The fate of deoxynivalenol and fumonisins in wheat and maize during commercial breakfast cereal production

World Mycotoxin Journal, November 2008; 1(4): 437-448 Wageningen g Academic P u b l i s h e r s The fate of deoxynivalenol and fumonisins in wheat and maize during commercial breakfast cereal production K.A. Scudamore 1 and S. Patel 2 1 KAS Mycotoxins, 6 Fern Drive, Taplow, Maidenhead, Berks, SL6 0JS, United Kingdom; 2 RHM Technology, Premier Foods, The Lord Rank Centre, Lincoln Road, High Wycombe, HP12 3QR, United Kingdom; kasmyc@aol.com Abstract 1. Introduction Cereals are invaded in the field and after harvest by fungi that may produce mycotoxins and pose potential problems for human or animal health. The European Community has addressed this by introducing legislation (EC, 2006a, 2007) and methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs (EC, 2006b). It is important for the cereal industry that the fate of mycotoxins in the food chain is understood, to avoid situations arising where products manufactured from raw cereals pass legislation on intake but fail to meet the regulatory limits after processing. Received: 20 May 2008 / Accepted: 11 June 2008 2008 Wageningen Academic Publishers A study of the changes undergone by Fusarium mycotoxins present in maize and wheat at intake during the processing of commercial grain samples into breakfast cereals was carried out. Natural concentrations of deoxynivalenol and zearalenone in wheat were mostly low although higher levels of fumonisins occurred in maize. An exhaustive cleaning regime was used for wheat received from UK farms that reduced deoxynivalenol levels by about 50%, although this varied considerably between consignments. During processing to manufacture two commercial breakfast cereals, further loss of deoxynivalenol was minimal. However, this was significantly greater in a product from which excess aqueous cooker effluent was drained, suggesting that loss was due to solution of the mycotoxin in the cooking liquor rather than to hydrolysis. It is estimated that deoxynivalenol present at the EC maximum limit of 1,250 µg/kg in intake wheat would be reduced to about 700 and 400 µg/kg respectively for the 2 types of whole-wheat breakfast cereals examined during processing. Maize flaking grits were inherently low in mycotoxin concentrations compared to the raw maize so that the mycotoxin levels in the cereal ingredient for cornflakes used in this manufacturing process were unlikely to approach EC regulatory levels. In processing these grits, fumonisins were reduced further by at least 93% although no reduction of deoxynivalenol occurred. It is estimated that fumonisins and deoxynivalenol at the EC maximum limits in raw maize of 4,000 µg/kg and 1,750 µg/kg would be reduced to about 17 µg/kg and 288 µg/kg respectively in corn flakes made by the traditional cooking process used in the UK. Keywords: Fusarium mycotoxins, milling, processing, legislation, cereals Different cereals such as wheat, maize and oats are used to produce breakfast cereals. These cereals are, for example, milled, cooked, extruded, dried, rolled, shredded or toasted to produce a comprehensive range of products, many of which are household names. Mycotoxins are usually concentrated in the bran and outer layers of grains and reduced in the endosperm (Abbas et al., 1985; Chelkowski and Perkowski, 1982; Scott et al. 1984; Scudamore et al. 2003; Seitz et al. 1985; Trigo-Stockli et al. 1996). This suggests that breakfast cereals based on bran or wholemeal grains have the potential to contain higher concentrations of certain mycotoxins than those manufactured from flours or grits milled from the grain endosperm. However, the use of whole-wheat grains, for example, retains the nutrient ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2008.1059 437

K.A. Scudamore and S. Patel rich bran so that minimising the mycotoxin content may then become more important. Cornflakes are another popular breakfast cereal produced from maize made by several processors and marketed under a number of brand names. It is not well recognised that there are two different manufacturing processes used for cornflake production. One uses the extrusion of maize flour while the other cooks maize flaking grits, before drying and toasting. Most cornflakes consumed in the UK use a traditional cooking method and are produced from high quality flaking grits. It might be predicted that the fate of mycotoxins would be very different in the two processes, especially with mycotoxins that are chemically reactive. There are several publications of relevance. De Girolamo et al. (2001) discussed the effect of processing on fumonisin concentrations in corn flakes. In that case these were produced by an extrusion technique. One finding was that the solvent for extraction of fumonisins from manufactured cornflakes rather than from spiked maize flour is extremely important and could greatly affect the numerical figure obtained. Certain additives in the products could interfere with the operation of