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Occurrence and risk assessment of zearalenone through broa consumption, typical maize bread from Portugal
Accepted Manuscript Occurrence and risk assessment of zearalenone through broa consumption, typical maize bread from Portugal Nuno M.C. Ribeiro, Liliana J.G. Silva, Angelina Pena, Celeste M. Lino PII:
S0956-7135(15)00218-2
DOI:
10.1016/j.foodcont.2015.03.043
Reference:
JFCO 4398
To appear in:
Food Control
Received Date: 13 December 2014 Revised Date:
20 March 2015
Accepted Date: 25 March 2015
Please cite this article as: Ribeiro N.M.C., Silva L.J.G., Pena A. & Lino C.M., Occurrence and risk assessment of zearalenone through broa consumption, typical maize bread from Portugal, Food Control (2015), doi: 10.1016/j.foodcont.2015.03.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Occurrence and risk assessment of zearalenone through broa
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consumption, typical maize bread from Portugal
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Nuno M. C. Ribeiro, Liliana J.G. Silva, Angelina Pena, Celeste M. Lino∗
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LAQV, REQUIMTE, Group of Bromatology, Pharmacognosy and Analytical Sciences,
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Faculty of Pharmacy, University of Coimbra, Polo III, Azinhaga de Stª Comba, 3000-
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548 Coimbra, Portugal
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* Corresponding author:
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[email protected]; [email protected]
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Faculty of Pharmacy, University of Coimbra Pólo das Ciências da Saúde
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Azinhaga de Santa Comba
3000-548 Coimbra, Portugal
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Phone number: 00351239488477 Fax number: 00351239488503
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Abstract
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The occurrence of zearalenone (ZEA) in maize bread, broa, for human consumption,
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from the Portuguese market, was evaluated. Good analytical performance was obtained
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through extraction with acetonitrile:water (90:10), clean-up with immunoaffinity
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columns, and detection and quantification by liquid chromatograph with-tandem mass
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spectrometry. ZEA levels were determined in 52 samples to verify the compliance with
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the maximum permitted levels by the European legislation. One broa sample exceeded
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the maximum limit established. A higher contamination frequency was observed in
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samples in which maize and wheat were present. Overall, 13.5% of the samples were
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contaminated, at levels oscillating between 9.6 and 50.4 µg/kg. Considering the
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percentage of the tolerable daily intake (TDI) obtained, 0.6%, the risk assessment linked
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with the exposure to ZEA was considered very low for the studied population. This is
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the first study on the intake assessment of ZEA through maize bread consumption in
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Portugal.
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Keywords:
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Zearalenone; maize bread; risk assessment; Portuguese population.
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1. Introduction Zearalenone (ZEA), a metabolite primarily associated with several Fusarium
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species (F. culmorum, F. graminearum, F. sporotrichioides, F. cerealis, F. equiseti, F.
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crookwellense and F. semitectum) (Marasas, 1991 ; Zinedine, Soriano, Moltó, & Mañes,
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2007) and known as 6-(10-hydroxy-6-oxo-trans-1-undecenyl) - β-resorcylic acid-
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lactone (Marasas, 1991), is a phytoestrogenic compound (Diekman & Green, 1992)
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along with its alcohol metabolites, α-zearalenol (α-ZEN) and β-zearalenol (β-ZEN)
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(Cheeke, 1998, Kaushik, 2014). Despite being a non-steroidal estrogenic toxin, it was
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categorized in the group 3 (not classifiable as to its carcinogenicity to humans) by the
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International Agency for Research on Cancer (International Agency for Reserach on
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Cancer, 2002).
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ZEA is most commonly found in maize (Ryu, Hanna, Eskridge, & Bullerman,
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2003; Zinedine, Soriano, Moltó, & Mañes, 2007), as well as in its derivatives such as
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flour (Ramos Aldana , Silva, Pena, Mañes, & Lino, 2014). Broa, a traditional maize
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bread highly consumed in Portugal, especially in the north and central zones, has been
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studied for fumonisins B1 (FB1) and B2 (FB2) (Lino, Silva, Pena, Fernández, & Mañes,
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2007) but not for ZEA. As far as we know, few studies on the ZEA content in other
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traditional maize foods, such as tortillas (Hewitt, Flack, Kolodziejczyk, Chacon, &
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D’Ovidio, 2012), and polenta (MacDonald, Anderson, Brereton, Wood, & Damant,
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2005), typical maize-based products from Mexico and northern Italy, respectively, are
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available in the scientific literature. The results on the stability of the main Fusarium
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toxins, during traditional Turkish maize bread production, revealed that deoxynivalenol
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(DON), ZEA and FBs remained stable during the bread making, indicating that breads
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produced from highly contaminated maize may represent a potential risk to the
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population (Numanoglu, Uygun, Koksel, & Solfrizzo, 2010).
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The traditional processing of broa, as reported by Lino, Silva, Pena, Fernández, &
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Mañes (2007) consists of adding sieved maize flour, its principal ingredient, wheat
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flour, hot water, yeast and leavened dough from the late broa. After mixing, working up
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and leavening, the dough is baked in a wood-fired oven. Previous studies on maize-based products for human consumption in Portugal,
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ragarding mycotoxins produced by Fusarium species, such as ZEA in flours and FB1
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and FB2 in broa, showed a higher prevalence in maize flour, 50%, than in wheat flour,
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31.6%, as well as the highest mean levels (Ramos Aldana , Silva, Pena, Mañes, & Lino,
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2014), and a high incidence of FB1 and FB2 in broa, 83%, with 27% of the samples
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exceeding the regulatory limits (Lino, Silva, Pena, Fernández, & Mañes, 2007).
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The European Commission (EC), through EC legislation Nº 1126/2007, set
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regulatory limits, as regards Fusarium toxins in maize and maize products, in order to
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protect public health and the maximum limit established for ZEA in bread is 50 µg/kg
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(European Commission, 2007). On the other hand, the European Food Safety Authority
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(EFSA) has published a scientific opinion on the risks to public health in 2011 for ZEA,
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and proposed a TDI of 0.25 µg/kg b.w./day based on more recent data on pig, but also
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taking into account comparisons between pigs and humans (EFSA, 2011).
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This first report on the incidence of ZEA in maize bread from Portugal was aimed
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to evaluate its levels in samples, obtained from the Portuguese market. Good analytical
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performance was obtained using extraction with acetonitrile:water (90:10), clean-up
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with immunoaffinity columns, followed by detection and quantification by liquid
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chromatography with tandem mass spectrometry. The occurrence and levels of ZEA
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were determined in 52 samples in order to verify the compliance with the maximum
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limits of the European legislation. This study also intended to present the results
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concerning the exposure of the Portuguese population, inhabiting the central zone,
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through the consumption of this typical bread, and evaluate the risk with regard to the
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international health-based guidance values.
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2. Materials and methods
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2.1. Sampling
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A total of 52 broa samples (15 in which composition yellow maize was the only
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cereal, and 37 composed by yellow maize and wheat) were analysed. The samples were
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purchased in commercially available size in different supermarkets, markets and
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groceries of Coimbra, central zone of Portugal, between September and October 2014.
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After purchase, samples were brought to the laboratory under ambient conditions, all
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the information available on the labels was assembled, and were frozen until their
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analysis.
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2.2. Chemical, reagents and equipment
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The reagents of HPLC grade used were acetonitrile (Merck, KGaA, Darmstadt,
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Germany) and methanol (Carlo Erba, Milan, Italy). Formic acid 98-100% was obtained
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from Merck (KGaA, Darmstadt, Germany), and sodium chloride from Pronolab
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(Lisboa, Portugal). Micro-glass fiber paper (150 mm, Munktell & Filtrak GmbH,
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Bärenstein, Germany), Whatman N°1 filter paper, polyamide membrane filters (0.2 µm,
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50 mm; Whatman GmbH, Dassel, Germany), and Durapore membrane filter (0.22 um,
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GVPP, Millipore, Ireland) were used. Immunoaffinity columns (IAC) ZearalaTestTM
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were from VICAM (Watertown, USA).
