germinating seeds per 1 ha. Nitrogen was added in the form of potassium nitrate (27%) in the dose equal to 30 kg N ha–1 (8 March 2013) and 50 kg N ha–1 (15 April 2013) and then in the form of DAM (ammonium nitrate 42.2% and 32.7% of urea) in the dose equal to 40 kg N ha–1. The trials was treated with growth regulators Retacel extra R 68 (chlormequat – chloride 720 g l–1) at a dose of 1.5 l ha–1 (23 April 2013) and Moddus (250 g l–1 trinexapac-ethyl) at a dose of 0.3 l ha–1 (10 May 2013). Fungicides were applied twice: 16 May 2013 – Hutton (spiroxamine 250 g l–1, prothioconazole 100 g l–1, tebuconazole 100 g l–1) at a dose of 0.8 l ha–1 and 07 June 2013 – Prosaro (prothioconazole 125 g l–1, tebuconazole 125 g l–1) at a dose of 0.75 l ha–1. Crop treatment and protection was the same as would be in a good farming practice appropriate to the area in order to achieve good grain quality. All trial plots were harvested by a small plot combine (1 August 2013 with the exception of Chevalier and Lupidur, which were not rape enough and were harvested on 8 August 2013); from harvested grain representative subsamples (approximately 1 kg each) for laboratory analyses were taken. Grains samples were milled using sieve of 0.5 mm. They were divided into plastic bags and kept at –20°C until mycotoxin analysis. Chemicals LC gradient-grade methanol, acetonitrile, isopropyl alcohol, hexane and MS-grade glacial acetic acid were purchased from Sigma Aldrich (Vienna, Austria; and Schnelldorf, Germany). A Purelab Ultra system (ELGA LabWater, Celle, Germany) was used for further purification of reverse osmosis water. All other reagents were of analytical or HPLC grade and were from Lach:ner (Neratovice, Czech Republic). Standards of lutein and trans-β-apo-8-carotenal were purchased from Sigma-Aldrich (Schnelldorf, Germany). Standards solutions of lutein and the internal standard (trans-β-apo-8-carotenal) were prepared in n-butanol (0.5 and 1 mg ml–1, respectively) and stored at –20°C. The working standards solutions were freshly prepared prior to the calibration by dilution. The internal standard spiking solution (25 ng µl) was stored inter-day analysis at –20°C. Standards of fungal and bacterial metabolites were obtained either as gifts from various research groups or from the following commercial sources: Romer Labs (Tulln, Austria), Sigma-Aldrich (Vienna, Austria), Iris Biotech GmbH (Marktredwitz, Germany), Axxora Europe (Lausanne, Switzerland), BioAustralis (Smithfiled, Australia), BiovioticaNaturstoffe GmbH (Dransfeld, Germany), Fermentek (Jerusalem, Israel) and
LGC Promochem GmbH (Wesel, Germany). Stock solutions of each analyte were prepared by dissolving the solid substance in acetonitrile (preferably), acetonitrile/water 1:1 (v/v), methanol, methanol/water 1:1 (v/v) or water. Thirty-four combined working solutions were prepared by mixing the stock solutions of the corresponding analytes for easier handling and were stored at –20°C. The final working solution was freshly prepared prior to spiking experiments by mixing of the combined working solutions.
Methods Sample preparation for carotenoids and mycotoxins determination ● Carotenoid determination. Carotenoids were extracted according to Panfili et al. (2003) with the modifications of Peterson et al. (2007). Thus, 0.5 g of wheat samples with 100 µl of internal standard (trans-β-apo-8-carotenal, 25 ng µl–1) were combined with 0.5 ml potassium hydroxide (600 g l–1), 0.5 ml of 95% ethanol, 0.5 ml of sodium chloride (10 g l–1) and 1.25 ml of pyrogallol (60 g l–1 ethanol) in screw dark tubes. They were incubated at 70ºC under nitrogen atmosphere for 25 min, then cooled in an ice water bath. After adding 3.75 ml of sodium chloride (10 g l–1), the samples were extracted with 3.75 ml of hexane/ethyl acetate (9:1, v/v) until colourless. The organic extracts were combined and evaporated to dryness under nitrogen. The residues were dissolved in 1 ml of the mobile phase (methanol/acetonitrile/isopropyl alcohol, 38:60:2). Then, 10 µl of the samples were analysed by an HPLC-DAD system. ● Mycotoxin determination. Ground wheat (5 g) was extracted with 20 ml of acetonitrile/water/acetic acid (79/20/1) for 90 min using a GFL 3017 rotary shaker (GFL, Burgwedel, Germany). Aliquots of 100 µl were transferred into glass vials and diluted with the same amount of a mixture containing acetonitrile/water/acetic acid (20/79/1). After mixing, 5 µl of the diluted extract were injected into the LC-MS/MS system.