the strong anion exchange (SAX) columns used in the analysis. A survey of mycotoxins in maize products carried out by the UK Food Standards Agency showed that most samples of cornflakes contained low levels of fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2 ) and fumonisin B 3 (FB 3 ) while a few also contained deoxynivalenol (DON). The method used for fumonisins was immunoaffinity column clean-up. A few samples, however, contained considerably higher levels of all three fumonisins and it is suggested that these cornflakes may have been produced from maize flour by extrusion. Another publication by Paepens et al. (2005) found that fumonisin levels in cornflake brands sampled in Belgium fell roughly into 2 populations and the authors speculated (incorrectly in our opinion) that this might relate to conventional versus organic products; the possibility of different manufacturing practices did not appear to be considered. Surveys of wheat in the United Kingdom have shown that the most common Fusarium mycotoxin found is DON, sometimes with lower concentrations of 15-acetyl- DON, 3-acetyl-DON, nivalenol, HT-2 toxin, T-2 toxin and zearalenone (ZEA). Maize may also contain these mycotoxins as well as fumonisin toxins, sometimes in high concentrations. The EC has set maximum permissible levels for DON, ZEA and FB 1 + FB 2 in cereals including wheat and maize, in the derived milled products, consumer foods and in infant cereal-based foods. Maximum limits for fumonisins based on the sum of FB 1 + FB 2 were introduced and operated from 1 July 2007 (EC, 2007). To summarise, maximum allowed limits for FB 1 + FB 2 are 4,000 µg/kg in unprocessed maize, 2,000 µg/kg in maize products with particle size <500 µ (e.g. flour), 1,400 µg/kg in maize products with particle size >500 µ (e.g. flaking grits), 800 µg/kg in breakfast cereals and 200 µg/kg in processed cereal-based foods and baby foods for infants and young children. These limits operated from 1 October 2007. Respective limits for DON are 1,750 µg/kg in raw maize, 1,250 µg/kg in maize products with particle size <500 µ (e.g. flour), 750 µg/kg in maize products with particle size >500 µ (e.g. flaking grits), 500 µg/kg in breakfast cereals and 200 µg/kg in processed cereal-based foods and baby foods for infants and young children. Most studies report DON and ZEA as being relatively stable during processing (Gilbert et al., 1984; Isohata et al., 1986; Kamimura et al., 1980; Lauren and Smith, 2001; Patey and Gilbert, 1989; Wolf-Hall et al., 1999) although it is essential to understand how DON or other mycotoxins are affected along the whole food chain and the typical reduction that can be expected to occur from the whole wheat to the product as eaten by the consumer. The most commonly occurring mycotoxin in maize is FB 1, which is found together with lesser amounts of FB 2 and FB 3. These compounds occur in most consignments of maize and can attain very high levels, particularly in hot dry climates but can be partially destroyed during processing (De Girolamo et al., 2001; Paepens et al., 2005; Castelo et al., 1998; Jackson et al., 1997), although hidden fumonisins may also be formed (Kim et al., 2003). DON also occurs frequently, but its formation is favoured by cooler weather and concentrations are often much lower than the fumonisins. ZEA is another mycotoxin that also occurs subject to legislation. These studies report on the change in concentration of Fusarium mycotoxins during wheat and maize processing to manufacture selected breakfast cereals at a full commercial scale over a 2-year period. It is clearly impossible to carry out commercial scale processing with cereals contaminated with mycotoxins at levels close to or in excess of statutory limits in order to protect the consumer and conform to regulatory limits. Care is then required when extrapolating results from these studies to the legislative level and these should ideally be backed up by results from pilot scale or laboratory studies. In practice, DON concentrations found in cereals used for human foods in the UK are usually well below current EC limits. Some additional studies with relatively high DON concentrations have thus been carried out at the pilot scale. Results here show the change in concentration of the mycotoxins at each stage, indicate their chemical stability and examine how closely values obtained from commercial operations are consistent with EC regulations. Data for this latter purpose is presented on an as is basis (that is, without consideration of moisture content or the presence of other ingredients). 438 World Mycotoxin Journal 1 (4)

