Journal Food and Environment Safety of the Suceava University – FOOD ENGINEERING, Year IX, No1 - 2010 68 STUDY ON THE CONTENT OF ZEARALENONE FROM WHEAT AND DERIVATIVES Sonia GUTT 1, Georg GUTT1, Mariana MAZAREANU2 1Stefan cel Mare University of Suceava, Faculty of Food Engineering, 13 Universitatii Street, 720229, Suceava, Romania, e-mail: gutts @usv.ro 2 Animal Sources Food Safety Department-Suceava Abstract: In food and fodder naturally contaminated with fungus there are in high concentrations seven groups of mycotoxins: aflatoxins, ochratoxin, trichothecenes, zearalenone, patulin, citrinine and acid penicilic. Mycoestrogens are estrogens produced by fungi. The most important mycoestrogen is zearalenone, produced by Fusarium species of fungi. Zearalenone and its metabolites exert their estrogenic effects through binding to estrogen receptors[1]. Zearalenone is the main phyto-oestrogen and it is the primary toxin causing infertility, abortion or other breeding problems, especially in swine. Chickens fed with contaminated feed showed zearalenone residues in muscles and liver. The concentrations of zearalenone and its metabolites, α- and β-zearalenol, were determined quantitatively by high-performance liquid chromatography (HPLC), in placenta, in maternal liver and spleen rats, α- and β-zearalenol were transferred into the foetus [2]. The paper analyzed raw materials used in the bakery industry, samples of grain, flour and bran in order to determine the content of zearalenone Specific limits for zearalenone have been regulated in 2007 by the European Union, ranging from 20 µg/kg for infants and small children foods to 75 µg /kg for maize flour (EU Commission Regulation (EC No 1126/2007). Amounts of mycotoxins have been highlighted by ELISA. In analyzed samples were detected exceeded limits for a number of one sample of wheat and one sample of bran, the other samples containing zearalenone below the maximum allowable limit, the analysed raw materials considered as safe for bakery. 1.Introduction Mycotoxins are secondary metabolites of fungi which contaminate feed and food. Mycotoxins presented in food products and animal feeds are an important problem concerning food and feed safety and significant economic losses are associated with their impact on human and animal [3]. Vegetable products are convenient substrates for fungi development and mycotoxins production[4]. During storage they are favorable substrates if they have a high moisture content above 80% and a temperature above 20°C. The main types of fungi producing mycotoxins are: Aspergillus, Penicillium, Fusarium şi Alternaria. It should be noted that a fung can produce more mycotoxins and that a particular mycotoxin may be produced by several fungi; it can lead to phenomena of synergism and potentiation of toxic action. Zearalenone is heat-stable and is found worldwide in a number of cereal crops, such as maize, barley, oats, wheat, rice, and sorghum and also in bread. Zearalenone has a chemical structure similar to steroid hormones and it is produced by fungi of the genus Fusarium (F. graminearum, F. avenacum F. Equisetti). Zearalenone belongs to the group of phytoestrogens have an estrogen- mimetic effect, but it is not a true, it is not a plant product (soy, alfalfa, clover) but a fungical one. Zearalenone is a lactone of resorcylate acid, it is carcinogenic, mutagenic and genotoxic in a concentration 1.5 mM/L. Journal Food and Environment Safety of the Suceava University – FOOD ENGINEERING, Year IX, No1 - 2010 69 Zearalenone induces cell proliferation in estrogen-dependent tissues (carcinogenesis), and contributes to endometrial hyperplasia and adenocarcinoma formation. 2. 2. Materials and methods A method for determination of zearalenone in cereal flour has been developed applying pressurized liquid extraction (PLE) using methanol/acetonitrile (50:50 v/v) as the solvent extraction[5]. The extracted samples is analyzed with liquid chromatography coupled to mass spectrometry (LC–MS) with an electro spray ionisation interface (ESI). Conventional chromatographic methods are generally intensive time and capital consuming and therefore have been developed and marketed a series of rapid analysis methods mostly based on immunological principles. Method used in paper is based on immunoenzymatic reactions in heterogeneous system (ELISA - Enzymes Linked Immuno Sorbent Assay) that used for determination both of antigen and of antibody. Antigenic structures, usually in the form of liquid suspension can have microbial origin (bacteria, viruses, fungi). Antibodies can be represented by non-immune sera or purified polyclonal immunoglobulins. In the technique used, the specific antigen or antibody is attached to a solid support that can bind antigen or antibody by passive adsorption or covalent binding. Passive adsorption of macromolecules consists of immobilization on a surface of glass or plastic.The vast majority of protein diluted (1-10 mg / ml) in an alkaline solution (pH - 9.6) are adsorbed on plastic in few hours. As a solid support can be used propylene, polystyrene, polycarbonate or latex. In the tests were used microplates of polystyrene, with fully automated work advantage.Covalent binding allows irreversible connections between reactants and solid support so that quantitative assessments have a maximum precision. Calitatea fixării structurilor proteice pe suportul solid depinde de concentraţia proteinei, de natura suportului utilizat şi de intervenţia unor agenţi chimici de fixare. Quality of protein structures are fixed on solid support depends on protein concentration, the nature of substrate used and the occurrence of chemical fixation. Enzyme conjugate there should be a couple antigen - enzyme or antibody - enzyme, the latter being the most frequently used form. 2.1 Materials The sample must be kept cold, avoid light. A quantity of 5 g of ground sample is mixed with 25 ml of methanol / water (70/30); shaked vigorously for 3 minutes; centrifuged 10 minutes at 3500 rpm. at room temperature or filter; the supernatant or filtrate are diluted 1: 7 with a buffer for diluting the sample and using 50 µl per well. Reagents. It works with reagents at room temperature, prepared before use . Microtitre plate. Wells are coated with antibodies. Conjugate solution. Enzyme conjugate is concentrated and for reconstitution the enzyme conjugate is diluted 1: 11 in buffer (eg. 200 µl concentrate + 2.0 ml of buffer solution, sufficient for four wells). After use, the vial is kept at 2°C to 8°C in the dark. Solution substrate / chromogen. Solution substrate/chromogen is ready to use and tends to precipitate at 4°C. It brings the bottle to room temperature and kept in the dark, stirring before pipetting.. Chromogen staining is an indicator of deterioration and reagents should be discarded. Wash buffer. Wash buffer is diluted before use. Samples are needed to achieve the next steps. 1. Pipette 100 µl standard dilution buffer, which is the reagents blank (wells A1 and A2). Journal Food and Environment Safety of the Suceava University – FOOD ENGINEERING, Year IX, No1 - 2010 70 2. Pipette 50 µl standard S0 (wells B1 şi B2). 3. Pipette 50 µl standard, in duplicate (S1…S6). 4. Pipette 50 µl sample solution in duplicates, in the remaining wells (P1, P2 ....P40). Tabel 1 Order in which pipette standards and samples in microplate 1 2 3 4 5 6 7 8 9 10 11 12 A Blank Blank P1 P1 P9 P9 P17 P17 P25 P25 P33 P33 B S0 S0 P2 P2 P10 P10 P18 P18 P26 P26 P34 P34 C S1 S1 P3 P3 P11 P11 P19 P19 P27 P27 P35 P35 D S2 S2 P4 P4 P12 P12 P20 P20 P28 P28 P36 P36 E S3 S3 P5 P5 P13 P13 P21 P21 P29 P29 P37 P37 F S4 S4 P6 P6 P14 P14 P22 P22 P30 P30 P38 P38 G S5 S5 P7 P7 P15 P15 P23 P23 P31 P31 P39 P39 H S6 S6 P8 P8 P16 P16 P24 P24 P32 P32 P40 P40 5. Add 50 µl enzyme conjugate into each well. Cover the plate with aluminum foil, stir the stirrer of the microplate (60 rpm) and incubated for two hours at room temperature in the dark. 6. Discard the liquid and beat with power plate face down on absorbent paper to remove traces of liquid. Add 250 µl washing solution (PBS and distilled water) and discard the liquid. Repeat washing step twice. 7. Add 50 µl substrate and 50 µl chromogento each well. Mix rotating plate and incubated 30 min at room temperature (20-25 ° C), from darkness. Blue color intensity obtained is inversely proportional to the concentration of mycotoxins in the sample or standard. 