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Water was daily obtained from Milli-Q System (Millipore, Bedford, MA, USA) and
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the ZEA standard, a white powder with a purity degree ≥99.0%, was purchased from
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Sigma-Aldrich (St. Louis, MO, USA). A mobile phase (40% water with 0.1% formic
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acid and 60% acetonitrile) at 200 µL/min, was used. All liquid chromatographic
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reagents were degassed for 15 minutes in an ultrasonic bath. ZEA standard stock solution was prepared at 5 mg/mL, diluting 10 mg of ZEA in 2
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mL of acetonitrile, and stored at -20ºC. The intermediate solution was prepared by
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diluting the stock solution at 50 µg/mL, in acetonitrile, and a working standard solution,
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at 1 µg/mL in acetonitrile, was prepared by diluting the intermediate solution. They
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were stored in darkness, at 4 ºC, until the analysis. The calibration curve standard
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solutions, in mobile phase, were prepared between 10 and 100 ng/mL (10, 20, 50, 75,
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100 ng/mL). For the matrix-matched calibration curve, standards were prepared
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between 10 and 100 µg/kg (10, 20, 50, 75, 100 µg/kg).
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A 3–16 k Sigma centrifuge (Reagente 5, Porto, Portugal), a Braun MR 5000 M
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multiquick/minipimer 500W (220–230 V, 50–60 Hz) (Esplungues del Llobregat,
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Spain), a vacuum manifold of Macherey-Nagel (Düren, Germany), a Dinko pump (mol.
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D-95, 130 W, 220 V), a magnetic stirrer (Agimatic-S, Selecta, Barcelona, Spain), a
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Retsh vortex mixer (Haan, Germany), and a Sonorex RK 100 ultrasonic bath (Berlin,
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Germany) were employed.
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2.3. Sample extraction and clean-up Extraction and clean-up were performed according to Ramos Aldana , Silva, Pena,
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Mañes, & Lino (2014). Briefly, 20 g of sample, mixed with NaCl, were extracted twice
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with acetonitrile:water (90:10), and the supernatants were mixed with Milli-Q water and
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filtered. An aliquot (10 mL) of the filtered was cleaned through the ZearalaTestTM IAC
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attached onto a vacuum manifold. After column washing with water, an elution with
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methanol was performed. After drying, at 42 ºC under a gentle nitrogen flow, the dried
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extract was stored at -20 ºC.
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For liquid chromatography coupled to tandem mass spectrometry (LC/MSn)
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analysis, the dried extracts were dissolved in 1.0 mL mobile phase (40% water with
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0.1% formic acid and 60% acetonitrile).
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2.4. LC/MSn conditions
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LC/MS2 analyses were performed using a Liquid Chromatograph of High
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Performance (Thermo Finnigan, San Jose, California, USA) coupled to a LCQ
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Advantage MAX Quadrupole Ion Trap Mass Spectrometer (Thermo Finnigan, San Jose,
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California, USA). The LC column (Waters Spherisorb ODS2; 3 µm, 150x2.1 mm i.d;
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Waters Corporation, Milford, U.S.A.) was preceded by a guard cartridge (Waters
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Spherisorb ODS2; 5 µm, 10x4.6 mm i.d.; Waters Corporation, Milford, U.S.A.). A 20
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µL (partial loop) injection volume was used with the mobile phase (40% water with
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0.1% formic acid and 60% acetonitrile) flow maintained at 200 µL/min.
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The MS was operated in the negative electrospray ionization (ESI) mode using
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selected reaction monitoring (SRM) acquisition. Nitrogen was used as nebulizing gas,
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with a sheath gas flow of 80 (arbitrary unit) and the auxiliary sweep gas flow of 20
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(arbitrary unit). Source and capillary temperatures were set at 0ºC and 270ºC and
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voltages at 4.5 and 10V, respectively. Collision gas was helium with a normalized
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collision energy of 35%. Three precursor-to-fragment transitions were acquired: m/z
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317(|M-H|-)> m/z 273 (for quantification purpose); m/z 317(|M-H|-)> m/z 175 and m/z
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317(|M-H|-)> m/z 149 (for confirmation purposes).
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2.5. Recovery studies
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Recoveries were determined by spiking ZEA – maize bread at three different levels,
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20, 50, and 100 ng/g, using three replicates for each level, according to the maximum 7
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foods and processed maize-based foods for infants and young children, bread (including
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small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals, excluding
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maize-snacks and maize-based breakfast cereals, and maize intended for direct human
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consumption, maize-based snacks and maize-based breakfast cereals, respectively.
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2.6. Calculation of estimated daily intake
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The estimated daily intake (EDI) was calculated through a deterministic method
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(IPCS, 2009) using the equation EDI = (Σc) (CN-1 D-1 K-1), where Σc is the sum of ZEA
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in the analyzed samples (µg/kg), C is the mean annual intake estimated per person, N is
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the total number of analyzed samples, D is the number of days in a year, and K is the
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body weight. The 2012 latest assessment of the bread consumption in Portugal is 39.78
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kg/inhabitant (INE, 2013). Assuming that maize bread consumption is about 25% of the
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total bread consumption (Lino, Silva, Pena, Fernández, & Mañes, 2007).), this
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corresponds to 9.95 kg/inhab./year for maize bread consumption. The mean body
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weight considered for the Portuguese adult population was 69 kg (Arezes, Barroso,
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Cordeiro, Costa, & Miguel, 2006).
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3. Results and discussion 3.1. Analytical performance Several experimental conditions were tested in order to obtain the adequate
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analytical performance. Different mobile phases were evaluated, 30% water with 0.1%
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acetic acid and 70% acetonitrile, 40% water with 1.0% acetic acid and 60% acetonitrile,
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40% water with 1.0% formic acid and 60% acetonitrile, and 40% water with 0.1%
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formic acid and 60% acetonitrile. The first three showed low recoveries, ranging from
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51%, for fortification levels at 20 µg/kg, to 71% for levels at 50 µg/kg. As seen below,
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good analytical performance was obtained using a mobile phase consisting of 40%
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water with 0.1% formic acid and 60% acetonitrile using a flow rate of 200 µL/min. The mixture acetonitrile:water (90:10) showed a high extraction efficiency, as
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previously described for ZEA in different type of flours (maize, wheat and mixed)
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(Ramos Aldana, Silva, Pena, Mañes, & Lino, 2014). Various extraction mixtures of
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acetonitrile:water and methanol:water have been reported to extract ZEA from cereals
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(Juan, Ritieni, & Mañes, 2012).
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methanol:water mixture was used (Sulyok, Berthiller, Krska, & Schuhmacher, 2006).
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Some authors found low recoveries when the
Linearity, both on standard solutions (10-100 ng/mL) and on matrix-matched
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assays (10-100 ng/g), was adequate, r2=0.9998 and r2=0.9995, respectively. Matrix and
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standard calibration curves were used to calculate the matrix effect (ME) (Rubert,
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Soriano, Mañes, & Soler, 2011). The obtained value, 97.6%, can be considered
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negligible.
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The LOD and LOQ were calculated through the matrix-matched calibration curve
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as |3.3Sy/x|/b and |10Sy/x|/b, respectively, where b is the slope and Sy/x is the residual
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standard deviation of the linear function. LOD and LOQ were 3.19 and 9.6 µg/kg,
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respectively. These values are very satisfactory considering the maximum limits
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established by the Commission Directive, 2007/1126/EC of the European Commission
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(European Commission, 2007) and similar to that obtained by Rodríguez-Carrasco,
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Moltó, Berrada, & Mañes (2014), when maize was analyzed by GC-MS-MS (LOQ=10
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µg/kg).
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Recovery values, for fortification levels at 20, 50 and 100 µg/kg, ranged between
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89.8 and 99.7 % for 20 µg/kg and 100 µg/kg, respectively (Table 1). The intra-day
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repeatability varied between 0.7% and 1.2% for the levels at 50 and 20 µg/kg,
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respectively, and the inter-day repeatability oscillated between 1.7% and 3.8% for 100
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and 20 µg/kg, respectively. The validation results comply with the requirements
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established by the EC directive 401/2006 (European Commission, 2006).
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3.2. Surveillance results
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ZEA content was evaluated in 52 samples of bread maize, broa, the second most
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consumed type of bread in Portugal. Seven samples (13.5%) were contaminated, with
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levels oscillating between 9.6 and 50.4 µg/kg, and mean levels of 28.2 µg/kg, similar to
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those found in maize flours in Portugal (Ramos Aldana , Silva, Pena, Mañes, & Lino,
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2014). One broa sample, contaminated with 50.4 µg/kg, exceeded the limit of 50 µg/kg
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proposed by the EC legislation No 1126/2007 (European Commission, 2007) for bread
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(including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals,
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excluding maize-snacks and maize-based breakfast cereals.