HPLC analysis ● Carotenoids analysis (HPLC-DAD). Chromatography was performed using a Shimadzu HPLC 20AD (Kyoto, Japan) Prominence analytical system equipped with an automatic sampler. Separation was done on a Kinetex Phenomenex C18 column
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Food Additives & Contaminants: Part A
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Figure 2. Chromatogram of wheat extracts with the added internal standard (trans-β-apo-8-carotenal) at 445 nm. Retention times of lutein and trans-β-apo-8-carotenal were 3.346 and 4.851 min, respectively.
(250 × 4.6 mm i.d., particle size = 5 µm) (Torrance, CA, USA). Spectrophotometric detection was achieved by means of a diode array detector SPDM20A (λ = 445 nm). The mobile phase was methanol/acetonitrile/isopropyl alcohol (38/60/2) at a flow rate of 1.5 ml min–1. Lutein was identified through comparison of its retention time with those of known available standard solutions and quantified on the basis of calibration curves of standard solutions (r2 > 0.99) (Figure 2). Data were processed by LC Solution version 1.21 SP1 (Torrance, CA, USA). ● Mycotoxins analysis (LC-MS/MS). Detection and quantification was performed with a QTrap5500 LC-MS/MS System (Applied Biosystems, Foster City, CA) equipped with a TurboIonSpray electrospray ionisation (ESI) source and an 1290 Series UHPLC System (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25°C on a Gemini® C18-column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a C18 security guard cartridge, 4 × 3 mm i.d. (all Phenomenex). Elution was carried out in binary gradient mode. Both mobile phases contained 5 mM ammonium acetate and were composed of methanol/water/acetic acid 10:89:1 (v/v/v; eluent A) and 97:2:1 (v/v/v; eluent B), respectively. After an initial time of 2 min at 100% A, the proportion of B was increased linearly to 50% within 3 min. Further linear increase of B to 100% within 9 min was followed by a hold time of 4 min at 100% B and 2.5 min column re-equilibration at 100% A. The flow rate was 1000 μl min–1. ESI-MS/MS was performed in the scheduled multiple reaction monitoring (sMRM) mode in both positive and negative polarities in two separate chromatographic runs. The sMRM detection window of each analyte was
set to the respective retention time ±27 s and ±42 s in positive and in negative mode, respectively. The target scan time was set to 1 s. Confirmation of positive analyte identification is obtained by the acquisition of two sMRMs per analyte (with the exception of moniliformin and 3-nitropropionic acid, which exhibited only one fragment ion). This yielded 4.0 identification points according to Commission Decision 2002/657/EC (EU 2002). LOD was defined as three times the signal/noise ratio (Table 1). Analyst® software version 1.5.1 (AB Sciex, Foster City, CA, USA) was used to control the LC-MS/MS instrument, as well as for automatic and manual integration of the peak.
Statistical analysis The extraction was carried out in triplicate and data were reported as mean ± SD. Analysis of variance (ANOVA) was carried out and a Fisher least significant difference (LSD) test was used to describe means with 95% confidence. The Spearman correlation coefficients were calculated by STATISTICA Cz 10 statistical software (StatSoft CR, Prague, Czech Republic) at a probability level of p < 0.05.