Fusarium mycotoxins in breakfast cereals 2. Materials and methods Sampling and manufacture of whole-wheat breakfast cereals Five truckloads of wheat (often from different origins but normally straight from the farm) were delivered by lorry into a dirty wheat silo to provide a batch of about 140 tons in a partly filled silo. Each intake lorry was sampled routinely for quality purposes using an automatic sampler (8 probe points inserted across the whole surface of the load and sampling to full depth). Incremental samples were combined to provide a composite sample. One kg of this bulk sample from each lorry was combined to give an aggregate sample of 5 kg that was considered to provide an acceptable representation of the mean mycotoxin concentration in the wheat in the whole silo. The uncleaned grain was transferred from the delivery silo to a similar-sized holding silo before going through a comprehensive cleaning procedure comprising a separator/ classifier that sieves the grain, indented cylinders that remove foreign seeds and a destoner. In addition, each step includes an aspiration process to remove dust. Five 1 kg grab samples of cleaned wheat were taken at the cleaning plant exit regularly on a timed basis. These were combined to give the aggregate sample of cleaned wheat (this may or may not relate closely to mean levels in the whole silo but should relate accurately to the samples being examined during processing). The wheat at this stage is considered to be consistent with that specified in the regulations for unprocessed cereals. The wheat is then returned to a different silo that feeds the production lines. Wheat is withdrawn from this silo for various processes including those under study. Further mixing occurs as the silo empties. It takes between 10 and 30 hours to use the contents of the silo depending on the products being manufactured. As the mean toxin content of the raw wheat (140 tons) in the starting silo and in the cleaned wheat can be determined and the wheat batch be traced through processing by timing, the end product is sampled at regular intervals over the complete time that the wheat is used. For example 5 1 kg aliquots of each end product sample comprises the composite sample. In this study two products were manufactured and are referred to as whole-wheat breakfast cereals 1 and 2 respectively. These differ only in the details of processing and their final form and are breakfast cereals familiar to the UK and European consumer. The identity of the products and full details of the process cannot be disclosed, as these are confidential. The wheat studied was in routine commercial use by the manufacturer and was subject to the normal quality checks and legal requirements for contaminants in food required in the UK. 700 kg batches of wheat added to a cooking vessel with about 800 l quantities of hot water were used to manufacture each product. To produce breakfast cereal 1, wheat at 13.5% moisture content was cooked at 100 C under high pressure for about 40 minutes with sugar, malt, salt and vitamins. The outgoing dough had a moisture content of approx. 40% when the cooker was emptied. To produce breakfast cereal 2, wheat was added to hot water and then live steam was injected at 100 C for 28-32 minutes at atmospheric pressure. The aqueous effluent was then drained off. Samples of the effluent from product 2 were collected from some runs to test for the presence of DON. Both products were run through a cooling process before holding. The products were then shredded and dried in successive operations. Prior to packaging, a 1 kg grab sample of either product was taken from each of 5 process runs and combined. Thus each composite sample was 5 kg comprising 5 1 kg incremental samples and related by timing to the feed to the production line. Sampling and manufacture of cornflakes Maize flaking grits were supplied on contract by a miller and confined to specific silos from which batches were withdrawn for processing. A simplified flow chart for the manufacture of cornflakes is shown as Figure 1. The progress of each batch was tracked through the process by timing. Flaking grits of 4-6 mm in size together with other ingredients, which included approximately 1% of a smaller size maize grit added for flavouring purposes, were placed in a cooker of about 1,000 kg capacity. This was heated under steam pressure above 100 C for about 1 hour to soften the grits. After emptying the cooker, the wet product is dried to 10-14% moisture in hot air after which the flakes are rolled flat and elongated. The flakes are then toasted in a very hot rotating oven for about 30 seconds. The flakes are then sprayed with vitamins to give the finished product. Details of the process are subject to commercial confidentiality. Samples of the raw grits and cornflakes were collected from each run. Additional samples of the Flaking grits from silo Cooker Drier Rolling mills Toasting oven Packing lines Flavour components Vitamin addition Figure 1. Flow diagram for cornflake manufacture. World Mycotoxin Journal 1 (4) 439