8. Add 100 µl stop solution to each well. Mix gently rotating plate and measured at 450 nm against air blank site. Not to exceed 60 min after adding stop solution. Using the optical densities (OD) of the standard, the calibration curve is plotted against the concentrations of other standards, and the amount of of mycotoxin in the sample is extrapolated from standard curve. Results and discussion Mycotoxicological samples analysed by ELISA were represented by samples of wheat flour, samples of bran and samples of wheat. Table 2 Zearalenone concentrations for each analysed sample No. Sample Zearalenone ppb No. Sample Zearalenone ppb No Sample Zearalenone ppb 1 Wheat 33.40 1 Bran 21.12 1 Wheat flour 18.12 2 Wheat 42.07 2 Bran 38.53 2 Wheat flour 16.22 3 Wheat 53.56 3 Bran 58.10 3 Wheat flour 19.36 4 Wheat 23.60 4 Bran 43.67 4 Wheat flour 22.02 5 Wheat 47.11 5 Bran 52.13 5 Wheat flour 14.21 6 Wheat 44.63 6 Bran 40.49 6 Wheat flour 16.41 7 Wheat 46.34 7 Bran 44.88 7 Wheat flour 11.12 8 Wheat 48.19 8 Bran 54.41 8 Wheat flour 13.14 9 Wheat 61.45 9 Bran 24.62 9 Wheat flour 10.84 10 Wheat 111.76 10 Bran 46.42 10 Wheat flour 19.41 11 Wheat 27.75 11 Bran 53.53 11 Wheat flour 52.63 12 Wheat 39.39 12 Bran 41.43 12 Wheat flour 28.41 13 Wheat 45.35 13 Bran 31.11 13 Wheat flour 18.74 14 Wheat 68.29 14 Bran 37.88 14 Wheat flour 31.02 Journal Food and Environment Safety of the Suceava University – FOOD ENGINEERING, Year IX, No1 - 2010 71 15 Wheat 33.98 15 Bran 51.63 15 Wheat flour 24.51 16 Wheat 67.22 16 Bran 42.64 16 Wheat flour 15.47 17 Wheat 28.20 17 Bran 76.87 17 Wheat flour 25.74 18 Wheat 82.22 18 Bran 19.41 18 Wheat flour 33.43 19 Wheat 76.14 19 Bran 32.01 19 Wheat flour 57.96 20 Wheat 51.13 20 Bran 51.07 20 Wheat flour 12.64 21 Bran 48.96 22 Bran 54.11 Analyzing the data presented in Table 2 derived from zearalenone determination it found that:  in one wheat sample, (5%) of the total number of samples, zearalenone is presented in an amount greater than the maximum limit permitted by applicable law (100 ppb);  in two wheat samples (10% of total samples), zearalenone is presented in a quantity of 76-100 ppb and falls below the maximum allowed by the applicable law;  the five wheat samples (25% of total samples), zearalenone is presented in an amount between 51-75 ppb and falls below the maximum permitted by applicable law;  the nine wheat samples (45% of total samples), zearalenone is presented in an amount between 31-50 ppb and falls below the maximum permitted by applicable law;  the three wheat samples (15% of the total number of samples), zearalenone is presented in an amount between 1-30 ppb and falls below the maximum permitted by applicable law; The frequency of wheat samples with zearalenone values is shown in Fig .1 0,00% 5,00% 10,00% 15,00% 20,00% 25,00% 30,00% 35,00% 40,00% 45,00% 1-30 ppb 31-50 ppb 51-75 ppb 76- 100 ppb peste 101 ppb 15,00% 45,00% 25,00% 10,00% 5% Fig.1. Graphical representation of samples of wheat contamination with zearalenone  From the analyzing samples of bran is resulted the following values for zearalenone content  in a single sample of bran (4.54%) of the total number of samples, zearalenone is presented in an amount greater than the maximum permitted by applicable law (ppb 75);  in seven samples of bran (31.81% of total samples), zearalenone is presented in an amount of 51-75 ppb and falls below the maximum permitted by applicable law;  în 11 probe tărâţe (50% din numărul total de probe) ZEA este prezentă într-o cantitate cuprinsă în intervalul 31 – 50 Journal Food and Environment Safety of the Suceava University – FOOD ENGINEERING, Year IX, No1 - 2010 72 ppb şi se încadrează sub limita maximă admisă de legislaţia în vigoare;  in eleven samples of bran (50% of the total number of samples), zearalenone is presented in an amount of 31-50 ppb and falls below the maximum permitted by legislation;  in three samples of bran (13.65% of total samples), zearalenone is presented in an amount of 1-30 ppb and falls below the maximum permitted by applicable law; 13,65% 50% 31,81% 4,54% 0,00% 5,00% 10,00% 15,00% 20,00% 25,00% 30,00% 35,00% 40,00% 45,00% 50,00% 1-30 ppb 31-50 ppb 51-75 ppb peste 76 ppb Fig. 2. Graphical representation of samples of bran contamination with zearalenone The results of analysis of flour samples are these:  in two samples of wheat flour (10% of total samples), zearalenone is presented in an amount of 31-50 ppb and falls below the maximum permitted by applicable law;  in eighteen samples of wheat flour (90% of total samples), zearalenone is presented in an amount of 1-30 ppb and falls below the maximum permitted by applicable law Conclusions From the analysis of samples represented by wheat flour, bran and wheat using ELISA method was found that a single sample of wheat, and one of bran contained zearalenone amount over the limit while the vast majority of samples (90% flour wheat, 63.65% samples of bran and 35% samples of wheat ) are below the permissible content of zearalenone and other samples are well below the maximum allowed. It can be concluded that these raw materials can be used to obtain reliable bakery food. References 1. J. FINK-GREMMELS ∗, H. 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