Results and discussion Lutein content in common and durum wheat cultivars Figure 3 shows the lutein content determined by HPLCDAD for seven durum and two common wheat cultivars from our trial. Significant differences were found in lutein content between all cultivars both for durum and common
6.0 0.1 1.0 6.9 9.1 18.2 0.3 0.0
± ± ± ± ± ± ± ± ± 3.3 ± 1.2
0.1 13.9 1.4 0.9
± ± ± ±
± 19.2 ± 2.9
Akteur
Claviceps Elymoclavine 0.5 Ergine 0.08 Ergocornine 0.8 Ergocorninine 0.2 Ergocristine 1.0 Ergocristinine 0.1 Ergocryptine 0.4 Ergocryptinine 0.06 Ergometrinine 0.03 Ergosin 0.80 Ergotamine 0.3 Ergovalin 0.5 Chanoclavine 0.008 Secalonic acid D 4.0 Fusarium 15-Hydroxyculmorin 3 136.9e 15-Hydroxyculmoron n.d. 26.2c 5-Hydroxyculmorin n.d. Apicidin 0.1 1.0ef Aurofusarin 1.0 144.1cd Beauvericin 0.002 6.1bc Culmorin 0.8 34.2h Diacetoxyscirpenol 0.15 Don 0.5 116.4h Don glucoside n.d. 2.4f Enniatin A 0.01 3.9f Enniatin A1 0.02 37.8e Enniatin B 0.01 157.8f Enniatin B1 0.02 133.5f Enniatin B2 0.06 8.6f Enniatin B3 0.001 0.1g Equisetin 0.20 HT2 toxin 16 Moniliformin 0.5 127.5e Monoacetoxyscirpenol 1.5 13.2b
LOD
68.0f ± 2.1 16.7a ± 0.6
53.1i ± 1.9 1.6g ± 0.1 2.8f ± 0.2 27.5e ± 4.5 102.1g ± 3.8 85.1g ± 1.3 5.1g ± 0.1 0.1g ± 0.0 ± ± ± ± ± ± ± ±
27.9 158.6e ± 13.3 6.4 34.9c ± 1.7 9.3 61.3e ± 12.3 0.1 4.5c ± 0.8 63.0 203.3c ± 29.4 0.5 4.2d ± 1.0 8.4 202.4f ± 10.0
Logidur
± ± ± ± ± ± ± ±
23.8 0.8 0.8 3.3 22.1 14.8 0.8 0.0
9.2 5.5 3.3 0.2 43.7 0.8 9.4
283.4c ± 12.0 9.5cd ± 1.1
582.7e 6.5d 23.1c 147.4c 454.5c 392.7d 25.8c 0.2e
± ± ± ± ± ± ±
Lunadur
464.2a ± 28.6 167.4e 87.8a ± 12.0 25.4c a 242.2 ± 13.7 158.0bc 2.2d ± 0.2 1.6de a 603.9 ± 24.2 305.1b 4.5d ± 0.4 4.7cd a 667.6 ± 10.8 240.2e
Auradur
13.0 460.1f ± 16.1 1275.7a ± 61.1 0.2 4.1e ± 0.2 12.4a ± 0.6 f 2.3 2.4 ± 0.4 12.8de ± 1.2 33.3 31.7e ± 5.0 163.7c ± 31.2 11.5 239.1e ± 23.9 619.5a ± 5.0 19.2 154.0f ± 15.7 564.1b ± 14.9 1.5 13.8e ± 1.5 41.2a ± 0.7 0.0 0.1f ± 0.0 0.4b ± 0.0 c 1.6c ± 0.2 4.6 ± 1.1 39.9a ± 4.3 19.5b ± 1.3 262.9c ± 5.7 192.3d ± 5.3 417.2a ± 51.4 8.8cd ± 0.1 7.3ef ± 0.4 10.1c ± 1.3
309.8g 4.2e 26.9b 197.2b 525.8b 529.6c 31.1b 0.3c
0.6f ± 0.1 22.2e ± 5.2 4.9cd ± 0.9 46.1h ± 5.7
± ± ± ± ± ± ±
0.9c ± 0.1
1.4d ± 0.1 1.2c ± 0.2
Pentadur
216.0d 24.5c 92.7d 1.5de 360.6b 6.9b 167.5g
0.8a ± 0.2 0.8a ± 0.1 462.7b ± 22.8 365.4a ± 16.5 276.9a ± 49.1 88.4a ± 12.3 260.1a ± 28.6 67.6a ± 2.8 253.0a ± 14.4 126.6a ± 5.6 65.4a ± 7.3 31.6a ± 3.3 14.2a ± 2.2 1792.0a ± 61.4
Chevalier
Mycotoxins level (ppb) in common and durum wheat classified by mycotoxin producer.
