Natural occurrence of deoxynivalenol in wheat, wheat ftour and bakery products in Argentina

Food Additives and Contaminants, 1997, Vol. 14, No. 4, 327-331 s Natural occurrence of deoxynivalenol in wheat, wheat ftour and bakery products in Argentina Á. M. PacinH, s. L. Resnikt,M. s. NeiraH, G. Moltó and E. Martínez t Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Argentina; t Centro de Investigación en Micotoxinas, Universidad Nacional de Luján, Argentina; Universidad de Buenos Aires, Argentina (Received 30 June 1996; revised 18 November 1996; accepted 23 November 1996) The objective of this study was to evaluate the natural occurrence of deoxynivalenol (DN) in wheat, wheat flour and different kinds of breads and pastries widely consumed by the population in Argentina. f 60 wheat samples analysed, 93,3% were contaminated. The average DN contamination level over al! samples was 1798 J-1.g/kg, and the minimum and maximum values were 100 J-1.g/kgand 9250 J-1.g/kg,respectively. The wheat flour samples (61 samples) were contaminated with DN at levels ranging from 250 J-1.g/kgto 9000 J-1.g/kgwith an average of 1309 J-1.g/kg.The frequency of DN contamination over 42 samples of different bakery products was 92'8%, with levels ranging from 200 J-1.g/kgto 2800 J-1.g/kgwith an average of 464 J-1.g/kg.These results suggest a high risk for consumers of wheat products and the need to monitor final products before consumption. Keywords: deoxyniva1eno1, DN, mycotoxins, natural occurrence, wheat contamination Introduction graminearum (Marasas 1992). DN-contaminated feeds cause decreased feed consumption, dermatological1esions, gastrointestina1 disorders and haemato- 10gica1 changes in most of the animal species studied (Veno 1983, Pacin 1992). The immunotoxic effects of DN were also demonstrated in laboratory assays (Tryphonas et al. 1984). Many data of the natural occurrence of DN in wheat in different countries have been reported by Veno (1983), y oshizawa (1992), and other authors (Je1inek et al. 1989, Perkowski et al. 1990, Tanaka et al. 1990, Abouzied et al. 1991, Hietaniemi and Kumpulainen 1991, Luo et al. 1992, Yoshizawa 1992, Stratton et al. 1993, Trigo-Stockli et al. 1995, Trucksess et al. 1995). In Argentina, wheat products are consumed to a great extent by the population (Gallo et al. 1992) and wheat is one of the most important grains in the export trade. The natural occurrence of DN has been assessed in a survey conducted from 1985 to 1992 (Quiroga et al. 1995). It was suggested that when meteorological conditions could favour contamination it is necessary to carry out strict control prior to storage. Due to a rainy season in some regions, the 1993/94 wheat crop showed high levels of infection by Fusarium, which caused a reduction in harvest yield, decreased hectolitre weight and significantly increased grain damage (Luqui and Gómez 1995). Taking into account the above information a survey of DN contamination in the 1993/94 wheat crop, as well as flour and bakery products, was carried out. Deoxynivalenol (DN) is a naturally-occurring trichothecene mycotoxin of cereal grains produced by several species of the genus Fusarium, especially F. To whom correspondence should be addressed at: Centro de Investigación en Micotoxinas (C.LM.), Universidad Nacional de Luján, c.c. 221, (6700) Luján, Pcia. de Buenos Aires, Argentina. Materials and methods Samples Wheat samples (60) were collected from several fields located in the main production are a in Argentina 0265-203Xj97 $12,00 «:J 1997 Taylor & Franeis Ltd

