Vomitoxin and Zearalenone Content of Soft Wheat Flour Milled by Different Methods

Transcription

1 5 Journal of Food Protection, Vol. 70, No. 2, 20, Pages 5 5 Copyright, International Association for Food Protection Research Note Vomitoxin and Zearalenone Content of Soft Wheat Flour Milled by Different Methods VALENTINO PALPACELLI, 1 * LUCA BECO, 1 AND MAURIZIO CIANI 2 1 Studio Analisi Alimentari, Via XXV aprile 2, 0 Montone (PG), Italy; and 2 Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, via Brecce Bianche, 6 Ancona, Italy MS -297: Received 31 May 20/Accepted August 20 ABSTRACT Given the prominence and the growing importance of mycotoxins in human and animal health, and particularly of vomitoxin and zearalenone in people who use wheat and wheat products as their staple diet, we investigated two different types of wheat milling. Wheat produced according to good manufacturing practice related to mycotoxin risks (from sowing to harvesting) was used to compare the vomitoxin and zearalenone content of soft, following the use of two different types of milling, traditional milling with a stone mill and modern milling with a roller mill. Moreover, the vomitoxin and zearalenone content was also evaluated in commercial stone-milled and roller-milled flours. Our results show that stone milling reduced vomitoxin and zearalenone content in flours, compared with the use of the roller-mill system. Wheat and wheat products form the staple diet of about half of the world s population (6), and for this reason, the nutritional and toxicological properties of wheat are particularly important to the human diet. Mycotoxins are the primary food risk in wheat (9), and vomitoxin and zearalenone are mycotoxins that are produced by a well-known wheat pathogen: the fungi of the Fusarium genus (). In Western countries, the risks associated with the mycotoxins appear to be related to chronic exposure rather than to those of acute exposure (as seen in the developing countries) (7). It has been shown that vomitoxin is able to superinduce cytokine gene expression and secretion. This superinduction of the cytokines is associated with many allergic conditions and autoimmune diseases (7, 8,, ), and it appears that chronic exposure to low levels of this toxin can produce immunological effects. Zearalenone appears to be immunotoxic and associated with various diseases of the reproductive system (1,,, ). Vomitoxin and zearalenone are generally found in the external layers of the wheat kernels (, 19); however, under particular climatic conditions the infection can be systemic, and the contamination can extend through the whole kernel (5). Indeed, the milling phase initially included the elimination of the external layers of the wheat kernels, thus reducing vomitoxin and zearalenone levels. Before the 20th century, the milling operation was conducted exclusively by the stone-mill method (). However, in the 19th century, with the introduction of hard wheat varieties in Europe that produced lower flour yields with the stone-mill method, the practice of high grinding was developed. This consisted of milling with the stone more widely separated than * Author for correspondence. Tel: ; Fax: ; palpacelli@tin.it. usual, so it became possible to produce middlings, which were then further reduced by a low-grinding phase. This represented the first step toward the introduction of roller mills, which supplanted the stone-mill grinding in Europe, followed the rest of the world (except for some rural areas of developing countries and in some typical products in Western countries). In this context and in view of the fact that vomitoxin and zearalenone show widespread diffusion in wheat products (7), we carried out this study to investigate the effects of the introduction of the rollermill method on the content of these mycotoxins in wheat flours. MATERIALS AND METHODS preparation. Beginning November 20, to obtain the necessary wheat an experimental plan and protocol was followed to reduce the potential for Fusarium infection to a minimum (4): Bolero variety, late seeding, seed dressing, fungicide treatment at anthesis, correct soil tillage, and reduced nitrogen fertilization. In July 20, the mechanical harvesting was carried out, and ca. 6,000 kg of sound kernels were obtained. These were stored in one semiunderground masonry bin located on a private farm at about 400 m above sea level, under a rooftop. Hermetic storage with periodic fumigation was adopted. Between July 20 and December 20, 36 samples of wheat kernels were taken at regular intervals to both an ancient stone mill located in Bevagna (Perugia, Italy), and experimental roller mills. The resulting flour samples were analyzed for their vomitoxin and zearalenone content. In the stone mill, the wheat kernels were loaded into a seed box and then passed through: (i) the grain sieve to eliminate the cereal straw, stones, etc.; (ii) the trimming machine with an aspirator to clean the surface layer of the grain and eliminate dust generated by this operation; and (iii) the seed winnower to eliminate vetch seed and other nonwheat seeds and to further clean

