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Journal of Food Protection, Vol. 70, No. 2, 2007, Pages 509–513 Copyright ᮊ, International Association for Food Protection

Research Note Vomitoxin and Content of Soft Flour Milled by Different Methods

VALENTINO PALPACELLI,1* LUCA BECO,1 AND MAURIZIO CIANI2

1Studio Analisi Alimentari, Via XXV aprile 2, 06014 Montone (PG), Italy; and 2Dipartimento di Scienze degli Alimenti, Universita` Politecnica delle Marche, via Brecce Bianche, 60121 Ancona, Italy Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/509/1678933/0362-028x-70_2_509.pdf by guest on 01 October 2021 MS 06-297: Received 31 May 2006/Accepted 10 August 2006

ABSTRACT

Given the prominence and the growing importance of 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 risks (from sowing to harvesting) was used to compare the vomitoxin and zearalenone content of soft wheat flour, 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 usual, so it became possible to produce middlings, which half of the world’s population (6), and for this reason, the were then further reduced by a low-grinding phase. nutritional and toxicological properties of wheat are partic- This represented the first step toward the introduction ularly important to the human diet. Mycotoxins are the pri- of roller mills, which supplanted the stone-mill grinding in mary food risk in wheat (9), and vomitoxin and zearalenone Europe, followed the rest of the world (except for some are mycotoxins that are produced by a well-known wheat rural areas of developing countries and in some typical pathogen: the fungi of the Fusarium genus (11). In Western products in Western countries). In this context and in view countries, the risks associated with the mycotoxins appear of the fact that vomitoxin and zearalenone show widespread to be related to chronic exposure rather than to those of diffusion in wheat products (7), we carried out this study acute exposure (as seen in the developing countries) (7). to investigate the effects of the introduction of the roller- It has been shown that vomitoxin is able to superinduce mill method on the content of these mycotoxins in wheat cytokine gene expression and secretion. This superinduc- flours. tion of the cytokines is associated with many allergic con- ditions and autoimmune diseases (7, 8, 13–16, 18), and it MATERIALS AND METHODS appears that chronic exposure to low levels of this Sample preparation. Beginning November 2001, to obtain can produce immunological effects. Zearalenone appears to the necessary wheat an experimental plan and protocol was fol- be immunotoxic and associated with various diseases of the lowed to reduce the potential for Fusarium infection to a mini- reproductive system (1, 12, 13, 16). mum (4): Bolero variety, late seeding, seed dressing, fungicide Vomitoxin and zearalenone are generally found in the treatment at anthesis, correct soil tillage, and reduced nitrogen external layers of the wheat kernels (17, 19); however, un- fertilization. In July 2002, the mechanical harvesting was carried der particular climatic conditions the infection can be sys- out, and ca. 6,000 kg of sound kernels were obtained. These were temic, and the contamination can extend through the whole stored in one semiunderground masonry bin located on a private farm at about 400 m above sea level, under a rooftop. Hermetic kernel (5). Indeed, the milling phase initially included the storage with periodic fumigation was adopted. Between July 2002 elimination of the external layers of the wheat kernels, thus and December 2004, 36 samples of wheat kernels were taken at reducing vomitoxin and zearalenone levels. Before the 20th regular intervals to both an ancient stone mill located in Bevagna century, the milling operation was conducted exclusively (Perugia, Italy), and experimental roller mills. The resulting flour by the stone-mill method (10). However, in the 19th cen- samples were analyzed for their vomitoxin and zearalenone con- tury, with the introduction of hard wheat varieties in Europe tent. that produced lower flour yields with the stone-mill method, In the stone mill, the wheat kernels were loaded into a seed the practice of ‘‘high grinding’’ was developed. This con- box and then passed through: (i) the grain sieve to eliminate the sisted of milling with the stone more widely separated than cereal straw, stones, etc.; (ii) the trimming machine with an as- pirator to clean the surface layer of the grain and eliminate dust * Author for correspondence. Tel: ϩ39-075-9306479; Fax: ϩ39-075- generated by this operation; and (iii) the seed winnower to elim- 9306479; E-mail: [email protected]. inate vetch seed and other nonwheat seeds and to further clean 510 PALPACELLI ET AL. J. Food Prot., Vol. 70, No. 2 the wheat. Finally, the cleaned wheat kernels were passed to the 80%. Data were not corrected for recovery. Analyses were carried mill stones. The millstone grinder comprises two large circular out in triplicate, and the data are given in the tables as means Ϯ stones that are placed one on the top of the other. The lower stone SD. 