328 A. M. Pacin et al. during harvest (crop 1993/1994), wheat flour samp1es (61) were purchased at milis, and bakery products (42) were chosen, taking into account their formu1ations and processing, from 10w-technology bakeries. Each sample was milled and its moisture content (me) was determined in triplicate. Well-mixed 2 g samples were heated in a vacuum oyen at 60 C until two successive weight loss measurements, performed every 2 h, showed less than 0,05% me difference. The rest of the sample (approximately 1 kg) was stored in a sealed plastic bag at -18 C until DN quantification. Deoxnivalenol analysis Ground wheat and flour samples (25 g) were blended with 100 mi of acetonitrile : water (84 : 16) for 3 min at high speed. Prior to the acetonitri1e : water extraction, doughs, fermented doughs and bakery products were defatted with three successive extractions with 100, 50 and 25 mi of n-hexane. The water component of the extraction (acetonitrile : water) mixture was adjusted to balance the water content of each sample. The extract was filtered through Whatman No. 4 paper. Approximately 8 mi of the filtrate was placed in a 15 x 85 mm culture tube and 4 mi of the filtrate was passed through a Mycosep 225 column (Romer Labs, Inc.). The purified extract was then transferred to another tube and evaporated to dryness in a 60 C water bath under vacuum. Thin-layer chromatography The residue was dissolved with 100 111 of 2: 1 acetone-methanol and vortexed for 30 s. A 25 111 Hamilton syringe was used for spotting 20 111of each sample, along with 1, 5, 10 and 20 111 of DN standard (50 111/ml) on a Silica gel 60 thin-layer chromatography p1ate (Merck cat. No. 5553, 20 cm x 20 cm) which was then placed into a developing tan k containing 100 mi of 1 : 2 toluene-acetone until the solvent travelled to 1.0 cm from the top of the plate. The plate was then removed and allowed to air dry, sprayed with 20% aluminium chloride (ethanol-water, 1: 1) and heated at 150 C for 10 min and viewed under long wave UV light. The levels of DN were estimated visually by comparison to standard spots (R[ DN 0'5). Gas chromatography. Derivatization. To the dried residue in a test tube was added 1 I1g HT-2 toxin as interna1 standard and 500111 of catalyst solution (4-N,N-dimethylaminopyridine, 2 mg/ml in toluene: acetonitrile 95: 5). The tube was vortexed for 1 min, 35 111 of heptafluorobutyric acid the tube was vortexed anhydride was added for another minute and then p1aced in a sand bath at vortexed for 1 mino The 60 C for 30 min reaction mixture and was allowed to cool to room temperature for 3-5 mino Excess derivatizing agent was destroyed by adding 1 mi of an aqueous 4% (w/v) sodium bicarbonate solution and vortexing for 2 mino The sample was centrifuged for 1 min at 350 g to separate the layers. The toluene layer was transferred to a vial prior to GC analysis. Chromatography. A Hewlett-Packard gas chromatograph model 5890 Series 11 was used for the confirmation of DN under the following conditions: capiliary column Hewlett-Packard HP-5 25 m by 0.2 mm (inside diameter) and 0,5 11mfilm thickness; carrier, nitrogen gas at a flow rate of 1 m1/ min and the nitrogen make-up gas at a flow rate of 50 ml/min; column temperature, 1 min at 120 C, then 40 C/min to 200 C held 2 min, 5 Cjmin to 250 C and held 11 mino The injector and electro n capture detector temperatures were 250 C and 300 C, respectively. Method recovery was determined by spiking DN standards at 1evels of 100, 250 and 500 I1g/kg to homemade bread, French bread and their corresponding doughs. Recovery average s were 102'0, 87,0 and 92-4% respectively. The results were expressed on a dry basis. ResuIts and discussion Figure 1 shows box-plots of DN contamination for wheat, flour and products. It may be observed that the contamination level of bakery products was less than those of the wheat or flour. A1so, the DN contamination values of bakery products were 1ess dispersed than the others. There were mild outliers, indicated by an asterisk and severe outliers indicated by a circle. Due to the presence of outliers we decided

Deoxynivalenol in wheat and wheat products 329 Box and Whisker Plot,-., J).::a::: -- ŌJ) :::t "-'........... Ō - " '2... i>< 10000 I 4000 2000 o 8000 j 6000. I * o Q * WHEAT FLUR PRDUCTS Figure l. Deoxynivalenol contamination of wheat and products (1993/1994). to use a non-parametric method to test hypotheses about the differences between DN contamination 1eve1s.Using the median test the hypothesis of equa1- ity of median levels of flour and wheat was not rejected (p 0,2517). With respect to flour and bakery products and wheat and bakery products, the hypothesis of equality of median levels was rejected in both cases (p < 0,0001). We could not analyse bakery products manufactured with the same wheat flour sampled in the study, but we suspect that the observed reduction in DN contamination was probably due to the dilution in dough preparation, the fermentation and baking process. Further studies will be needed in this field. Wheat contamination (ranging among positive samples from 100 to 9250 lg of DN/kg) showed an average of 1798 lg/kg on a dry weight basis. ther studies have been carried out in Argentina using wheat samples at the same harvest-time. The main wheat production area in Argentina includes region 11 (south-east of Córdoba Province, south of Santa Fe Province and north and south-east of Buenos Aires Province) and region V (central, north-west and south of Córdoba Province, south-west of Buenos Aires Province and La Pampa Province); and each region could be divided into subregions North and South. Rizzo et al. (1994) analysed 19 samples from region 11 North (11 from silo-storage and 8 from harvest) and detected DN contamination with an average of 13000 lg/kg; and 14 freshly harvested wheat samples of region 11 South, with an average of 1260 lg/kg. It is not possible to compare the work of Rizzo et al. with our data because they reported only total average values without discrimination between stored and freshly harvested samples. Luqui and Gomez (1995) reported DN-contaminated wheat from the 1993/94 crop. In 232 samples they found an average of 1473 lg/kg on a wet basis. We tested the hypothesis of equal DN contamination distribution between these data and our data using the 'two samples Kolmogorov-Smirnov test' (Conover 1980), and this hypothesis was not rejected (p 0,3863). As both data sets belong to the main production area, the result of the test allowed us to consider our data as a representative sample ofthe 1993/94 wheat crop. Sixty-one flour samples were analysed and all of these were contaminated by DN (ranging from 250 lg/kg to 9000 lg/kg) with a mean value of 1309 lg/kg and a median value of 950 lg/kg (figure 1). ther available data in Argentina showed that 54 flour samples,