2 5 PALPACELLI ET AL. J. Food Prot., Vol. 70, No. 2 the wheat. Finally, the cleaned wheat kernels were passed to the mill stones. The millstone grinder comprises two large circular stones that are placed one on the top of the other. The lower stone is fixed, whereas the upper stone rotates. The internal surfaces of the stones are scored, and in the middle of the stones there is a hole. The wheat entered through this hole and passed between the stones, where it was ground into smaller and smaller fragments, thus obtaining the whole meal that went into a sack. Whole meal can be sold directly or, as in this case, it was manually put into a separator (a simple system of sieves) to obtain the bran, the flour mixed with bran, and the white flour. White stone-mill flour is typically like semolina, with larger granulation than roller-mill flour. For the experimental roller mills, the wheat kernels were sieved to remove the unwanted material, such as stones, dust, and weed seeds, and then they were put into a wheat bin, and 3% water was added to them (tempering for 48 h to provide to % moisture content). The wheat kernels were then milled in the experimental roller mills, with the separation of bran, germ, and endosperm. The first set of mills (the breaking rollers) separated the bulk of the bran and the germ from the endosperm. The resulting product was then passed through the sieves to separate the various constituents, giving the bran, the wheat germ, the endosperm chunks, and the animal-feed byproducts. The endosperm chunks, together with small residual amounts of bran and germ, were passed through two series of rollers (the reducing rollers) to produce the flour. For each kind of milling, we obtained bran, flour mixed with bran, and white flour (about 50 kg of in 1-kg packaging). Thirty-six commercial flours were analyzed ( stone-mill flours and roller-mill flours), s were obtained over 3 months from five different sales points. analysis. (i) Sampling methods. Sampling of flours was carried out according to the official AOAC International methods (2). For each analysis, sacks were sampled, and 0 g was taken from each sack to obtain the 1-kg samples. (ii) Moisture analysis. s were analyzed for moisture following the official AOAC International methods, using the airoven method (3). Analyses were carried out in triplicate, and the data are given in the tables as means standard deviation (SD). (iii) Vomitoxin analysis. s were analyzed for vomitoxin with a JASCO HPLC system with a UV detector, using an immunoaffinity column (VICAM, Watertown, Mass.) according to manufacturer instructions (21). Briefly, 50 g of each flour sample was added to g of PEG 8000 and 200 ml of purified water, and then blended at high speed for ca. 1 min. The samples were then filtered through the vomitoxin test fluted filter paper, and then filtered again through a microfiber filter. The filtered extract (5 ml) was then applied to the vomitoxin test VICAM IAC column. The column was washed with 5 ml of distilled water, and the vomitoxin was eluted with 1 ml of methanol (HPLC grade). The eluate was collecting in a glass cuvette and evaporated to dryness. The dried samples were reconstituted in 1 ml mobile phase acetonitrile-water (:90, vol/vol, degassed) and injected (200 l) into the isocratic HPLC system (reverse-phase C column: flow rate, 1.0 ml/min; loop, 0 l; UV detection, 220 nm). The same procedure (evaporation and reconstitution) was repeated with the vomitoxin standard of 200 ppm vomitoxin in ethyl acetate methanol (95:5, vol/vol) (Supelco, Bellefonte, Pa.). The software used was JASCO Borwin HPLC software, version The limit of quantification was 50 ppb, with a mean recovery rate of 80%. Data were not corrected for recovery. Analyses were carried out in triplicate, and the data are given in the tables as means SD. (iv) Zearalenone analysis. s were analyzed for zearalenone with a JASCO HPLC system with a fluorimetric detector, using an immunoaffinity column (VICAM) according to manufacturer instructions (20). Briefly, 20 g of each flour sample was added to 2 g of NaCl and 50 ml of acetonitrile-water (90:, vol/ vol), and then blended at high speed for ca. 2 min. The samples were then filtered through fluted filter paper, and then filtered again through a microfiber filter. The filtered extract (5 ml) was