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 (iv) Zearalenone analysis. Samples were analyzed for zear- hole. The wheat entered through this hole and passed between the alenone with a JASCO HPLC system with a fluorimetric detector, stones, where it was ground into smaller and smaller fragments, using an immunoaffinity column (VICAM) according to manu- thus obtaining the ‘‘whole meal’’ that went into a sack. Whole facturer instructions (20). Briefly, 20 g of each flour sample was meal can be sold directly or, as in this case, it was manually put added to2gofNaCl and 50 ml of acetonitrile-water (90:10, vol/ into a separator (a simple system of sieves) to obtain the bran, the vol), and then blended at high speed for ca. 2 min. The samples flour mixed with bran, and the white flour. were then filtered through fluted filter paper, and then filtered White stone-mill flour is typically like semolina, with larger again through a microfiber filter. The filtered extract (5 ml) was granulation than roller-mill flour. then applied to the VICAM IAC column. The column was washed For the experimental roller mills, the wheat kernels were with 5 ml of distilled water, and the zearalenone was eluted with sieved to remove the unwanted material, such as stones, dust, and 1.5 ml of methanol (HPLC grade). The eluate was collected in a Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/509/1678933/0362-028x-70_2_509.pdf by guest on 01 October 2021 weed seeds, and then they were put into a wheat bin, and 3% glass cuvette, and 1.5 ml of water (HPLC grade) was added. The water was added to them (tempering for 48 h to provide 15 to samples were then injected (200 ␮l) into the isocratic HPLC sys- 16% moisture content). The wheat kernels were then milled in the tem (reverse-phase C18 column: mobile phase, acetonitrile-water- experimental roller mills, with the separation of bran, germ, and methanol [46:46:8; vol/vol/vol, degassed]; flow rate, 1.0 ml/min; endosperm. The first set of mills (the breaking rollers) separated loop, 100 ␮l; detection wavelength, 274-nm excitation and 440- the bulk of the bran and the germ from the endosperm. The re- nm emission). The zearalenone standard was 50 ppm in acetoni- sulting product was then passed through the sieves to separate the trile (Supelco). The software was JASCO Borwin HPLC software, various constituents, giving the bran, the wheat germ, the endo- version 1.50. The limit of quantification was 1 ppb, with a mean sperm chunks, and the animal-feed byproducts. The endosperm recovery rate of 85%. Data were not corrected for recovery. Anal- chunks, together with small residual amounts of bran and germ, yses were carried out in triplicate, and the data are given in the were passed through two series of rollers (the reducing rollers) to tables as means Ϯ SD. produce the flour. Statistical analysis. Normality testing was performed using For each kind of milling, we obtained bran, flour mixed with GraphPad InStat, version 3.0a, for Macintosh. None of the sam- bran, and white flour (about 50 kg of wheat flour in 1-kg pack- ples of both of the analyses passed the normality tests aging). Thirty-six commercial flours were analyzed (18 stone-mill (D’Agostino and Pearson omnibus normality test, alpha ϭ 0.05), flours and 18 roller-mill flours), Samples were obtained over 3 so we performed a nonparametric test (Mann-Whitney test) using months from five different sales points. GraphPad Prism, version 4.00, for Macintosh (GraphPad Soft- Sample analysis. (i) Sampling methods. Sampling of flours ware, San Diego, Calif.). For the commercial flour screening, we was carried out according to the official AOAC International assumed a non–Gaussian distribution (due to the small numbers methods (2). of samples) and performed a nonparametric test (Mann-Whitney For each analysis, 10 sacks were sampled, and 100 g was test) using GraphPad Prism, version 4.00, for Macintosh. taken from each sack to obtain the 1-kg samples. RESULTS (ii) Moisture analysis. Samples were analyzed for moisture following the official AOAC International methods, using the air- The flours obtained from these unique samples from oven method (3). Analyses were carried out in triplicate, and the wheat with the different milling procedures showed mois- data are given in the tables as means Ϯ standard deviation (SD). ture contents lower than 13%, a safe level for storage of the grain and flour (Table 1) (10). Thus, the wheat kernels (iii) Vomitoxin analysis. Samples were analyzed for vomi- and flours were correctly stored, and we can exclude any toxin with a JASCO HPLC system with a UV detector, using an significant effects due to the moisture contents. immunoaffinity column (VICAM, Watertown, Mass.) according to Both the vomitoxin and zearalenone contents were sig- manufacturer instructions (21). Briefly, 50 g of each flour sample nificantly lower in the stone-mill flours when compared was added to 10 g of PEG 8000 and 200 ml of purified water, and then blended at high speed for ca. 1 min. The samples were with the roller-mill flours (Table 2). This was confirmed by then filtered through the vomitoxin test fluted filter paper, and then the statistical analysis conducted using the rank sum test filtered again through a microfiber filter. The filtered extract (5 (Mann-Whitney test), the results of which are given in the ml) was then applied to the vomitoxin test VICAM IAC column. full statistical breakdown in Table 4. The mean vomitoxin The column was washed with 5 ml of distilled water, and the content in the stone-mill flours was 170 ppb, compared with vomitoxin was eluted with 1 ml of methanol (HPLC grade). The 360 ppb for the roller-mill flours; the zearalenone content eluate was collecting in a glass cuvette and evaporated to dryness. showed a similar distribution, with 6 ppb and 13 ppb, re- The dried samples were reconstituted in 1 ml mobile phase— spectively. acetonitrile-water (10:90, vol/vol, degassed)—and injected (200 The analyses of the randomized commercial flours con- ␮l) into the isocratic HPLC system (reverse-phase C18 column: firmed the results of these experimental trials (Table 3). flow rate, 1.0 ml/min; loop, 100 ␮l; UV detection, 220 nm). The same procedure (evaporation and reconstitution) was repeated Thus, the mean vomitoxin content in the stone-mill flours with the vomitoxin standard of 200 ppm vomitoxin in ethyl ace- was 245 ppb versus 945 ppb in the roller-mill flours, with tate–methanol (95:5, vol/vol) (Supelco, Bellefonte, Pa.). The soft- zearalenone showing 1.7 ppb and 6.0 ppb, respectively. ware used was JASCO Borwin HPLC software, version 1.50. The Again, the statistical analysis confirmed the significance of limit of quantification was 50 ppb, with a mean recovery rate of this comparison (stone-mill flour versus roller-mill flour), J. Food Prot., Vol. 70, No. 2 MYCOTOXINS IN FOOD 511