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With regard to the type of maize bread, as shown in Table 2, those in which
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composition of yellow maize and wheat were used displayed the highest frequency,
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16.2%, vs those made only with yellow maize, 6.7%. The first also revealed a mean
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level of 24.5 µg/kg. Nonetheless, only one sample of yellow maize was contaminated
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with a level of 50.4 µg/kg, exceeding the limit proposed by the EC.
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The comparison with other European countries is somehow difficult, if not
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impossible, regarding the absence of reports on ZEA in this kind of bread. As broa is a
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typical Portuguese maize-based food, the comparison with other similar products all
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over the world is complex. Although the cooking processes of tortilla and polenta are
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completely different from broa, they are also typical maize-based foods, from Mexico
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and northern Italy, respectively, nonetheless, as far as we know also only two studies
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were reported. One of them resulted of a collaborative study for polenta, with reported
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(MacDonald, Anderson, Brereton, Wood, & Damant, 2005)). The other is about tortillas
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consumed by Hispanic population in and around San Diego, USA, made with white
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(n=7) and yellow (n=8) maize. The white maize tortilla showed a range between 0.05
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and 3.1 µg/kg and a mean level of 1.2 µg/kg, while the yellow tortilla presented a mean
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level of 2.0 and a range of 0.78-6.8 µg/kg (Hewitt, Flack, Kolodziejczyk, Chacon, &
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D’Ovidio, 2012).
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Numanoglu, Uygun, Koksel, & Solfrizzo (2010), when studied the fate of DON,
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ZEA, and FBs during the traditional Turkish maize bread production, in the Black Sea
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region, verified that, in total, the toxin levels in 30% of the maize samples were higher
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than the limits established by the European Union, and a high stability of Fusarium
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toxins. After the bread processing, no significant reduction of those mycotoxins was
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measured in the bread crust and crumb. According to the mass balance of mycotoxins
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measured in maize flour and relevant maize bread only 2.1%, 0.1% and 3.1% of the
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total initial amounts of DON, ZEA and FBS, respectively, were lost.
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ZEA is heat-stable and, in the absence of reaction to form conjugates, cannot be
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expected a substantial degradation during moderate thermal processing (Maragos,
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2010). Since ZEA is a heat-stable compound, despite its large lactone ring (Ryu, Hanna,
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Eskridge, & Bullerman, 2003; Bullerman & Bianchini 2007), it has been estimated that
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about 60% of the ZEA survives after bread baking (Numanoglu, Yener, Gokmen,
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Uygun, & Koksel, 2013). These authors found maximum reduction (28%) at 250°C
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after 15 min, during bread baking with naturally contaminated maize flour with ZEA
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(Numanoglu, Yener, Gokmen, Uygun, & Koksel, 2013). Lauren & Smith (2001) also
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showed that ZEN content in ground maize was slightly reduced even by 12 days of
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heating at 110 ºC, after treatment with a sodium bicarbonate solution. Reductions were
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between 13% (water addition) and 35% (bicarbonate). In general, the mycotoxins were stable during bread-making and baking process
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(210 ºC, 60 min). For ZEA a statistically significant reduction was observed in crust
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(12.1%) while no reduction was observed in crumb. In particular, mean ZEA level in
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the flour was 838 µg/kg, while those in the crust and crumb of the bread were 737 and
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878 µg/kg, respectively, being 99% of initial amount ZEA recovered in crumb and
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crust, confirming the stability of ZEA during maize bread production (Numanoglu,
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Uygun, Koksel, & Solfrizzo, 2010). Scudamore (2008) also report that baking at 170 ºC
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did not degrade nivalenol and ZEA.
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These low ZEA reductions were also observed in wheat bread making procedures,
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either after fermentation with Saccharomyces cerevisiae or after baking at 200 °C for 20
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min (Cano-Sancho, Sanchis, Ramos, & Marin, 2013). However, Heidari, Milani, Seyed,
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& Nazari (2014) verified effective reduction in the levels of ZEA during fermentation
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by Saccharomyces cerevisiae (Baker's yeast) and baking at 180 °C for 25 min. In the
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bread baking process when using wheat flour with a ZEA concentration of 1 to 20
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mg/kg, 34 to 40% of ZEA were degraded at approximately 200 °C for 30 min.
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3.3. Estimated daily intake and risk assessment
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This is the first study on the intake assessment of ZEA through the broa
consumption, by the Portuguese population.
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As maize clearly represents a large potential contributor to ZEA exposure
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(Maragos, 2010), and previous studies in maize flour revealed an exposure of 0.013
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µg/kg b.w./day for adults in Portugal (Ramos Aldana , Silva, Pena, Mañes, & Lino,
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2014), which represented 5.2% of the TDI, the exposure evaluation through the broa
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consumption would be the next and the most important step. Being the average sample contamination of positive samples of 28.2 µg/kg,
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assuming that the estimation of intake of bread, in 2012, of Portuguese population was
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39.78 kg per inhabitant (INE, 2013), and considering that the consumption of broa
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represents a quarter of the total consumption of bread (Lino, Silva, Pena, Fernández, &
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Mañes, 2007), broa consumption was, in 2012, 27.3 g/ inhabitant/day. Therefore, the
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EDI of ZEA for an adult whose body weight is 69 kg reached, in average, 1.5 x 10-3
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µg/kg body weight/day.
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The resultant risk assessment from broa consumption is very low, since the EDI
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represents 0.6% of the TDI proposed by EFSA, in 2011, 0.25 µg/kg b.w./day. Due to the lack of data about the risk assessment resulting from the broa
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consumption, a comparison between the results of this study with other countries is
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impossible. However comparing with tortilla consumption by the Hispanic population
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in and around San Diego, USA, the Portuguese consumption is very low. Hewitt, Flack,
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Kolodziejczyk, Chacon, & D’Ovidio (2012) found a total ZEA exposure of 0.550 µg
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ZEA/day/person, for the Hispanic population, which is significantly lower than 500
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µg/kg (60 kg average) per day.
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was estimated as <0.98 µg/day for 60 kg adults (<0.016 µg/kg b.w./day), and for
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Denmark, Sweden, Finland, and Norway as 0.48 µg/day, 1.2 µg/day, 1.3 µg/day, and 1.5
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µg/day, respectively. In the United States of America, for 20 to 39 year old men, the
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EDI was lower than 2.1 µg/day. The intake estimates ranged from 1.9 ng/kg b.w./day
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(Italian adults) to 116.3 ng/kg b.w./day (Austrian adults). Mean exposure for adults (15
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years and older), based on the French total diet study, was estimated as 33 ng/kg
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The mean intake for the Swiss population was estimated to be <1 µg per capita/day
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(<0.02 µg/kg b.w./day). In Côte d’Ivoire (Ivory Coast), the ZEA weekly intake was
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estimated at 655 µg, or 1.56 µg/kg b.w./day, assuming a body weight of 60 kg. This
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level of exposure (93.5 µg/day) is substantially higher than in the various estimates for
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Europe, Canada, USA and New Zealand, which generally were below 2 µg/day. The
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mean exposure for French adults was estimated between 5.9 ng/kg b.w./day and 25.5
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ng/kg b.w./day (Sirot, Fremy, & Leblanc, 2013).
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Conclusions
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The performed analytical methodology fulfilled the requirements established by the EC directive 401/2006.
The study highlights the fact that the levels of ZEA in the broa collected from
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Portuguese local markets were not totally suitable with regard to the current EC
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legislation. It also emphasizes that there was no concern about intake of the ZEA
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through its consumption by adult population in the area surveyed.
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This study is the first report of ZEA occurrence in maize bread, broa, in Portugal
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and further studies are needed to elucidate the extent of ZEA contamination in different
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years and in different regions of the country.
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Acknowledgements
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The authors gratefully acknowledge the Portuguese governmental FCT through
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PTDC/AAC-AMB/120889/2010 the financial support, and the post-PhD fellowship
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granted to L.J.G. Silva (SFRH/BPD/62877/2009). The authors are also grateful to the
337
Laboratory of Mass Spectrometry (LEM) of the Node CEF/UC integrated in the
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National Mass Spectrometry Network (RNEM) of Portugal, for the MS analysis, and to
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Fátima Nunes for all the assistance.
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References
342
Arezes, P. M., Barroso, M. P., Cordeiro, P., Costa, L. G., & Miguel, A. S. (2006).
343
Estudo Antropométrico da População Portuguesa (1 st.). Lisboa: Instituto para a
344
Segurança, Higiene e Saúde no Trabalho.