K.A. Scudamore and S. Patel cooked maize and the dried cooked maize grits were taken from a few of the consignments. A 1 kg sample of maize grits entering each of 10 cookers was collected and combined to give a 10 kg composite. The additional small grit component was kept for testing as necessary. Manufacture was carried out as a continuous process and sampling was performed at regular intervals. At each sampling stage, a 1 kg quantity was collected from each cooker, drier or toasting oven, combining the 10 incremental samples to give the composite sample. The final cornflake product or intermediate material was linked to the corresponding cooker batches on a time basis. The flaking grits and cornflakes for retailing were those in routine commercial use by the manufacturer and are subject to the normal quality checks and legal requirements for contaminants in food required in the UK. Analytical methods Analysis for fumonisins, DON and ZEA was carried out at RHM Technology, High Wycombe. On receipt in the laboratory, the whole of each cereal ingredient or product was ground using an 0.8 mm screen and mixed for 30 minutes in a Gardner horizontal mixer to ensure homogeneity. Samples were stored at -20 C if not analysed immediately. All reagents were purchased from commercial sources and were of analytical grade. Solvents were of HPLC grade. Mycotoxin standards of DON and ZEA were purchased from Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK. DON, and ZEA were quantified prior to use by UV spectrometry. FB 1, FB 2 and FB 3 were standards obtained from PROMEC Unit, MRC, P.O. Box 19070, Tygerberg 7505, Cape Town, South Africa. The methods used were UKAS (ISO 17025) accredited and followed the UK Food Standards Agency Requirements of External Contracts (Thompson and Wood, 1995). Deoxynivalenol DON (and other related trichothecene mycotoxins) was determined following the method of Patel et al. (1996) as used by Scudamore et al. (2007). Ground samples (20 g) were extracted with 100 ml acetonitrile/water (84:16) by shaking for 2 hours on a wrist action shaker. A 5 ml aliquot of the extract was applied to a pre-washed charcoal/ alumina column and this was then washed with 40 ml acetonitrile:water (84:16). The total eluate was taken to dryness and transferred to a vial with acetonitrile. The residue was derivatised to form the trichothecene-trimethyl silyl derivatives and determined by GC/MS operating in selected ion mode, using 4 ions for confirmation. Zearalenone ZEA was determined by HPLC with fluorescence detection as used by Scudamore et al. (2007). Ground samples (25 g) were extracted with 125 ml of acetonitrile/water (75/25) by shaking for 30 minutes. The extract was filtered and 10 ml of the extract diluted with 40 ml of Phosphate Buffered Saline (PBS) solution. The diluted extract was passed through an EASI-EXTRACT ZEA immunoaffinity column (R Biopharm Rhone) under gravity. The column was washed with 10 ml water. ZEA was eluted under gravity with 2 ml of acetonitrile. The eluate was evaporated to dryness and transferred to a vial with chloroform. The chloroform was evaporated to dryness and dissolved in 1 ml of mobile phase of which 20 µl were used for the detection of mycotoxins. The equipment consisted of a Gilson 307 pump, Gilson 231/401 auto-injector and a Jasco fluorescence detector. Liquid chromatography was performed on a Kromasil C8 (Hichrom) 5 µ particle size 25 cm 3.2 mm i.d., operated at 0.5 ml/min with 1% acetic acid in water:acetonitrile (55:45) as mobile phase. The excitation wavelength of the fluorescence detector was set at 274 nm and the emission wavelength was 440 nm. Fumonisins The method for fumonisins was a modification of the method of Shephard et al. (1990) as used by Scudamore and Patel (2000). A 25 g sample was extracted with 100 ml acetonitrile:water (50:50) by shaking for 120 minutes. The extract was then filtered. Aliquots of 5 ml of the resulting filtrates were adjusted to ph 5.8-6.5 and applied to Bond-Elut SAX cartridges, previously conditioned by the successive passage of 5 ml methanol and 5 ml methanol:water (75:25). The cartridge was washed with 8 ml methanol:water (75:25), with 3 ml methanol and finally the fumonisins were eluted with 10 ml 1% acetic acid in methanol. The eluate was evaporated to dryness at 40 C, by rotary evaporation, transferred to a vial with acetonitrile: water (50:50) and made up to 1 ml final volume. Fumonisins contained in a 150 µl aliquot of the purified extract were derivatised automatically with 150 µl orthoophthaldialdehyde (OPA) solution. This solution was prepared by adding 5 ml 0.1 M sodium tetraborate and 50 µl 2-mercaptoethanol to 1 ml methanol containing 40 mg OPA. The fumonisin OPA derivatives (20 µl) were separated and measured using reversed-phase HPLC with fluorescence detection. The column was a Spherisorb ODS 2 (25 cm 3.2 mm i.d.) with elution solvent of 0.1 M sodium dihydrogen orthophosphate:methanol (25:75) operated at 0.5 ml/min. Excitation wavelength of the fluorescence detector was set at 335 nm and the emission wavelength was 440 nm. 440 World Mycotoxin Journal 1 (4)