TABLE 1. Results of the analyses of the moisture content of the TABLE 2. Results of the analyses of vomitoxin and zearalenone stone-mill and roller-mill wheat flour samplesa content of the stone-mill and roller-mill wheat flour samplesa Moisture Vomitoxin Zearalenone

Stone-mill Roller-mill Stone-mill Roller-mill Stone-mill Roller-mill wheat flour wheat flour Sam- wheat flour wheat flour wheat flour wheat flour Sample (%, w/w) (%, w/w) ple (ppb) (ppb) (ppb) (ppb)

01 11.4 Ϯ 0.1 11.4 Ϯ 0.1 01 158.6 Ϯ 13.6 363.6 Ϯ 4.1 3.4 Ϯ 0.3 11.4 Ϯ 0.3 02 10.8 Ϯ 0.1 10.8 Ϯ 0.1 02 187.0 Ϯ 9.1 336.0 Ϯ 23.5 5.2 Ϯ 0.4 12.8 Ϯ 0.3 03 11.4 Ϯ 0.1 11.5 Ϯ 0.1 03 188.3 Ϯ 13.0 464.0 Ϯ 17.6 6.7 Ϯ 1.2 13.5 Ϯ 0.6 04 12.1 Ϯ 0.1 11.7 Ϯ 0.1 04 144.3 Ϯ 7.5 427.6 Ϯ 13.6 5.1 Ϯ 0.9 12.5 Ϯ 0.3 05 11.6 Ϯ 0.1 11.9 Ϯ 0.1 05 166.6 Ϯ 8.7 340.0 Ϯ 40.2 4.7 Ϯ 0.1 11.8 Ϯ 0.3 06 11.7 Ϯ 0.1 11.6 Ϯ 0.1 06 212.0 Ϯ 15.6 389.0 Ϯ 7.0 5.5 Ϯ 0.2 13.6 Ϯ 0.4