Mycotoxins
Table 1.
882.9c 7.4bc 13.5d 142.9c 614.1a 592.2a 40.9a 0.4a 123.6a 23.5b 320.2b 8.6de
361.8b 61.2b 181.6b 8.9b 312.7b 8.5a 562.1c
± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ±
3.6 0.5 0.6 10.0 20.3 19.4 1.0 0.0 4.5 1.8 15.3 0.1
9.0 9.6 11.3 0.8 42.7 1.0 10.5
0.0 11.7 9.9 1.4 1.6 8.2 1.6 2.5 3.1 0.8 0.2 0.2 70.7
Lupidur
0.1c 111.5c 90.0b 83.8b 25.2b 139.7b 22.4c 27.3c 45.8c 10.8b 1.5b 2.3c 442.4c
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± ± ± ± ± ± ±
22.4 6.5 16.0 0.1 26.5 0.6 5.1
± ± ± ± ± ± ± ±
297.9a 55.1b 179.3b 99.4b 4.5b 5.2b 7.3b 671.9b
33.8 5.9 23.8 0.5 17.9 0.2 15.4 0.1 42.1 0.3 1.9 10.0 10.4 3.0 1.5 0.0 0.7 1.3 5.8 0.9
24.7 2.1 33.4 11.9 0.5 0.6 1.4 79.3
0.2 0.0 55.3 64.5
(continued )
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ±
1.0a 0.5b 535.8a 419.3a
Selyemdur
292.8c 66.2b 152.0c 41.2a 85.5de 5.1cd 373.7d 1.5 974.4b ± 23.2 766.2d 7.7b ± 0.3 7.0cd e 11.4 ± 0.6 39.8a 91.8d ± 0.9 254.9a 425.0d ± 12.7 427.8d 345.6e ± 8.8 576.6ab 22.2d ± 0.8 25.0c 0.3d ± 0.0 0.2e c 1.9 ± 0.2 22.3b 14.4c bc 288.4 ± 9.7 221.5d 7.1f ± 0.7 13.6b
279.1c 61.5b 232.9a 0.3f 193.2c 5.3cd 623.1b
0.6c ± 0.0
Cliodur
1278 R.M. Delgado et al.
0.03 0.5 3.0 0.1 0.03 0.3
Monocerin Nivalenol T2 tetraol T2 toxin Zea-4-sulfate Zearalenon Alternaria Altenariol Altenariolmethylether Altertoxin I Infectopyron Macrosporin Tentoxin Aspergillus 3-Nitropropionic acid Asperloxin A Emodin Unspecific Tryptophol ± ± ± ± ±
0.0 0.1 9.8 0.4 0.2
37.4b ± 1.0
n.d.
1.0de ± 0.1
0.5de ± 0.3 1.6c ± 0.1 142.0c ± 6.8 9.5f ± 0.7 10.8c ± 0.2
49.6d ± 3.1 10.1b ± 1.5
Chevalier
± ± ± ± ±
0.1 0.3 6.8 7.7 0.0
4.1c ± 0.3 10.7b ± 0.4 0.9fg ± 0.1
0.5de 1.7c 245.3a 36.4e 5.7fg
1.5a ± 0.0 94.7a ± 5.4 30.7a ± 3.9
0.5c ± 0.1 67.3c ± 3.3
± ± ± ± ± ±
2.6 0.6 0.1 17.7 1.5 1.5
3.3 ± 0.2 0.8g ± 0.1
e
12.0a 4.9a 2.2b 225.4b 25.0ef 12.6b
0.5a ± 0.1
Logidur
Pentadur
11.7c ± 3.0 7.3d ± 0.4 0.9ef ± 0.0
0.2e ± 0.0 2.6a ± 0.2 74.2e ± 8.4 60.4d ± 4.7 4.5g ± 0.1
1.1b ± 0.3 45.5d ± 1.7 16.2b ± 2.8 7.6ab ± 1.1 1.1a ± 0.2 0.7a ± 0.4
Auradur
± ± ± ± ± ±
2.2 0.1 0.2 13.9 12.6 0.8 28.4b ± 6.5 16.3a ± 1.1 1.2ab ± 0.1
± ± ± ± ± ±
0.5a ± 0.1
31.6e ± 1.6 26.7a ± 3.7 8.1a ± 1.2
Lupidur
0.2 7.6b 0.2 1.6b 0.1 2.8a 8.6 221.6b 15.8 149.5bc 0.5 13.0b 10.3 ± 0.0 1.1bc ± 0.0
b
1.5c 1.3bc 2.1b 110.6d 346.1a 17.2a
0.4a ± 0.2
4.6d ± 0.3
1.5a ± 0.2 53.9d ± 5.9
Lunadur
Notes: Values are means ± SD. Means in the same row with different superscripts are significantly different (p < 0.05). LOD = limit of detection by LC-MS/MS.