Fusarium mycotoxins in breakfast cereals Recovery, limit of detection and determination All analyses were conducted with a spiked sample, i.e. a known amount of toxin was added to a sample of ground wheat or maize each day prior to extraction, clean-up and HPLC determination for each batch of 1-5 samples. These results were used to assess recovery and all reported results were corrected using the values obtained. Recoveries in the range 70-110% were considered acceptable. The spike level was 200 µg/kg for DON, 50 µg/kg for ZEA and 400 µg/kg for each fumonisin. In house reference material (wheat and maize) contaminated at 220 and 550 µg/kg of DON was spiked with 200 and 500 µg/kg respectively. Typical mean recovery was 89% with a coefficient of variability of 8.1% for 10 replicates. For fumonisins, maize in house reference material contaminated at 498 µg/kg was then spiked at 400 µg/kg. Typical mean recovery was 86% with a coefficient of variability of 6.5% for 10 replicates. For ZEA, ground maize in house reference material contaminated at 50 µg/kg of ZEA was spiked at 53 µg/kg. Typical mean recovery was 88% with a variability coefficient of 7.2% for 10 replicates. The limit of detection and limit of determination respectively for each mycotoxin were 5 µg/kg and 10 µg/ kg for DON and each fumonisin mycotoxin and 1.5 µg/kg and 3 µg/kg respectively for ZEA although no ZEA was detected in this study. The limit of detection is defined as 3 times the electronic baseline noise and the limit of determination as 6 times baseline noise. Calibration curves for each mycotoxin were plotted with the lowest calibration points respectively being equivalent to 10, 3 and 10 µg/kg for DON, ZEA and fumonisins. After analysis samples were retained and stored at -20 C. Testing of cooker effluent To determine DON, 16 ml of aqueous cooker effluent sample were extracted with 84 ml acetonitrile (84:16) by shaking for 2 hours on a wrist action shaker. A 5 ml aliquot of the extract was applied to a pre-washed charcoal/alumina column, this was then washed with 40 ml acetonitrile: water (84:16) and the combined eluate was processed as above. 3. Results All analytical results are corrected for recovery using spiked samples. Whole-wheat breakfast cereals The results of the study for the production of whole-wheat breakfast cereals are given in Table 1. Results for breakfast cereals are corrected for the change in moisture content by applying a factor to bring results to the same moisture level as the whole raw wheat. In total, 25 consignments of wheat were used to manufacture the 2 brands of breakfast cereals. On 18 occasions, samples of both the dirty, intake wheat and the corresponding cleaned wheat were supplied and then analysed. As described earlier a consignment could be used to manufacture both products. Product 1 was manufactured on 20 occasions and product 2 from all 25 wheat consignments. For 10 consignments, the cooker effluent drained from product 2 was collected and analysed for DON and the results for these are given in Table 2. Results have then been extrapolated to EC maximum limits for DON in unprocessed wheat to show what might be expected in the manufactured breakfast cereals. Results that are used for this extrapolation are labelled a and b in Table 1. Cornflakes The manufacture of cornflakes involves 2 distinct phases; the milling of maize to produce flaking grits and the manufacture of the corn flakes from the grits. Table 3 summarises results showing the reduction in mycotoxin concentration from intake maize to the flaking grits produced determined in a separate study (Scudamore, unpublished). Table 4 provides the results from each manufacturing run and gives the concentrations of DON, FB 1 and FB 2 in maize grits and in the cornflakes manufactured from them. Results for the cornflakes which have a moisture content of 3.5% are corrected to that of the flaking grits of 13.5%. Results have then been extrapolated to EC maximum limits for DON in unprocessed wheat to show what might be expected in the manufactured cornflakes from maize with that mycotoxin content. 4. Discussion To assess the fate of mycotoxins in industrial processes it is necessary to distinguish clearly between changes in concentrations and loss of mycotoxins. The EC Statutory limits for maximum permissible levels of mycotoxins refer to the product as is, that is without any correction for moisture content or other ingredients. Thus, for example, if DON present at a level of 1,000 µg/kg in wheat flour of 13% moisture content proves completely stable in bread manufacture this would result in a figure of approximately 790 µg/kg in bread (which is approximately 40-45% water) because of the additional water present and ignoring other ingredients. In addition, if DON should be lost, the value in bread would be reduced further. Conversely, the level of DON or fumonisins in cornflakes would be increased if the mycotoxin were stable as the product is drier than the maize flaking grits used, introducing a concentration effect. Analytical results in this study are corrected for changes in moisture and for the presence of other ingredients as indicated when the stability or loss of the mycotoxins is World Mycotoxin Journal 1 (4) 441