07 12.0 Ϯ 0.1 12.0 Ϯ 0.1 07 187.0 Ϯ 8.1 295.0 Ϯ 3.0 6.6 Ϯ 0.2 12.4 Ϯ 0.2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/509/1678933/0362-028x-70_2_509.pdf by guest on 01 October 2021 08 11.8 Ϯ 0.1 12.2 Ϯ 0.1 08 213.6 Ϯ 9.6 336.0 Ϯ 19.9 6.4 Ϯ 0.1 10.5 Ϯ 0.3 09 12.1 Ϯ 0.1 11.7 Ϯ 0.1 09 264.3 Ϯ 12.3 348.3 Ϯ 9.2 5.1 Ϯ 0.2 13.6 Ϯ 0.3 10 11.8 Ϯ 0.1 12.3 Ϯ 0.1 10 208.0 Ϯ 4.5 338.3 Ϯ 7.5 3.4 Ϯ 0.3 12.6 Ϯ 0.3 11 12.2 Ϯ 0.1 11.7 Ϯ 0.1 11 175.6 Ϯ 6.1 338.0 Ϯ 10.5 5.9 Ϯ 0.2 13.1 Ϯ 0.3 12 11.9 Ϯ 0.1 12.2 Ϯ 0.1 12 188.6 Ϯ 7.0 355.3 Ϯ 4.0 6.4 Ϯ 0.1 12.4 Ϯ 0.5 13 11.8 Ϯ 0.2 12.3 Ϯ 0.1 13 203.0 Ϯ 4.5 339.6 Ϯ 5.0 5.4 Ϯ 0.3 13.1 Ϯ 0.3 14 12.4 Ϯ 0.2 11.9 Ϯ 0.2 14 205.0 Ϯ 15.7 288.3 Ϯ 7.0 6.3 Ϯ 0.2 12.5 Ϯ 0.2 15 11.9 Ϯ 0.3 11.7 Ϯ 0.1 15 219.3 Ϯ 4.5 521.3 Ϯ 9.7 6.4 Ϯ 0.2 11.8 Ϯ 0.4 16 12.1 Ϯ 0.1 12.2 Ϯ 0.1 16 180.3 Ϯ 6.0 379.6 Ϯ 5.0 4.5 Ϯ 0.1 12.3 Ϯ 0.3 17 12.1 Ϯ 0.2 11.9 Ϯ 0.2 17 182.6 Ϯ 4.0 394.6 Ϯ 6.6 5.4 Ϯ 0.3 12.3 Ϯ 0.2 18 11.9 Ϯ 0.1 11.9 Ϯ 0.3 18 193.6 Ϯ 5.1 314.3 Ϯ 5.0 5.0 Ϯ 0.1 12.7 Ϯ 0.7 19 11.8 Ϯ 0.1 12.1 Ϯ 0.2 19 204.0 Ϯ 5.0 339.3 Ϯ 7.3 6.0 Ϯ 0.2 12.1 Ϯ 0.3 20 11.9 Ϯ 0.2 12.0 Ϯ 0.1 20 179.0 Ϯ 6.0 338.6 Ϯ 11.5 5.2 Ϯ 0.3 12.7 Ϯ 0.2 21 12.2 Ϯ 0.1 12.3 Ϯ 0.1 21 196.0 Ϯ 4.3 457.3 Ϯ 3.5 6.1 Ϯ 0.3 12.9 Ϯ 0.2 22 11.9 Ϯ 0.2 12.1 Ϯ 0.4 22 216.0 Ϯ 4.5 279.3 Ϯ 8.3 5.9 Ϯ 0.1 12.0 Ϯ 0.3 23 12.3 Ϯ 0.1 12.2 Ϯ 0.1 23 222.6 Ϯ 2.5 385.0 Ϯ 6.0 5.4 Ϯ 0.3 12.7 Ϯ 0.1 24 11.8 Ϯ 0.1 11.9 Ϯ 0.1 24 215.0 Ϯ 4.0 332.0 Ϯ 4.5 5.5 Ϯ 0.1 15.5 Ϯ 0.3 25 11.7 Ϯ 0.1 12.1 Ϯ 0.2 25 197.3 Ϯ 26.8 371.3 Ϯ 4.0 5.3 Ϯ 0.2 12.7 Ϯ 0.2 26 11.8 Ϯ 0.1 12.5 Ϯ 0.1 26 188.0 Ϯ 7.0 382.6 Ϯ 3.5 5.8 Ϯ 0.2 11.8 Ϯ 0.3 27 11.9 Ϯ 0.1 12.3 Ϯ 0.1 27 171.6 Ϯ 17.0 353.6 Ϯ 4.5 5.8 Ϯ 0.4 15.5 Ϯ 0.2 28 12.2 Ϯ 0.1 12.0 Ϯ 0.2 28 178.3 Ϯ 9.5 374.3 Ϯ 4.0 5.7 Ϯ 0.4 12.9 Ϯ 0.3 29 12.4 Ϯ 0.1 11.7 Ϯ 0.1 29 177.3 Ϯ 7.0 394.3 Ϯ 13.6 6.1 Ϯ 0.2 12.8 Ϯ 0.2 30 12.4 Ϯ 0.1 12.1 Ϯ 0.2 30 188.6 Ϯ 6.8 400.6 Ϯ 5.5 6.1 Ϯ 0.3 13.4 Ϯ 0.2 31 11.7 Ϯ 0.1 12.3 Ϯ 0.1 31 191.0 Ϯ 6.2 379.0 Ϯ 13.1 5.5 Ϯ 0.5 12.2 Ϯ 0.3 32 12.2 Ϯ 0.1 12.1 Ϯ 0.2 32 179.0 Ϯ 16.3 401.6 Ϯ 12.3 