0.6h ± 0.0
7.3c ± 0.5
0.1e 1.5c 90.2e 2.2f 6.3ef
86.4ab ± 10.0 14.3b ± 3.6
Akteur
1.0 0.4 0.2
0.3 0.02 0.3 n.d. 0.5 0.15
LOD
Mycotoxins
Table 1. Continued .
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± ± ± ± ± ±
± ± ± ± ±
0.2 0.2 0.3 5.3 23.4 0.8
0.2 1.9 1.8 0.3 0.2
60.6a ± 9.0
5.5c ± 0.7 8.9c ± 0.2 1.1cd ± 0.0
1.0c 0.9cd 2.7a 146.6c 154.4b 8.9d
0.7c 49.7d 11.9b 6.5bc 1.3a
Cliodur
± ± ± ± ± ±
± ± ± ±
0.0 0.1 0.2 5.9 25.1 0.5
0.4 5.7 6.7 0.5
48.6a ± 11.9 3.4e ± 0.1 1.2a ± 0.1
0.3c 0.4e 2.9a 133.1c 130.9c 6.9e
1.2ab 80.0b 29.9a 5.9cd
Selyemdur
Food Additives & Contaminants: Part A 1279
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R.M. Delgado et al.
Figure 3. Determination of lutein content by LC-DAD in common and durum wheat cultivars. Letters indicate significant difference (p < 0.05).
wheat. As expected, common wheat (Akteur and Chevalier) showed a lower lutein content (2.9 and 3.8 µg g–1, respectively) than most durum wheat (values in the range from 3.7 to 7.3 µg g–1 with the exception of the cultivar Auradur with the highest value, 12 µg g–1). These values are in accordance with earlier observations by Leenhardt et al. (2006) who explored the genetic variability of carotenoid concentration among cultivated diploid (einkorn, T. monococcum L.), tetraploid (durum wheat, T. turgidum L. subsp. durum (Desf.) Husn.) and hexaploid (common wheat, T. aestivum L.) wheat species. They reported a varietal variation in the lutein concentration of 5.7, 3.3 and 1.2 mg kg–1 DM for einkorn, durum and common wheat, respectively. Durum wheat is appreciated by its elevated yellow pigment which is provided by its lutein content. The findings of the current study provide information about the lutein variation based on the cultivar of durum wheat. These results are consistent with previous research which found that lutein was present in Canadian durum wheat in the amount ranging from 0.37 to 5.6 µg g–1 (Van Hung & Hatcher 2011). The most striking result to emerge from the data is that Auradur cultivar can reach lutein values of 12 µg g–1. The relevance of the lutein that contributes to the desirable yellowness of semolina and flours to make a healthy yellow alkaline noodle without artificial yellow agent (Van Hung & Hatcher 2011) provides an interest in cultivars such as Auradur with higher lutein content. Mycotoxins in common and durum wheat cultivars Wheat harvested in our trial was contaminated by toxins produced by fungi belonging to Fusarium, Alternaria, Aspergillus and Claviceps genera. In total, 50 fungal secondary metabolites were detected with most of them attributed to Fusarium (26 metabolites) were found,
followed by Claviceps (14 metabolites), Alternaria (six metabolites), Aspergillus (three metabolites), in addition to one unspecific metabolite (tryptophol) (Table 1). However, the presence of these mycotoxins depended on the cultivar under study. While Claviceps affected mainly one cultivar of common (Chevalier) and two cultivars of durum wheat (Lupidur and Selyemdur), Fusarium, Alternaria and Aspergillus mycotoxins were present in almost all the cultivars (common and durum wheat). Many durum cultivars are more susceptible to Fusarium infection and subsequent mycotoxin contamination in comparison with common wheat and other small cereal grains. This is supported by Clear et al. (2005) who found higher DON and moniliformin concentrations in durum samples grown in the same crop districts as common wheat in western Canada. Although our study only comprised two common wheat cultivars and no general statements can be