330 A. M. Pacin et al. Table l. Contamination by DN in different bakery products expressed on a dry basis ver positive samples (g/kg) Contaminated Average Median Products samples/total (g/kg) (g/kg) Minimum Maximum Croissant butter 8/8 453.1 395,5 326 648 Croissant fat 4/5 377-0 429.0 336 563 Figazzasd 8/8 851-4 387,0 212 2800 French bread 10/12 263.2 294.0 198 436 Homemade bread 4/4 326.5 326.5 269 384 'Libritos,b 4/4 581.0 545,5 210 1023 aeach kilogram of wheat flour is kneaded with 533 g of water, 156 g of fat and 20 g of salt. Malt (8 g) is added, fermented with yeasts (19 g). The dough is cooked for 10 min at 2l0 e. beach kilogram of wheat flour is kneaded with 667 g of water, 667 g of fat and 33 g of salt. Malt (10 g) is added, fermented with yeasts (19 g). The dough is cooked for 15 min at 210 e. produced from the same wheat harvest, presented an average DN contamination of 1210 Ilg/kg (Rizzo et al. 1994). Comparing our data with the latter, it should be highlighted that the percentage of samples under 2000 Ilg/kg (usual regulatory levels) in our wheat flour was 83,6% while in the data of Rizzo et al. (1994) it was 78,5%. We tested the hypothesis of equality of these two percentages and it was not rejected (p 0-4894). Bran bread (only one sample) had a DN contamination level of 378 Ilg/kg. DN contamination values in other bakery products analysed are presented in table l. As is shown, mean and median values were similar, except in the case of 'figazzas' which reached a level of 2800 Ilg/kg in one sample. The results show that it is necessary to continue the assessment not only ofmycotoxin occurrence in foodstuffs, but also contamination of bakery products. This is the first study to evaluate the bakery products most commonly purchased and consumed by the population in Argentina. nly by evaluating contamination could it be possible to minimize or avoid the risk of mycotoxin intoxication. Acknowledgements The authors acknowledge Mrs G. Cano for her technical assistance and Consejo Nacional de Investigaciones y Tecnicas, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Merck Química Argentina, Universidad de Buenos Aires for financial support. References ABUZIED, M. M., AzcoNA, 1 l., BRASELTN,W. E., and PESTKA, 1 1, 1991, lmmunochemical assessment ofmycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination. Applied and Environmental Microbiology, 57, 672-677. CNVER, W 1,1980, Practical Non-parametric Statistics (New York: Wiley), pp. 309-314. GALL, A., BURDlGNN, F., PACIN, A., BARBIERI,T., and RESNIK, S., 1992, Evaluation of food intake by means of 24 hour dietary recall in a town of the province of Buenos Aires, Argentina, Ecology 01 Food and Nutrition, 28, 299-317. HIETANIEMI, v., and KUMPULAINEN, 1, 1991, Contents of Fusarium toxins in Finnish and imported grains and feeds. Food Additives and Contaminants, 8, 171-182. JELINEK, e. F., PHLAND, A. E., and WD, G. E., 1989, Worldwide occurrence of mycotoxins in foods and feeds-an update. Journal 01 the Association 01 fficial Analytical Chemists, 72, 223-229. Luo, Y, YSHIZAWA,T., YANG, 1 S., ZHANG, S. Y, and ZHANG, B. 1, 1992, A survey of the occurrence of Fusarium mycotoxins (trichothecenes, zearalenone and fusarochromanone) in corn and wheat samples from Shaanxi and Shanxi Provinces. Mycotoxin Research, 8, 85-91. LUQUI, A. B., and GÓMEZ, M., 1995, Evaluación de la incidencia del porcentaje de granos dañados por Fusarium en el nivel de toxinas DN y T2 en trigo pan durante la campaña 1993/94, edited by IASCA V (Argentina: Secretaria de Agricultura, Ganaderia y Pesca), pp. 1-9. MARASAS, W F.., 1992, Toxigenic Fusaria. Mycotoxins and Animal Foods, edited by J. E. Smith and R. S. Henderson (Boca Raton, USA: CRC Press), pp. 119-139. PACIN, A., 1992, Las micotoxinas como factor de riesgo en la salud humana. Seminario Internacional sobre Micotoxinas, edited by Asociación Colombiana de Postcosecha de Granos (Colombia: IDEMA), pp. 87-108. PERKWSKI, 1, PLATTNER, R. D., GLINSKI, P, VESNDER, R. F., and CHELKWSKI, 1, 1990, Natural occurrence of deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, nivalenol, 4,7-dideoxynivalenol and zearalenone in Polish wheat. Mycotoxin Research, 6, 7-12. QUIRGA, N., RESNIK, S., PACIN, A., MARTÍNEZ, E., PAGAN,A., RIC- CBENE, 1., and NEIRA, S., 1995, National occurrence of

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