SC
RI PT
341
Bullerman, L.B., & Bianchini, A. (2007). Stability of mycotoxins during food
346
processing.International Journal of Food Microbiology, 119, 140-146.
347
Cano-Sancho, G., Sanchis, V. J., Ramos, A., & Marin, S. (2013). Effect of food
348
processing on exposure assessment studies with mycotoxins. Food Additives and
349
Contaminants: Part A, 30, 867-875.
TE D
M AN U
345
Cheeke, P. R. (1998). Mycotoxins in cereal grains and supplements. In: Cheeke, P.R.
351
(Ed.), Natural Toxicants in Feeds, Forages, and Poisonous Plants. Interstate
352
Publishers, Inc, Danville, IL, pp. 87–136.
354
355 356
Diekman, M. A., & Green, M. L. (1992). Mycotoxins and reproduction in domestic
AC C
353
EP
350
livestock. J. Anim. Sci. 70, 1615–1627.
EFSA, P. on C. in the F. C. (2011). Scientific Opinion on the risks for public health related to the presence of zearalenone in food. EFSA Journal, 9(6:2197), 1–124.
357
European Commission. (2006). COMMISSION REGULATION (EC) No 401/2006 of
358
23 February 2006 laying down the methods of sampling and analysis for the
15
ACCEPTED MANUSCRIPT 359
official control of the levels of mycotoxins in foodstuffs. Official Journal of the
360
European Union, L70, 12–34.
European Commission. (2007). COMMISSION REGULATION (EC) No 1126/2007 of
362
28 September 2007 amending Regulation (EC) No 1881/2006 setting maximum
363
levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize
364
and maize products. Official Journal of the European Union, L255, 14–17.
RI PT
361
Heidari S., Milani J., Seyed S., & Nazari J. (2014). Effect of the bread making process
366
on zearalenone levels. Food Additives & Contaminants: Part A, Accepted, DOI:
367
10.1080/19440049.2014.972472
M AN U
SC
365
Hewitt T. C., Flack C. L., Kolodziejczyk J. K., Chacon A. M., & D’Ovidio K. L. (2012)
369
Occurrence of zearalenone in fresh corn and corn products collected from local
370
Hispanic markets in San Diego County, CA. Food Control, 26, 300-304
372
INE (2013). Instituto Nacional de Estatística. Available from www.ine.pt/. Accessed on November, 2014.
EP
371
TE D
368
International Agency for Reserach on Cancer. (2002). Some traditional herbal
374
medicines, some mycotoxins, naphthalene and styrene. Monograph on the
375
AC C
373
evaluation of carcinogenic risk to humans. (vol. 82) (p. 601). Lyon: IARCPress.
376
IPCS, 2009. Dietary exposure assessment of chemicals in food. In W. H. Organization
377
(Ed.). Principles and methods for the risk assessment of chemicals in food (p. 98).
378
Geneva, Switzerland International Programme on Chemical Safety.
16
ACCEPTED MANUSCRIPT 379
Juan, C., Ritieni, A., & Mañes, J. (2012). Determination of trichothecenes and
380
zearalenones in grain cereal, flour and bread by liquid chromatography tandem
381
mass spectrometry. Food Chemistry, 134(4), 2389-2397.
Kaushik G. (2014). Effect of processing on mycotoxin content in grains. Critical
383
Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2012.701254
384
Lauren D. R. & Smith W. A. (2001) Stability of the Fusarium mycotoxins nivalenol,
385
deoxynivalenol and zearalenone in ground maize under typical cooking
386
environments. Food Additives & Contaminants, 18, 11, 1011-1016.
M AN U
SC
RI PT
382
387
Lino C. M., Silva L. J. G., Pena A., Fernández M., & Mañes J. (2007) Occurrence of
388
fumonisins B1 and B2 in broa, typical Portuguese maize bread. International
389
Journal of Food Microbiology 118, 79–82
MacDonald S. J., Anderson S., Brereton P., Wood R., & Damant A. (2005).
391
Determination of Zearalenone in Barley, Maize and Wheat Flour, Polenta, and
392
Maize-Based Baby Food by Immunoaffinity Column Cleanup with Liquid
393
Chromatography: Interlaboratory Study. Journal of AOAC International, 88, 6,
394
1733- 1740
396
EP
AC C
395
TE D
390
Maragos C. M. (2010). Zearalenone occurrence and human exposure. World Mycotox J., 3, 369–383
397
Marasas, W. F. O. (1991). Toxigenic Fusaria. In: Smith, J.E., Anderson, R.A. (Eds.),
398
Mycotoxins and Animal Foods. CRC Press, Boca Raton, FL, pp. 119–139.
17
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Numanoglu, E., Uygun, U., Koksel, H., & Solfrizzo, M. (2010). Stability of Fusarium
400
toxins during traditional Turkish maize bread production. Quality Assurance and
401
Safety of Crops & Foods, 2, 84–92.
Numanoglu, E., Yener, S., Gokmen, V., Uygun, U., & Koksel, H. (2013). Modelling
403
thermal degradation of zearalenone in maize bread during baking. Food Additives
404
and Contaminants: Part A, 30, 3, 528-533.
RI PT
402
Ramos Aldana, J., Silva, L. J. G., Pena, A., Mañes, J. V., & Lino, C. M. (2014).
406
Occurrence and risk assessment of zearalenone in flours from Portuguese and
407
Dutch markets. Food Control, 45, 51-55.
M AN U
SC
405
Rodríguez-Carrasco, Y., Moltó, J. C., Berrada, H., & Mañes, J. (2014). A survey of
409
trichothecenes, zearalenone and patulin in milled grain-based products using GC–
410
MS/MS. Food Chemistry 146, 212-219.
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408
Rubert, J., Soriano, J. M., Mañes, J., & Soler, C. (2011). Rapid mycotoxin analysis in
412
human urine: a pilot study. Food and chemical toxicology : an international
413
journal published for the British Industrial Biological Research Association, 49(9),
414
2299–2304.
AC C
EP
411
415
Ryu, D., Hanna, M. A, Eskridge, K. M., & Bullerman, L. B. (2003). Heat stability of
416
zearalenone in an aqueous buffered model system. Journal of Agricultural and
417
418 419
Food Chemistry, 51, 1746-1748.
Scudamore, K. A. (2008) Fate of fusarium mycotoxins in the cereal industry: recent UK studies. World Mycotoxin Journal, 1(3), 315-323.
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ACCEPTED MANUSCRIPT Sirot, V., Fremy, J.-M., & Leblanc, J.-C. (2013). Dietary exposure to mycotoxins and
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health risk assessment in the second French total diet study. Food and chemical
422
toxicology : an international journal published for the British Industrial Biological
423
Research Association, 52, 1–11.
RI PT
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Sulyok, M., Berthiller, F., Krska, R. & Schuhmacher, R. (2006). Development and
425
validation of a liquid chromatography/tandem mass spectrometric method for the
426
determination of 39 mycotoxins in wheat and maize. Rapid Communications in
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Mass Spectrometry, 20, 2649-2659
SC
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Zinedine, A., Soriano, J. M., Moltó, J. C., & Mañes, J. (2007). Review on the toxicity,
429
occurrence, metabolism, detoxification, regulations and intake of zearalenone: an
430
oestrogenic mycotoxin. Food and chemical toxicology : an international journal
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published for the British Industrial Biological Research Association, 45(1), 1–18.
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Table 1. Accuracy and repeatability intra and inter-day.
Exactitude
Intra-day
Inter-day
(µg/kg)
(%)
repeatability
repeatability
(% RSD)
(% RSD) 3.8
89.8
1.2
50
97.3
0.7
100
99.7
1.1
2.8
1.7
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Fortification levels
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Table 2. Frequency and levels of ZEA in broa.
Sample
Frequency
Range
Mean ± SD
composition
size
(%)
(µg/kg)
(µg/kg)
Maize and wheat
37
6 (16.2)
9.6-42.5
Yellow maize
15
1 (6.7)
nd-50.4
TOTAL
52
7 (13.5)
9.6-50.4
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Sample
24.5±13.1 -
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28.2±15.4
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Occurrence and risk assessment of zearalenone through broa consumption, typical maize bread from Portugal
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HIGHLIGHTS: -Different Portuguese maize bread, broa, was investigated for zearalenone.
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-A higher contamination frequency was observed in samples with maize and wheat.
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-The ZEA levels in broa were not totally suitable with regard to the EU legislation. -There is no concern about intake of ZEA through its consumption by adult
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population.