made, the trend of our data is consistent with those previous results. Thus, Fusarium metabolite production was higher in all durum than in common wheat cultivars mainly in enniatins, DON, aurofusarin, culmorin and its derivates, and moniliformin. European Union legislation establishes legal limits for DON in unprocessed cereals other than durum wheat, oats and maize at the level of 1250 µg kg–1. However, the limit is higher for unprocessed durum wheat, maize and oats (1750 µg kg–1) (EU 2006). All our durum wheat samples showed a DON content between 53 and 1275 µg kg–1 which is under the appropriate limit of legislation. The highest value was found in durum cultivar Auradur (1275 µg kg–1), while the lowest was found in common wheat (Akteur (116 µg kg–1) and Chevalier (53 µg kg–1)). Besides DON, DON-3-glucoside was detected in all samples being also higher in durum than in common wheat. Recent studies have pointed out that lactic acid bacteria commonly found in the digestive tract of mammals are able to cleave a significant proportion of DON-3-glucoside
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Food Additives & Contaminants: Part A releasing DON in vitro (Berthiller et al. 2011). Thus, the total bioavailable amount of DON in contaminated cereal could be increased by the presence of DON-3-glucoside. However, DON-3-glucoside showed a considerably lower toxicological relevance than DON in rats in vivo (Nagl et al. 2012). Furthermore, even if we consider the conversion of DON-3-glucoside in DON, the total amount of bioavailable DON would not surpass the European Commission guidance value in any sample. As with DON, durum wheat showed a higher content of culmorin and hydroxyculmorin derivates than common wheat with the exception of Pentadur in 15-hydroxyculmoron content. A correlation was found between DON and culmorin (r = 1.000) as well as DON with hydroxylated analogues of culmorin such as 5-hydroxyculmorin (r = 0.929). This co-occurrence, which has also been pointed out by other authors (Uhlig et al. 2013), might be due to F. graminearum being a producer of both mycotoxins, DON and culmorin (Desjardins 2006). The content of depsipeptide compounds (the sum of enniatins A, A1, B, B1, B2 and beauvericin) showed a higher content in durum wheat as well (Lupidur (1412 µg kg–1), Auradur (1406 µg kg–1), Selyemdur (1329 µg kg–1), Pentadur (1317 µg kg–1), Lunadur (1048 µg kg–1), Cliodur (901 µg kg–1), Logidur (445 µg kg–1)) than in common wheat (Akteur (347 µg kg–1) and Chevalier (227 µg kg–1)). The results of the current study are within the range determined in previous research. Thus, mean and median concentrations of the sum of beauvericin and enniatins A, A1, B, B1 were 4150 and 215 µg kg–1 for wheat harvested in 2001 in Finland (Jestoi et al. 2004). Furthermore, Uhlig et al. (2006) showed that the content of enniatin B was the highest of the depsipeptide compounds followed by enniatin B1 in Norwegian wheat harvested in 2000–02. The same trend was found in our study where enniatin B followed by enniatin B1 was the major depsipeptide in all the varieties under study, with the exception of Selyemdur and Pentadur. According to our previous results, we found a higher content of moniliformin in durum wheat peaked at 417 µg kg–1 in Auradur than in common wheat being the minimum at 68 µg kg–1 in Chevalier. Different to other studies, while in our findings all the samples showed values of moniliformin above the LOD, only the 47% of Canada western amber durum harvested in Canada in 2000–02 contained moniliformin in the range from