K.A. Scudamore and S. Patel Table 1. Reduction of DON (in µg/kg) during wheat cleaning and the production of breakfast cereals, values corrected for moisture change. Consignment Wheat % reduction by cleaning Product 1 % reduction from: Product 2 % reduction from: Dirty Clean Dirty wheat Clean wheat Dirty wheat Clean wheat 1 a,b 35 ns - 23 34 - <10 >86-2 a,b 50 ns - 40 20-19 66-3 a,b 27 ns - 15 44 - <10 >82-4 a,b 149 55 63 51 66 7 24 85 60 5 a,b 84 36 57 58 31-61 28 70 31 6 ns 17-17 - -0 <10 - >71 7 ns 39-36 - 8 <10 - >87 8 b 33 <10 - ns - - <10 >85-9 a,b 10 <10 - <10 >50 - <10 >50-10 a,b 28 12 61 12 57 0 <10 >82 >58 11 a,b 39 24 38 36 8-50 15 64 42 12 ns <10 - <10 - - <10 - - 13 ns <10 - <10 - - <10 - - 14 <10 <10-10 - - <10 - - 15 a,b 15 11 27 <10 >67 - <10 >33 >54 16 a,b 12 13-8 13-8 0 <10 >58 >61 17 a,b 52 17 67 20 61-18 11 81 41 18 a,b 64 14 78 26 59-86 <10 >85 >64 19 a,b 68 36 47 21 69 42 <10 >93 >86 20 <10 16-17 - -6 <10 - >69 21 a,b 58 19 67 14 76 26 <10 >86 >74 22 b 321 159 50 ns - - 15 96 97 23 b 61 36 41 ns - - 32 53 19 24 b 122 32 74 ns - - 26 81 28 25 b 37 32 13 ns - - 24 43 34 Mean 61 c 27 c 48 24 c 49 a -11 11 c 76 b 57 (Mean a ) 49 (Mean b ) 67 Median 39 17 54 17 57 0 24 81 60 Range <10 to 321 <10 to159-8 to 78 <10 to 58-8 to 76-86 to 42 <10 to 32 43 to 96 19 to 97 ns= no sample. a,b Samples providing the values on which extrapolation to EC maximum permitted levels is carried out. a is for product 1, b is for product 2. c Values <10 given value of 5 µg/kg. assessed but are uncorrected when relating concentrations found to the legislative limits. Studies of foods in commercial production present a number of problems not encountered in laboratory experimentation. Firstly, mycotoxins in the cereal ingredients must be within regulatory limits. Secondly, the actual level of contamination of a consignment or the mycotoxins present may not be known until the resulting manufactured food is analysed, as it is often a continuous flow process. This means that not all runs will yield useful results and consignments cannot be pre-selected. Details of recipes or processing conditions are often commercially sensitive and need to remain confidential to avoid providing these to competitors. This requires trust and cooperation between industry and the scientists involved. Because concentrations of mycotoxins were low in quite a few samples, particularly DON in raw wheat and maize and fumonisins in cornflakes, a nominal value of half the quantitative limit has been assigned to those samples in which concentration values are below these limits. This enables mean mycotoxin concentrations to be calculated in all intake and cleaned samples in Tables 1 and 4 and 442 World Mycotoxin Journal 1 (4)

Fusarium mycotoxins in breakfast cereals Table 2. DON concentration in aqueous effluent run off during the manufacture of product 2. No. Raw wheat µg/kg Clean wheat µg/kg makes it possible to confidently assert that the reduction in a particular sample is at least a certain amount. Analytical calibration standards were linear at low concentrations for both DON and fumonisins. Whole-wheat breakfast cereals Product 2 µg/kg Effluent µg/l 13 ns <10 <10 <10 14 <10 <10 <10 <10 15 15 11 <10 <10 19 68 36 <10 19 20 <10 16 <10 <10 21 58 19 <10 <10 22 321 159 15 15 23 61 36 32 14 24 122 32 26 12 25 37 32 24 <10 Note: ns = no sample. During this study the concentrations of DON present in wheat were low (Table 1) the maximum value found in intake wheat was 321 µg/kg with mean and median values of 61 µg/kg and 39 µg/kg respectively. In some consignments the levels were at or below the analytical quantitative level. As the aggregate sample is a composite from 5 lorry loads, often originating from different sources, the maximum value in any individual lorry before mixing, in theory, could have been as high as 1,600 µg/kg if the other four loads were all free from detectable DON. Such a value would exceed the regulatory maximum for DON in wheat. Detectable Table 3. Mycotoxin content in flaking grits compared to that in intake maize. amounts of ZEA, HT-2, T-2 and nivalenol occurred in a few samples but levels were too low to allow their fate to be followed through the processing stages. Although these low mycotoxin levels are welcome for the industry, it did mean that some consignments were unsuitable for determining the loss of DON. During this study, the UK cereal industry became increasingly aware of the problem of DON in wheat and the Home Grown Cereals Authority provided its levy payers with a risk assessment programme that allows a risk factor to be applied to grain. Lorries containing wheat at high risk were then sampled and analysed for DON, thus allowing exclusion of the most contaminated grain from the human cereal food chain. The company collaborating uses a comprehensive cleaning regime and this is reflected in a mean clean-up value for DON of 48% being achieved (Table 1). This value is derived from the mean of those 14 individual pairs of intake and cleaned wheat in which the DON concentrations measured in both samples were above the qualitative limit. However, the clean-up achieved was very variable between consignments as might be expected