then applied to the VICAM IAC column. The column was washed with 5 ml of distilled water, and the zearalenone was eluted with 1.5 ml of methanol (HPLC grade). The eluate was collected in a glass cuvette, and 1.5 ml of water (HPLC grade) was added. The samples were then injected (200 l) into the isocratic HPLC system (reverse-phase C column: mobile phase, acetonitrile-watermethanol [46:46:8; vol/vol/vol, degassed]; flow rate, 1.0 ml/min; loop, 0 l; detection wavelength, 274-nm excitation and 440- nm emission). The zearalenone standard was 50 ppm in acetonitrile (Supelco). The software was JASCO Borwin HPLC software, version The limit of quantification was 1 ppb, with a mean recovery rate of 85%. Data were not corrected for recovery. Analyses were carried out in triplicate, and the data are given in the tables as means SD. Statistical analysis. Normality testing was performed using GraphPad InStat, version 3.0a, for Macintosh. None of the samples of both of the analyses passed the normality tests (D Agostino and Pearson omnibus normality test, alpha 0.), so we performed a nonparametric test (Mann-Whitney test) using GraphPad Prism, version 4.00, for Macintosh (GraphPad Software, San Diego, Calif.). For the commercial flour screening, we assumed a non Gaussian distribution (due to the small numbers of samples) and performed a nonparametric test (Mann-Whitney test) using GraphPad Prism, version 4.00, for Macintosh. RESULTS The flours obtained from these unique samples from wheat with the different milling procedures showed moisture contents lower than %, a safe level for storage of the grain and flour (Table 1) (). Thus, the wheat kernels and flours were correctly stored, and we can exclude any significant effects due to the moisture contents. Both the vomitoxin and zearalenone contents were significantly lower in the stone-mill flours when compared with the roller-mill flours (Table 2). This was confirmed by the statistical analysis conducted using the rank sum test (Mann-Whitney test), the results of which are given in the full statistical breakdown in Table 4. The mean vomitoxin content in the stone-mill flours was 0 ppb, compared with 360 ppb for the roller-mill flours; the zearalenone content showed a similar distribution, with 6 ppb and ppb, respectively. The analyses of the randomized commercial flours confirmed the results of these experimental trials (Table 3). Thus, the mean vomitoxin content in the stone-mill flours was 245 ppb versus 945 ppb in the roller-mill flours, with zearalenone showing 1.7 ppb and 6.0 ppb, respectively. Again, the statistical analysis confirmed the significance of this comparison (stone-mill flour versus roller-mill flour),

3 J. Food Prot., Vol. 70, No. 2 MYCOTOXINS IN FOOD 5 TABLE 1. Results of the analyses of the moisture content of the stone-mill and roller-mill samples a Moisture TABLE 2. Results of the analyses of vomitoxin and zearalenone content of the stone-mill and roller-mill samples a Vomitoxin Zearalenone (%, w/w) (%, w/w) using the Mann-Whitney test (Table 4), confirming the results from the experimental trial. DISCUSSION The results obtained in this comparative study show that stone milling results in a reduction of about 40 to 50% in the vomitoxin and zearalenone contents of s. This was also confirmed with the commercial flour samples, where again the stone-mill commercial flours show lower amounts of vomitoxin and zearalenone than do the rollermill flours. The main considerations toward an explanation of these results would be as follows: (i) The stone-mill system used for the experimental samples has a trimming machine with an aspirator, and thus it can at least partially eliminate the external layer of the wheat kernels, where most of the mycotoxins are generally located; and (ii) the roller-mill system has a series of reduction rollers that are designed to extract the residual flours from the bran, and this will probably also further extract the mycotoxins. This last observation is in accordance with those of Scott et al. (5), who noted that there was little difference in the mycotoxin content between flour streams (using roller mills). In addition, debranning before milling (preprocessing) was used successfully in experimental trials, and it was seen that this can produce flours that have a lower mycotoxin content than those produced as roller-mill flours (without debranning) (5). At the same time, the change in the milling system could bring about an increase in the potential exposure of consumers to the mycotoxins. Such an increased exposure