S0956-7135(15)00218-2
DOI:
10.1016/j.foodcont.2015.03.043
Reference:
JFCO 4398
To appear in:
Food Control
Received Date: 13 December 2014 Revised Date:
20 March 2015
Accepted Date: 25 March 2015
Please cite this article as: Ribeiro N.M.C., Silva L.J.G., Pena A. & Lino C.M., Occurrence and risk assessment of zearalenone through broa consumption, typical maize bread from Portugal, Food Control (2015), doi: 10.1016/j.foodcont.2015.03.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Occurrence and risk assessment of zearalenone through broa
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consumption, typical maize bread from Portugal
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Nuno M. C. Ribeiro, Liliana J.G. Silva, Angelina Pena, Celeste M. Lino∗
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LAQV, REQUIMTE, Group of Bromatology, Pharmacognosy and Analytical Sciences,
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Faculty of Pharmacy, University of Coimbra, Polo III, Azinhaga de Stª Comba, 3000-
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548 Coimbra, Portugal
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* Corresponding author:
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[email protected]; [email protected]
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Faculty of Pharmacy, University of Coimbra Pólo das Ciências da Saúde
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Azinhaga de Santa Comba
3000-548 Coimbra, Portugal
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Phone number: 00351239488477 Fax number: 00351239488503
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Abstract
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The occurrence of zearalenone (ZEA) in maize bread, broa, for human consumption,
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from the Portuguese market, was evaluated. Good analytical performance was obtained
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through extraction with acetonitrile:water (90:10), clean-up with immunoaffinity
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columns, and detection and quantification by liquid chromatograph with-tandem mass
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spectrometry. ZEA levels were determined in 52 samples to verify the compliance with
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the maximum permitted levels by the European legislation. One broa sample exceeded
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the maximum limit established. A higher contamination frequency was observed in
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samples in which maize and wheat were present. Overall, 13.5% of the samples were
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contaminated, at levels oscillating between 9.6 and 50.4 µg/kg. Considering the
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percentage of the tolerable daily intake (TDI) obtained, 0.6%, the risk assessment linked
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with the exposure to ZEA was considered very low for the studied population. This is
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the first study on the intake assessment of ZEA through maize bread consumption in
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Portugal.
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Keywords:
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Zearalenone; maize bread; risk assessment; Portuguese population.
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1. Introduction Zearalenone (ZEA), a metabolite primarily associated with several Fusarium
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species (F. culmorum, F. graminearum, F. sporotrichioides, F. cerealis, F. equiseti, F.
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crookwellense and F. semitectum) (Marasas, 1991 ; Zinedine, Soriano, Moltó, & Mañes,
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2007) and known as 6-(10-hydroxy-6-oxo-trans-1-undecenyl) - β-resorcylic acid-
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lactone (Marasas, 1991), is a phytoestrogenic compound (Diekman & Green, 1992)
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along with its alcohol metabolites, α-zearalenol (α-ZEN) and β-zearalenol (β-ZEN)
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(Cheeke, 1998, Kaushik, 2014). Despite being a non-steroidal estrogenic toxin, it was
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categorized in the group 3 (not classifiable as to its carcinogenicity to humans) by the
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International Agency for Research on Cancer (International Agency for Reserach on
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Cancer, 2002).
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ZEA is most commonly found in maize (Ryu, Hanna, Eskridge, & Bullerman,
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2003; Zinedine, Soriano, Moltó, & Mañes, 2007), as well as in its derivatives such as
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flour (Ramos Aldana , Silva, Pena, Mañes, & Lino, 2014). Broa, a traditional maize
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bread highly consumed in Portugal, especially in the north and central zones, has been
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studied for fumonisins B1 (FB1) and B2 (FB2) (Lino, Silva, Pena, Fernández, & Mañes,
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2007) but not for ZEA. As far as we know, few studies on the ZEA content in other
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traditional maize foods, such as tortillas (Hewitt, Flack, Kolodziejczyk, Chacon, &
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D’Ovidio, 2012), and polenta (MacDonald, Anderson, Brereton, Wood, & Damant,
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2005), typical maize-based products from Mexico and northern Italy, respectively, are
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available in the scientific literature. The results on the stability of the main Fusarium
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toxins, during traditional Turkish maize bread production, revealed that deoxynivalenol
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(DON), ZEA and FBs remained stable during the bread making, indicating that breads
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produced from highly contaminated maize may represent a potential risk to the
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population (Numanoglu, Uygun, Koksel, & Solfrizzo, 2010).
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The traditional processing of broa, as reported by Lino, Silva, Pena, Fernández, &
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Mañes (2007) consists of adding sieved maize flour, its principal ingredient, wheat
66
flour, hot water, yeast and leavened dough from the late broa. After mixing, working up
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and leavening, the dough is baked in a wood-fired oven. Previous studies on maize-based products for human consumption in Portugal,
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ragarding mycotoxins produced by Fusarium species, such as ZEA in flours and FB1
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and FB2 in broa, showed a higher prevalence in maize flour, 50%, than in wheat flour,
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31.6%, as well as the highest mean levels (Ramos Aldana , Silva, Pena, Mañes, & Lino,
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2014), and a high incidence of FB1 and FB2 in broa, 83%, with 27% of the samples
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exceeding the regulatory limits (Lino, Silva, Pena, Fernández, & Mañes, 2007).
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The European Commission (EC), through EC legislation Nº 1126/2007, set
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regulatory limits, as regards Fusarium toxins in maize and maize products, in order to
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protect public health and the maximum limit established for ZEA in bread is 50 µg/kg
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(European Commission, 2007). On the other hand, the European Food Safety Authority
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(EFSA) has published a scientific opinion on the risks to public health in 2011 for ZEA,
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and proposed a TDI of 0.25 µg/kg b.w./day based on more recent data on pig, but also
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taking into account comparisons between pigs and humans (EFSA, 2011).
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This first report on the incidence of ZEA in maize bread from Portugal was aimed
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to evaluate its levels in samples, obtained from the Portuguese market. Good analytical
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performance was obtained using extraction with acetonitrile:water (90:10), clean-up
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with immunoaffinity columns, followed by detection and quantification by liquid
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chromatography with tandem mass spectrometry. The occurrence and levels of ZEA
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were determined in 52 samples in order to verify the compliance with the maximum
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limits of the European legislation. This study also intended to present the results
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concerning the exposure of the Portuguese population, inhabiting the central zone,
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through the consumption of this typical bread, and evaluate the risk with regard to the
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international health-based guidance values.
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2. Materials and methods
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2.1. Sampling
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A total of 52 broa samples (15 in which composition yellow maize was the only
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cereal, and 37 composed by yellow maize and wheat) were analysed. The samples were
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purchased in commercially available size in different supermarkets, markets and
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groceries of Coimbra, central zone of Portugal, between September and October 2014.
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After purchase, samples were brought to the laboratory under ambient conditions, all
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the information available on the labels was assembled, and were frozen until their
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analysis.
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2.2. Chemical, reagents and equipment
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The reagents of HPLC grade used were acetonitrile (Merck, KGaA, Darmstadt,
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Germany) and methanol (Carlo Erba, Milan, Italy). Formic acid 98-100% was obtained
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from Merck (KGaA, Darmstadt, Germany), and sodium chloride from Pronolab
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(Lisboa, Portugal). Micro-glass fiber paper (150 mm, Munktell & Filtrak GmbH,
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Bärenstein, Germany), Whatman N°1 filter paper, polyamide membrane filters (0.2 µm,
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50 mm; Whatman GmbH, Dassel, Germany), and Durapore membrane filter (0.22 um,
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GVPP, Millipore, Ireland) were used. Immunoaffinity columns (IAC) ZearalaTestTM
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were from VICAM (Watertown, USA).
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Water was daily obtained from Milli-Q System (Millipore, Bedford, MA, USA) and
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the ZEA standard, a white powder with a purity degree ≥99.0%, was purchased from
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Sigma-Aldrich (St. Louis, MO, USA). A mobile phase (40% water with 0.1% formic
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acid and 60% acetonitrile) at 200 µL/min, was used. All liquid chromatographic
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reagents were degassed for 15 minutes in an ultrasonic bath. ZEA standard stock solution was prepared at 5 mg/mL, diluting 10 mg of ZEA in 2
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mL of acetonitrile, and stored at -20ºC. The intermediate solution was prepared by
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diluting the stock solution at 50 µg/mL, in acetonitrile, and a working standard solution,
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at 1 µg/mL in acetonitrile, was prepared by diluting the intermediate solution. They
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were stored in darkness, at 4 ºC, until the analysis. The calibration curve standard
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solutions, in mobile phase, were prepared between 10 and 100 ng/mL (10, 20, 50, 75,
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100 ng/mL). For the matrix-matched calibration curve, standards were prepared
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between 10 and 100 µg/kg (10, 20, 50, 75, 100 µg/kg).