6.1 Ϯ 0.3 13.1 Ϯ 0.2 33 11.7 Ϯ 0.1 11.9 Ϯ 0.1 33 178.3 Ϯ 10.2 374.6 Ϯ 7.0 6.4 Ϯ 0.3 13.1 Ϯ 0.3 34 12.2 Ϯ 0.1 11.8 Ϯ 0.1 34 142.0 Ϯ 6.5 279.0 Ϯ 13.2 3.5 Ϯ 0.3 12.2 Ϯ 0.3 35 11.7 Ϯ 0.1 12.2 Ϯ 0.1 35 173.3 Ϯ 4.1 413.0 Ϯ 4.5 5.8 Ϯ 0.2 12.4 Ϯ 0.5 36 12.4 Ϯ 0.1 11.8 Ϯ 0.2 36 176.0 Ϯ 3.6 349.3 Ϯ 11.0 5.5 Ϯ 0.3 13.1 Ϯ 0.3 a Values are mean Ϯ SD. a Values are mean Ϯ SD. using the Mann-Whitney test (Table 4), confirming the re- where most of the mycotoxins are generally located; and sults from the experimental trial. (ii) the roller-mill system has a series of reduction rollers that are designed to extract the residual flours from the DISCUSSION bran, and this will probably also further extract the myco- The results obtained in this comparative study show . that stone milling results in a reduction of about 40 to 50% This last observation is in accordance with those of in the vomitoxin and zearalenone contents of wheat flours. Scott et al. (5), who noted that there was little difference This was also confirmed with the commercial flour samples, in the mycotoxin content between flour streams (using roll- where again the stone-mill commercial flours show lower er mills). In addition, debranning before milling (prepro- amounts of vomitoxin and zearalenone than do the roller- cessing) was used successfully in experimental trials, and mill flours. it was seen that this can produce flours that have a lower The main considerations toward an explanation of mycotoxin content than those produced as roller-mill flours these results would be as follows: (i) The stone-mill system (without debranning) (5). used for the experimental wheat flour samples has a trim- At the same time, the change in the milling system ming machine with an aspirator, and thus it can at least could bring about an increase in the potential exposure of partially eliminate the external layer of the wheat kernels, consumers to the mycotoxins. Such an increased exposure 512 PALPACELLI ET AL. J. Food Prot., Vol. 70, No. 2