from bulked wheat of different history and origin and ranged from -8% to 78%. The values for mean and median of all 22 samples of cleaned wheat were 27 µg/kg and 17 µg/kg respectively (note that there were only 21 intake wheats sampled). Published information about the effectiveness of cleaning for reducing mycotoxins in wheat is limited and also very variable although the reduction in mycotoxin levels reported are usually less than found here. As supplies were usually obtained direct from farms, previous cleaning is unlikely to have been carried out. Lower values generally reported elsewhere could be due to the unknown history of the wheat prior to intake, allowing for the possibility that some initial cleaning might already been carried out. Cleaning here is also carried out using a very vigorous regime. A number of factors may affect the condition of the wheat before Mycotoxin Sample Mycotoxin, concentration, µg/kg % reduction in flaking grits Mean Median Range Mean Median Range DON intake 89 74 16-220 85 83 3-93 grits 13 5 <10-87 FB 1 intake 1,426 1,405 345-3,813 93.5 93 86-98 grits 92 90 28-222 FB 2 intake 484 408 101-1,230 95 95 82-98 grits 24 23 <10-72 FB 3 intake 249 229 74-711 94 93 79-98 grits 15 15 <10-27 FB 1 + FB 2 intake 1,990 1,661 505-5,002 94 93 85-98 grits 116 115 33-206 World Mycotoxin Journal 1 (4) 443

K.A. Scudamore and S. Patel Table 4. Change of FB 1, FB 2 and DON (in µg/kg) during production of cornflakes (flakes), values are corrected for moisture change. Consignment DON FB 1 a FB 2 a Grits Flakes Apparent change Grits Flakes % reduction Grits Flakes % reduction 1 <10 22-276 <10 >98 69 <10 >97 2 16 27 +62 181 <10 >97 41 <10 >96 3 <10 <10-98 <10 >95 24 <10 >93 4 13 27 +108 144 <10 >96 46 <10 >96 5 23 15-35 128 <10 >96 34 <10 >95 6 <10 13-51 <10 >90 16 <10 >89 7 <10 13-89 <10 >94 24 <10 >93 8 43 14-67 76 <10 >93 20 <10 >91 9 25 32 +28 49 <10 >90 15 <10 >89 10 <10 44-107 <10 >95 26 <10 >93 11 16 25 +56 107 <10 >95 30 <10 >94 12 <10 10-62 <10 >92 16 <10 >89 13 12 15 +25 97 <10 >95 28 <10 >94 14 <10 <10-115 13 >89 28 <10 >94 15 <10 <10-28 <10 >82 <10 <10-16 12 31 +158 <10 <10 - <10 <10-17 <10 <10-83 <10 >93 22 <10 >93 18 <10 <10-105 <10 >95 26 <10 >93 19 <10 <10-106 <10 >95 25 <10 >93 20 32 <10 >-84 145 <10 >96 34 <10 >95 Mean 12.3 16.1 +28% b 102.7 5.4 >93 26.8 1.7 >93 Median 5 13.5 +43% b 101.5 5 >94 25.5 1.7 >93 Range <10 to 43 <10 to 44-67 to +158 <10 to 276 <10 to 69 <<10-<10 a Values <10 given value of 5 or 1.7 µg/kg for FB 1 and FB 2 respectively. b The SD for the mean and median values are extremely high. harvest, where the mycotoxin is concentrated in the grain seeds, or even the ability of the combine to clean during harvesting. The EC maximum levels set for unprocessed cereals apply to cereals placed on the market for Firststage processing, i.e. any physical or thermal treatment, other than drying, of or on the grain. Cleaning, sorting and drying procedures are not considered to be first stage processing insofar as no physical action is exerted on the grain kernel itself and the whole grain remains intact after cleaning and sorting. Therefore the clean wheat used for processing and not the intake wheat is subject to the 1,250 µg/kg limit for DON. The low concentrations of DON in some of the wheat samples and breakfast cereals make it difficult to determine the loss of DON accurately during breakfast cereal production, so the approach discussed earlier was adopted. Results were calculated both as the overall reduction achieved from intake wheat to breakfast cereal and the reduction from cleaned wheat to breakfast cereal. The breakfast cereal results have been adjusted for the final moisture content of the product (Table 1). The moisture of the cereal product is approximately 4% compared to 14% in the starting wheat. The overall reduction from intake wheat to finished product was 49% for breakfast cereal 1 (14 sample pairs, of which one pair assumed a value of 5 µg/kg for the product) and 76% for breakfast cereal 2 (19 sample pairs, of which 10 pairs assumed a value of 5 µg/kg for the product). The change from clean wheat to finished product showed a nominal increase of 11% in DON for product 1 (12 pairs) and a loss of 57% for product 2 (17 pairs of which 9 pairs assumed a value of 5 µg/kg for the product). The reason for different values for the 2 products is not entirely clear but the processes vary in detail. In those samples in which DON appears to increase between cleaning and manufactured product this was only equivalent to about 444 World Mycotoxin Journal 1 (4)