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A 3–16 k Sigma centrifuge (Reagente 5, Porto, Portugal), a Braun MR 5000 M
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multiquick/minipimer 500W (220–230 V, 50–60 Hz) (Esplungues del Llobregat,
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Spain), a vacuum manifold of Macherey-Nagel (Düren, Germany), a Dinko pump (mol.
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D-95, 130 W, 220 V), a magnetic stirrer (Agimatic-S, Selecta, Barcelona, Spain), a
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Retsh vortex mixer (Haan, Germany), and a Sonorex RK 100 ultrasonic bath (Berlin,
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Germany) were employed.
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2.3. Sample extraction and clean-up Extraction and clean-up were performed according to Ramos Aldana , Silva, Pena,
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Mañes, & Lino (2014). Briefly, 20 g of sample, mixed with NaCl, were extracted twice
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with acetonitrile:water (90:10), and the supernatants were mixed with Milli-Q water and
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filtered. An aliquot (10 mL) of the filtered was cleaned through the ZearalaTestTM IAC
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attached onto a vacuum manifold. After column washing with water, an elution with
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methanol was performed. After drying, at 42 ºC under a gentle nitrogen flow, the dried
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extract was stored at -20 ºC.
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For liquid chromatography coupled to tandem mass spectrometry (LC/MSn)
140
analysis, the dried extracts were dissolved in 1.0 mL mobile phase (40% water with
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0.1% formic acid and 60% acetonitrile).
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2.4. LC/MSn conditions
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LC/MS2 analyses were performed using a Liquid Chromatograph of High
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Performance (Thermo Finnigan, San Jose, California, USA) coupled to a LCQ
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Advantage MAX Quadrupole Ion Trap Mass Spectrometer (Thermo Finnigan, San Jose,
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California, USA). The LC column (Waters Spherisorb ODS2; 3 µm, 150x2.1 mm i.d;
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Waters Corporation, Milford, U.S.A.) was preceded by a guard cartridge (Waters
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Spherisorb ODS2; 5 µm, 10x4.6 mm i.d.; Waters Corporation, Milford, U.S.A.). A 20
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µL (partial loop) injection volume was used with the mobile phase (40% water with
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0.1% formic acid and 60% acetonitrile) flow maintained at 200 µL/min.
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The MS was operated in the negative electrospray ionization (ESI) mode using
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selected reaction monitoring (SRM) acquisition. Nitrogen was used as nebulizing gas,
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with a sheath gas flow of 80 (arbitrary unit) and the auxiliary sweep gas flow of 20
155
(arbitrary unit). Source and capillary temperatures were set at 0ºC and 270ºC and
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voltages at 4.5 and 10V, respectively. Collision gas was helium with a normalized
157
collision energy of 35%. Three precursor-to-fragment transitions were acquired: m/z
158
317(|M-H|-)> m/z 273 (for quantification purpose); m/z 317(|M-H|-)> m/z 175 and m/z
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317(|M-H|-)> m/z 149 (for confirmation purposes).
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2.5. Recovery studies
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Recoveries were determined by spiking ZEA – maize bread at three different levels,
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20, 50, and 100 ng/g, using three replicates for each level, according to the maximum 7
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166
foods and processed maize-based foods for infants and young children, bread (including
167
small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals, excluding
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maize-snacks and maize-based breakfast cereals, and maize intended for direct human
169
consumption, maize-based snacks and maize-based breakfast cereals, respectively.
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2.6. Calculation of estimated daily intake
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The estimated daily intake (EDI) was calculated through a deterministic method
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(IPCS, 2009) using the equation EDI = (Σc) (CN-1 D-1 K-1), where Σc is the sum of ZEA
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in the analyzed samples (µg/kg), C is the mean annual intake estimated per person, N is
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the total number of analyzed samples, D is the number of days in a year, and K is the
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body weight. The 2012 latest assessment of the bread consumption in Portugal is 39.78
177
kg/inhabitant (INE, 2013). Assuming that maize bread consumption is about 25% of the
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total bread consumption (Lino, Silva, Pena, Fernández, & Mañes, 2007).), this
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corresponds to 9.95 kg/inhab./year for maize bread consumption. The mean body
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weight considered for the Portuguese adult population was 69 kg (Arezes, Barroso,
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Cordeiro, Costa, & Miguel, 2006).
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3. Results and discussion 3.1. Analytical performance Several experimental conditions were tested in order to obtain the adequate
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analytical performance. Different mobile phases were evaluated, 30% water with 0.1%
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acetic acid and 70% acetonitrile, 40% water with 1.0% acetic acid and 60% acetonitrile,
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40% water with 1.0% formic acid and 60% acetonitrile, and 40% water with 0.1%
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formic acid and 60% acetonitrile. The first three showed low recoveries, ranging from
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51%, for fortification levels at 20 µg/kg, to 71% for levels at 50 µg/kg. As seen below,
191
good analytical performance was obtained using a mobile phase consisting of 40%
192
water with 0.1% formic acid and 60% acetonitrile using a flow rate of 200 µL/min. The mixture acetonitrile:water (90:10) showed a high extraction efficiency, as
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previously described for ZEA in different type of flours (maize, wheat and mixed)
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(Ramos Aldana, Silva, Pena, Mañes, & Lino, 2014). Various extraction mixtures of
196
acetonitrile:water and methanol:water have been reported to extract ZEA from cereals
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(Juan, Ritieni, & Mañes, 2012).
198
methanol:water mixture was used (Sulyok, Berthiller, Krska, & Schuhmacher, 2006).
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Some authors found low recoveries when the
Linearity, both on standard solutions (10-100 ng/mL) and on matrix-matched
200
assays (10-100 ng/g), was adequate, r2=0.9998 and r2=0.9995, respectively. Matrix and
201
standard calibration curves were used to calculate the matrix effect (ME) (Rubert,
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Soriano, Mañes, & Soler, 2011). The obtained value, 97.6%, can be considered
203
negligible.
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The LOD and LOQ were calculated through the matrix-matched calibration curve
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as |3.3Sy/x|/b and |10Sy/x|/b, respectively, where b is the slope and Sy/x is the residual
206
standard deviation of the linear function. LOD and LOQ were 3.19 and 9.6 µg/kg,
207
respectively. These values are very satisfactory considering the maximum limits
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established by the Commission Directive, 2007/1126/EC of the European Commission
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(European Commission, 2007) and similar to that obtained by Rodríguez-Carrasco,
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Moltó, Berrada, & Mañes (2014), when maize was analyzed by GC-MS-MS (LOQ=10
211
µg/kg).
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Recovery values, for fortification levels at 20, 50 and 100 µg/kg, ranged between
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89.8 and 99.7 % for 20 µg/kg and 100 µg/kg, respectively (Table 1). The intra-day
214
repeatability varied between 0.7% and 1.2% for the levels at 50 and 20 µg/kg,
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respectively, and the inter-day repeatability oscillated between 1.7% and 3.8% for 100
216
and 20 µg/kg, respectively. The validation results comply with the requirements
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established by the EC directive 401/2006 (European Commission, 2006).
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3.2. Surveillance results
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ZEA content was evaluated in 52 samples of bread maize, broa, the second most
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consumed type of bread in Portugal. Seven samples (13.5%) were contaminated, with
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levels oscillating between 9.6 and 50.4 µg/kg, and mean levels of 28.2 µg/kg, similar to
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those found in maize flours in Portugal (Ramos Aldana , Silva, Pena, Mañes, & Lino,
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2014). One broa sample, contaminated with 50.4 µg/kg, exceeded the limit of 50 µg/kg
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proposed by the EC legislation No 1126/2007 (European Commission, 2007) for bread
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(including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals,
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excluding maize-snacks and maize-based breakfast cereals.
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With regard to the type of maize bread, as shown in Table 2, those in which
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composition of yellow maize and wheat were used displayed the highest frequency,
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16.2%, vs those made only with yellow maize, 6.7%. The first also revealed a mean
231
level of 24.5 µg/kg. Nonetheless, only one sample of yellow maize was contaminated
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with a level of 50.4 µg/kg, exceeding the limit proposed by the EC.