4 5 PALPACELLI ET AL. J. Food Prot., Vol. 70, No. 2 TABLE 3. Results of the analyses of the vomitoxin and zearalenone content of the commercial stone-mill and roller-mill wheat flour samples a Vomitoxin , , , , , Zearalenone can also be caused by the tempering operation, where the wheat kernels are brought to to % moisture content to provide for a better quality for the baking industry, combined with a longer shelf life (up to to months). In previous centuries, the wheat was brought to the stone mills and then consumed shortly after milling, removing the need for long-term storage. Thus, this increased moisture content ( to %) that is achieved through the tempering of the wheat kernels, together with a longer shelf life of the resulting flour, could represent another cause of this increase in mycotoxin and mold content. In conclusion, our results demonstrate that the rollermill system can result in a greater exposure to vomitoxin and zearalenone in populations where wheat is used as the staple diet, even if this process has brought about great improvements in the milling efficiency of the wheat. REFERENCES 1. Alldrick, A. J Zearalenone, p In N. Magan and M. Olsen (ed.), Mycotoxins in food: detection and control. Woodhead Publishing, Ltd., Cambridge, UK. 2. AOAC International. 20. Method Official methods of analysis, th ed., 2nd rev. AOAC International, Gaithersburg, Md. 3. AOAC International. 20. Method Official methods of analysis, th ed., 2nd rev. AOAC International, Gaithersburg, Md. 4. Bilgrami, S. K., and A. K. Choudhary Mycotoxins in preharvest contamination of agricultural crops, p In K. K. Sinha and D. Bhatnagar (ed.), Mycotoxins in agriculture and food safety. Marcel Dekker, Inc., New York. 5. Dexter, J. E., and T. W. Nowicki. 20. Safety assurance and quality assurance issues associated with Fusarium Head Blight in wheat, p In K. J. Leonard and W. R. Bushnell (ed.), Fusarium Head Blight of wheat and barley. American Phytopathological Society, St. Paul, Minn. 6. Food and Agriculture Organization of the United Nations. 20. Food outlook. Report no. 1. Available at: 0/J24e/j24e00.htm. 7. Galvano, F., A. Ritieni, G. Piva, and A. Pietri. 20. Mycotoxins in the human food chain, p In T. D. Diaz (ed.), The mycotoxin blue book. Nottingham University Press, Nottingham, UK. 8. Krska, R Mycotoxins of growing interest: trichothecenes. Third Joint FAO/WHO/UNEP International Conference on Mycotoxins, Tunis, Tunisia. 9. Malmauret, L., D. Parent-Massin, J. L. Hardy, and P. Verger. 20. Contaminants in organic and conventional foodstuffs in France. Food Addit. Contam. 19: Matz, S. A The chemistry and technology of cereals as food and feed, 2nd ed. Pan-Tech International, Inc., McAllen, Tex.. McMullen, M., R. Jones, and D. Gallenberg Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Dis. 81: Mirocha, C. J Mycotoxins of growing interest: zearalenone. Third Joint FAO/WHO/UNEP International Conference on Mycotoxins, Tunis, Tunisia.. Peraica, M., B. Radic, A. Lucic, and M. Pavlovic Toxic effects of mycotoxins in humans. Bull. WHO 77: Pitt, J. I Toxigenic fungi and mycotoxins. Br. Med. Bull. 56: Riley, R. T Mechanistic interactions of mycotoxins: theoretical considerations, p In K. K. Sinha and D. Bhatnagar (ed.), Mycotoxins in agriculture and food safety. Marcel Dekker, Inc., New York. TABLE 4. Full overview of the statistical differences (as evaluated by the Mann-Whitney test) for the vomitoxin and zearalenone levels across all of the stone-mill and roller-mill samples comparison P value Exact or approximate P value Significantly different medians? (P 0.) Vomitoxin stone mill vomitoxin roller mill (Table 2) 0.00 Gaussian approximation Yes Zearalenone stone mill zearalenone roller mill (Table 2) 0.00 Gaussian approximation Yes Vomitoxin stone mill commercial samples vomitoxin roller-mill commercial samples (Table 3) 0.00 Gaussian approximation Yes Zearalenone stone mill commercial samples zearalenone roller mill commercial samples (Table 3) 0.00 Gaussian approximation Yes

5 J. Food Prot., Vol. 70, No. 2 MYCOTOXINS IN FOOD 5. Riley, R. T., and J. Pesta. 20. Mycotoxins: metabolism, mechanisms and biochemical markers, p In T. D. Diaz (ed.), The mycotoxin blue book. Nottingham University Press, Nottingham, UK.. Seitz, L. M., W. T. Yamazaki, R. L. Clements, H. E. Mohr, and L. Andrews Distribution of deoxynivalenol in soft wheat mill steams Cereal Chem. 62: Surai, P. F., and J. E. Dvorska. 20. Effects of mycotoxins on antioxidant status and immunity, p In T. D. Diaz (ed.), The mycotoxin blue book. Nottingham University Press, Nottingham, UK. 19. Trigo-Stockli, D. M., C. W. Deyoe, R. F. Satumbaga, and J. R. Pedersen Distribution of deoxynivalenol and zearalenone in milled fractions of wheat. Cereal Chem. 73: VICAM, L.P Zearala test instruction manual. VICAM, L.P., Watertown, Mass. 21. VICAM, L.P Vomitoxin test HPLC instruction manual. VI- CAM, L.P., Watertown, Mass.