TABLE 3. Results of the analyses of the vomitoxin and zearale- staple diet, even if this process has brought about great none content of the commercial stone-mill and roller-mill wheat improvements in the milling efficiency of the wheat. flour samplesa Vomitoxin Zearalenone REFERENCES 1. Alldrick, A. J. 1994. Zearalenone, p. 353–366. In N. Magan and M. Stone-mill Roller-mill Stone-mill Roller-mill Olsen (ed.), Mycotoxins in food: detection and control. Woodhead Sam- wheat flour wheat flour wheat flour wheat flour Publishing, Ltd., Cambridge, UK. ple (ppb) (ppb) (ppb) (ppb) 2. AOAC International. 2003. Method 925.08. Official methods of analysis, 17th ed., 2nd rev. AOAC International, Gaithersburg, Md. 01 79.6 Ϯ 5.5 719.0 Ϯ 20.5 2.5 Ϯ 0.2 8.7 Ϯ 0.4 Ϯ Ϯ Ϯ Ϯ 3. AOAC International. 2003. Method 925.10. Official methods of 02 105.3 5.8 1,769.0 57.0 0.0 0.0 9.5 0.3 analysis, 17th ed., 2nd rev. AOAC International, Gaithersburg, Md. Ϯ Ϯ Ϯ Ϯ 03 151.6 10.6 798.6 39.7 1.4 0.0 7.9 0.3 4. Bilgrami, S. K., and A. K. Choudhary. 1998. Mycotoxins in prehar- 04 382.3 Ϯ 8.3 462.6 Ϯ 11.0 1.8 Ϯ 0.2 0.0 Ϯ 0.0 vest contamination of agricultural crops, p. 1–43. In K. K. Sinha and 05 245.6 Ϯ 15.0 732.3 Ϯ 21.0 0.0 Ϯ 0.0 14.3 Ϯ 0.6 D. Bhatnagar (ed.), Mycotoxins in agriculture and food safety. Mar-

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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 wheat flour samples Exact or Significantly approximate different medians? Sample comparison P value P value (P Ͻ 0.05)

Vomitoxin stone mill vomitoxin roller mill (Table 2) Ͻ0.0001 Gaussian approximation Yes Zearalenone stone mill zearalenone roller mill (Table 2) Ͻ0.0001 Gaussian approximation Yes Vomitoxin stone mill commercial samples vomitoxin roller-mill commercial sam- ples (Table 3) Ͻ0.0001 Gaussian approximation Yes Zearalenone stone mill commercial sam- ples zearalenone roller mill commercial samples (Table 3) 0.0008 Gaussian approximation Yes J. Food Prot., Vol. 70, No. 2 MYCOTOXINS IN FOOD 513

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