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The comparison with other European countries is somehow difficult, if not
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impossible, regarding the absence of reports on ZEA in this kind of bread. As broa is a
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typical Portuguese maize-based food, the comparison with other similar products all
236
over the world is complex. Although the cooking processes of tortilla and polenta are
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completely different from broa, they are also typical maize-based foods, from Mexico
238
and northern Italy, respectively, nonetheless, as far as we know also only two studies
239
were reported. One of them resulted of a collaborative study for polenta, with reported
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ACCEPTED MANUSCRIPT mean levels of 66.5 µg/kg, and range comprised between 41.1 and 86.1 µg/kg
241
(MacDonald, Anderson, Brereton, Wood, & Damant, 2005)). The other is about tortillas
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consumed by Hispanic population in and around San Diego, USA, made with white
243
(n=7) and yellow (n=8) maize. The white maize tortilla showed a range between 0.05
244
and 3.1 µg/kg and a mean level of 1.2 µg/kg, while the yellow tortilla presented a mean
245
level of 2.0 and a range of 0.78-6.8 µg/kg (Hewitt, Flack, Kolodziejczyk, Chacon, &
246
D’Ovidio, 2012).
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Numanoglu, Uygun, Koksel, & Solfrizzo (2010), when studied the fate of DON,
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ZEA, and FBs during the traditional Turkish maize bread production, in the Black Sea
249
region, verified that, in total, the toxin levels in 30% of the maize samples were higher
250
than the limits established by the European Union, and a high stability of Fusarium
251
toxins. After the bread processing, no significant reduction of those mycotoxins was
252
measured in the bread crust and crumb. According to the mass balance of mycotoxins
253
measured in maize flour and relevant maize bread only 2.1%, 0.1% and 3.1% of the
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total initial amounts of DON, ZEA and FBS, respectively, were lost.
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ZEA is heat-stable and, in the absence of reaction to form conjugates, cannot be
256
expected a substantial degradation during moderate thermal processing (Maragos,
257
2010). Since ZEA is a heat-stable compound, despite its large lactone ring (Ryu, Hanna,
258
Eskridge, & Bullerman, 2003; Bullerman & Bianchini 2007), it has been estimated that
259
about 60% of the ZEA survives after bread baking (Numanoglu, Yener, Gokmen,
260
Uygun, & Koksel, 2013). These authors found maximum reduction (28%) at 250°C
261
after 15 min, during bread baking with naturally contaminated maize flour with ZEA
262
(Numanoglu, Yener, Gokmen, Uygun, & Koksel, 2013). Lauren & Smith (2001) also
263
showed that ZEN content in ground maize was slightly reduced even by 12 days of
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heating at 110 ºC, after treatment with a sodium bicarbonate solution. Reductions were
265
between 13% (water addition) and 35% (bicarbonate). In general, the mycotoxins were stable during bread-making and baking process
267
(210 ºC, 60 min). For ZEA a statistically significant reduction was observed in crust
268
(12.1%) while no reduction was observed in crumb. In particular, mean ZEA level in
269
the flour was 838 µg/kg, while those in the crust and crumb of the bread were 737 and
270
878 µg/kg, respectively, being 99% of initial amount ZEA recovered in crumb and
271
crust, confirming the stability of ZEA during maize bread production (Numanoglu,
272
Uygun, Koksel, & Solfrizzo, 2010). Scudamore (2008) also report that baking at 170 ºC
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did not degrade nivalenol and ZEA.
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These low ZEA reductions were also observed in wheat bread making procedures,
275
either after fermentation with Saccharomyces cerevisiae or after baking at 200 °C for 20
276
min (Cano-Sancho, Sanchis, Ramos, & Marin, 2013). However, Heidari, Milani, Seyed,
277
& Nazari (2014) verified effective reduction in the levels of ZEA during fermentation
278
by Saccharomyces cerevisiae (Baker's yeast) and baking at 180 °C for 25 min. In the
279
bread baking process when using wheat flour with a ZEA concentration of 1 to 20
280
mg/kg, 34 to 40% of ZEA were degraded at approximately 200 °C for 30 min.
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3.3. Estimated daily intake and risk assessment
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This is the first study on the intake assessment of ZEA through the broa
consumption, by the Portuguese population.
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As maize clearly represents a large potential contributor to ZEA exposure
286
(Maragos, 2010), and previous studies in maize flour revealed an exposure of 0.013
287
µg/kg b.w./day for adults in Portugal (Ramos Aldana , Silva, Pena, Mañes, & Lino,
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2014), which represented 5.2% of the TDI, the exposure evaluation through the broa
289
consumption would be the next and the most important step. Being the average sample contamination of positive samples of 28.2 µg/kg,
291
assuming that the estimation of intake of bread, in 2012, of Portuguese population was
292
39.78 kg per inhabitant (INE, 2013), and considering that the consumption of broa
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represents a quarter of the total consumption of bread (Lino, Silva, Pena, Fernández, &
294
Mañes, 2007), broa consumption was, in 2012, 27.3 g/ inhabitant/day. Therefore, the
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EDI of ZEA for an adult whose body weight is 69 kg reached, in average, 1.5 x 10-3
296
µg/kg body weight/day.
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The resultant risk assessment from broa consumption is very low, since the EDI
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represents 0.6% of the TDI proposed by EFSA, in 2011, 0.25 µg/kg b.w./day. Due to the lack of data about the risk assessment resulting from the broa
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consumption, a comparison between the results of this study with other countries is
301
impossible. However comparing with tortilla consumption by the Hispanic population
302
in and around San Diego, USA, the Portuguese consumption is very low. Hewitt, Flack,
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Kolodziejczyk, Chacon, & D’Ovidio (2012) found a total ZEA exposure of 0.550 µg
304
ZEA/day/person, for the Hispanic population, which is significantly lower than 500
305
µg/kg (60 kg average) per day.
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According to the review of Maragos (2010), the EDI of ZEA in Canadian adults
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was estimated as <0.98 µg/day for 60 kg adults (<0.016 µg/kg b.w./day), and for
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Denmark, Sweden, Finland, and Norway as 0.48 µg/day, 1.2 µg/day, 1.3 µg/day, and 1.5
309
µg/day, respectively. In the United States of America, for 20 to 39 year old men, the
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EDI was lower than 2.1 µg/day. The intake estimates ranged from 1.9 ng/kg b.w./day
311
(Italian adults) to 116.3 ng/kg b.w./day (Austrian adults). Mean exposure for adults (15
312
years and older), based on the French total diet study, was estimated as 33 ng/kg
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ACCEPTED MANUSCRIPT b.w./day, being the greatest exposure estimated to be from bread (28.7% of the total).
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The mean intake for the Swiss population was estimated to be <1 µg per capita/day
315
(<0.02 µg/kg b.w./day). In Côte d’Ivoire (Ivory Coast), the ZEA weekly intake was
316
estimated at 655 µg, or 1.56 µg/kg b.w./day, assuming a body weight of 60 kg. This
317
level of exposure (93.5 µg/day) is substantially higher than in the various estimates for
318
Europe, Canada, USA and New Zealand, which generally were below 2 µg/day. The
319
mean exposure for French adults was estimated between 5.9 ng/kg b.w./day and 25.5
320
ng/kg b.w./day (Sirot, Fremy, & Leblanc, 2013).
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Conclusions
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The performed analytical methodology fulfilled the requirements established by the EC directive 401/2006.
The study highlights the fact that the levels of ZEA in the broa collected from
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Portuguese local markets were not totally suitable with regard to the current EC
327
legislation. It also emphasizes that there was no concern about intake of the ZEA
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through its consumption by adult population in the area surveyed.
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This study is the first report of ZEA occurrence in maize bread, broa, in Portugal
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and further studies are needed to elucidate the extent of ZEA contamination in different
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years and in different regions of the country.
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Acknowledgements
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The authors gratefully acknowledge the Portuguese governmental FCT through
335
PTDC/AAC-AMB/120889/2010 the financial support, and the post-PhD fellowship
336
granted to L.J.G. Silva (SFRH/BPD/62877/2009). The authors are also grateful to the
337
Laboratory of Mass Spectrometry (LEM) of the Node CEF/UC integrated in the
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National Mass Spectrometry Network (RNEM) of Portugal, for the MS analysis, and to
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Fátima Nunes for all the assistance.
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References
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Arezes, P. M., Barroso, M. P., Cordeiro, P., Costa, L. G., & Miguel, A. S. (2006).
343
Estudo Antropométrico da População Portuguesa (1 st.). Lisboa: Instituto para a
344
Segurança, Higiene e Saúde no Trabalho.
SC
RI PT
341
Bullerman, L.B., & Bianchini, A. (2007). Stability of mycotoxins during food
346
processing.International Journal of Food Microbiology, 119, 140-146.
347
Cano-Sancho, G., Sanchis, V. J., Ramos, A., & Marin, S. (2013). Effect of food
348
processing on exposure assessment studies with mycotoxins. Food Additives and
349
Contaminants: Part A, 30, 867-875.
TE D
M AN U
345
Cheeke, P. R. (1998). Mycotoxins in cereal grains and supplements. In: Cheeke, P.R.
351
(Ed.), Natural Toxicants in Feeds, Forages, and Poisonous Plants. Interstate
352
Publishers, Inc, Danville, IL, pp. 87–136.
354
355 356
Diekman, M. A., & Green, M. L. (1992). Mycotoxins and reproduction in domestic
AC C
353
EP
350
livestock. J. Anim. Sci. 70, 1615–1627.
EFSA, P. on C. in the F. C. (2011). Scientific Opinion on the risks for public health related to the presence of zearalenone in food. EFSA Journal, 9(6:2197), 1–124.
357
European Commission. (2006). COMMISSION REGULATION (EC) No 401/2006 of
358
23 February 2006 laying down the methods of sampling and analysis for the
15
ACCEPTED MANUSCRIPT 359
official control of the levels of mycotoxins in foodstuffs. Official Journal of the
360
European Union, L70, 12–34.
European Commission. (2007). COMMISSION REGULATION (EC) No 1126/2007 of
362
28 September 2007 amending Regulation (EC) No 1881/2006 setting maximum
363
levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize
364
and maize products. Official Journal of the European Union, L255, 14–17.
RI PT
361
Heidari S., Milani J., Seyed S., & Nazari J. (2014). Effect of the bread making process
366
on zearalenone levels. Food Additives & Contaminants: Part A, Accepted, DOI:
367
10.1080/19440049.2014.972472
M AN U
SC
365
Hewitt T. C., Flack C. L., Kolodziejczyk J. K., Chacon A. M., & D’Ovidio K. L. (2012)
369
Occurrence of zearalenone in fresh corn and corn products collected from local
370
Hispanic markets in San Diego County, CA. Food Control, 26, 300-304
372
INE (2013). Instituto Nacional de Estatística. Available from www.ine.pt/. Accessed on November, 2014.
EP
371
TE D
368
International Agency for Reserach on Cancer. (2002). Some traditional herbal
374
medicines, some mycotoxins, naphthalene and styrene. Monograph on the
375
AC C
373
evaluation of carcinogenic risk to humans. (vol. 82) (p. 601). Lyon: IARCPress.
376
IPCS, 2009. Dietary exposure assessment of chemicals in food. In W. H. Organization
377
(Ed.). Principles and methods for the risk assessment of chemicals in food (p. 98).
378
Geneva, Switzerland International Programme on Chemical Safety.
16
ACCEPTED MANUSCRIPT 379
Juan, C., Ritieni, A., & Mañes, J. (2012). Determination of trichothecenes and
380
zearalenones in grain cereal, flour and bread by liquid chromatography tandem
381
mass spectrometry. Food Chemistry, 134(4), 2389-2397.
Kaushik G. (2014). Effect of processing on mycotoxin content in grains. Critical
383
Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2012.701254
384
Lauren D. R. & Smith W. A. (2001) Stability of the Fusarium mycotoxins nivalenol,
385
deoxynivalenol and zearalenone in ground maize under typical cooking
386
environments. Food Additives & Contaminants, 18, 11, 1011-1016.
M AN U
SC
RI PT
382
387
Lino C. M., Silva L. J. G., Pena A., Fernández M., & Mañes J. (2007) Occurrence of
388
fumonisins B1 and B2 in broa, typical Portuguese maize bread. International
389
Journal of Food Microbiology 118, 79–82
MacDonald S. J., Anderson S., Brereton P., Wood R., & Damant A. (2005).
391
Determination of Zearalenone in Barley, Maize and Wheat Flour, Polenta, and
392
Maize-Based Baby Food by Immunoaffinity Column Cleanup with Liquid
393
Chromatography: Interlaboratory Study. Journal of AOAC International, 88, 6,
394
1733- 1740
396
EP
AC C
395
TE D
390
Maragos C. M. (2010). Zearalenone occurrence and human exposure. World Mycotox J., 3, 369–383
397
Marasas, W. F. O. (1991). Toxigenic Fusaria. In: Smith, J.E., Anderson, R.A. (Eds.),
398
Mycotoxins and Animal Foods. CRC Press, Boca Raton, FL, pp. 119–139.
17
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Numanoglu, E., Uygun, U., Koksel, H., & Solfrizzo, M. (2010). Stability of Fusarium
400
toxins during traditional Turkish maize bread production. Quality Assurance and
401
Safety of Crops & Foods, 2, 84–92.
Numanoglu, E., Yener, S., Gokmen, V., Uygun, U., & Koksel, H. (2013). Modelling
403
thermal degradation of zearalenone in maize bread during baking. Food Additives
404
and Contaminants: Part A, 30, 3, 528-533.
RI PT
402
Ramos Aldana, J., Silva, L. J. G., Pena, A., Mañes, J. V., & Lino, C. M. (2014).
406
Occurrence and risk assessment of zearalenone in flours from Portuguese and
407
Dutch markets. Food Control, 45, 51-55.
M AN U
SC
405
Rodríguez-Carrasco, Y., Moltó, J. C., Berrada, H., & Mañes, J. (2014). A survey of
409
trichothecenes, zearalenone and patulin in milled grain-based products using GC–
410
MS/MS. Food Chemistry 146, 212-219.
TE D
408
Rubert, J., Soriano, J. M., Mañes, J., & Soler, C. (2011). Rapid mycotoxin analysis in
412
human urine: a pilot study. Food and chemical toxicology : an international
413
journal published for the British Industrial Biological Research Association, 49(9),
414
2299–2304.
AC C
EP
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415
Ryu, D., Hanna, M. A, Eskridge, K. M., & Bullerman, L. B. (2003). Heat stability of
416
zearalenone in an aqueous buffered model system. Journal of Agricultural and
417
418 419
Food Chemistry, 51, 1746-1748.
Scudamore, K. A. (2008) Fate of fusarium mycotoxins in the cereal industry: recent UK studies. World Mycotoxin Journal, 1(3), 315-323.
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health risk assessment in the second French total diet study. Food and chemical
422
toxicology : an international journal published for the British Industrial Biological
423
Research Association, 52, 1–11.
RI PT
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Sulyok, M., Berthiller, F., Krska, R. & Schuhmacher, R. (2006). Development and
425
validation of a liquid chromatography/tandem mass spectrometric method for the
426
determination of 39 mycotoxins in wheat and maize. Rapid Communications in
427
Mass Spectrometry, 20, 2649-2659
SC
424
Zinedine, A., Soriano, J. M., Moltó, J. C., & Mañes, J. (2007). Review on the toxicity,
429
occurrence, metabolism, detoxification, regulations and intake of zearalenone: an
430
oestrogenic mycotoxin. Food and chemical toxicology : an international journal
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published for the British Industrial Biological Research Association, 45(1), 1–18.
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Table 1. Accuracy and repeatability intra and inter-day.
Exactitude
Intra-day
Inter-day
(µg/kg)
(%)
repeatability
repeatability
(% RSD)
(% RSD) 3.8
89.8
1.2
50
97.3
0.7
100
99.7
1.1
2.8
1.7
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Fortification levels
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Table 2. Frequency and levels of ZEA in broa.
Sample
Frequency
Range
Mean ± SD
composition
size
(%)
(µg/kg)
(µg/kg)
Maize and wheat
37
6 (16.2)
9.6-42.5
Yellow maize
15
1 (6.7)
nd-50.4
TOTAL
52
7 (13.5)
9.6-50.4
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Sample
24.5±13.1 -
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28.2±15.4
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Occurrence and risk assessment of zearalenone through broa consumption, typical maize bread from Portugal
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HIGHLIGHTS: -Different Portuguese maize bread, broa, was investigated for zearalenone.
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-A higher contamination frequency was observed in samples with maize and wheat.
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-The ZEA levels in broa were not totally suitable with regard to the EU legislation. -There is no concern about intake of ZEA through its consumption by adult
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population.