Fusarium mycotoxins in breakfast cereals 20 µg/kg and this could merely be due to sampling and/or analytical error It could also indicate that DON is both being released more effectively by the cooking process while at the same time being hydrolysed during cooking. Alternatively the extraction process for the mycotoxins might be more effective for the cooked wheat than for the raw wheat. As the concentrations of DON were low, further studies with higher starting levels of DON are required to confirm these findings and to establish the cause. During this study, the maximum measured concentration of DON was 321 µg/kg which was reduced by cleaning to 159 g/kg and subsequently to 15 in product 2 (or 17 µg/kg before moisture correction), although product 1 was not manufactured from this wheat consignment. The highest value found for product 1 was 58 µg/kg (or 64 µg/kg before moisture correction) and this was manufactured from intake wheat with 84 µg/kg of DON. A consistently higher loss of DON occurred in product 2 although the cooking conditions for this could be regarded as less harsh. As the wheat is cooked in an aqueous solution it was postulated that some loss might be due to DON dissolving in this, rather than to hydrolysis, as excess liquid is subsequently drained off from the cooker containing product 2. To check this, the cooking effluent from product 2 from several runs was examined and the results are given in Table 2. In manufacture, 700 kg of wheat is cooked with 800 litres of water and this results in a moisture content of 45% in the dough after cooking, so that the volume of effluent will be about 500-600 litres. Thus, if the entire amount of DON was extracted into the water effluent the concentration would be of the same order as that in the original wheat. The results showed that when DON concentrations exceeded about 30 µg/kg it could be detected in this effluent. It is thus concluded that the main cause of loss of DON during manufacture of breakfast cereal 2 is dissolution in the aqueous cooking liquor and not loss due to hydrolysis, although this possibility cannot entirely be ruled out. While a consignment of unprocessed cereals (both intake wheat and cleaned wheat fall under this definition) permits levels of DON up to 1,250 µg/kg, the miller and processor must ensure that limits for subsequent products are also met. Thus, whole-wheat breakfast cereals manufactured from such a consignment must contain less than 500 µg/kg to be sold legally. This study indicates the extent to which DON values are changed during the manufacture of 2 specimen breakfast cereals Using those data presented in Table 1 (enables extrapolation to wheat at intake at the EC maximum value of 1,250 µg/kg. Thus, DON in product 1 is reduced by 49% but only by 44% on an as is basis and product 2 by 76% but by 68% as is. This suggests that product 1 could not be expected to meet the maximum limit of 500 µg/kg giving a mean reduction to 700 µg/kg while in contrast product 2 should do so giving a mean value of 400 µg/kg. Table 1 also indicates that cleaning protocols can reduce DON concentrations very effectively in most consignments thus building in a further, but variable, safety factor if intake wheat meets the 1,250 µg/kg criterion. In addition there is some suggestion that the reduction of DON through the processes may increase at higher contamination levels of DON although the naturally occurring concentrations of DON found in commercial supplies here did not allow this to be confirmed. The information found from these studies should provide valuable information to millers and processors in handling and monitoring wheat. It should assist with an informed purchasing policy to ensure that levels of wheat purchased will contain appropriate DON levels to manufacture breakfast cereal products that meet the EC limit of 500 µg/kg. Analytical results are also subject to some inherent uncertainty, and samples taken for enforcement purposes allow the appropriate value for this uncertainty to be taken into account before a trader can be considered negligent and liable to prosecution and then only in the absence of evidence to show that due diligence had been taken. Cornflakes As discussed earlier, cornflake manufacture involves 2 distinct stages: milling of raw maize to produce flaking grits and processing of the grits to manufacture cornflakes. Consignments of raw Argentinean maize were milled in a large UK mill over a 3-year period and samples of the intake maize and the resulting milling streams including the flaking grits were collected for analysis for DON and fumonisins using regular snatch samples during batch milling. Each composite sample or raw maize represented a day s throughput of at least 500 tons of maize unloaded from one hold of a large ship of 30,000 tons or more capacity. However, not all holds contained maize. In total a composite sample comprised 8 0.5 kg incremental samples. To calculate the mean reduction in mycotoxin concentration from intake maize to the flaking grits, only those results for consignments in which values were at or above the quantitative limit were selected. Because of the large reduction of mycotoxins in the grits compared to the intake maize and the relatively low levels of DON, the figures given in Table 3 are thus based on 8 milling runs for DON and 17 runs for fumonisin mycotoxins for which results were fully quantitative. A paper describing the distribution of mycotoxins in full-scale commercial milling operations is in preparation. There was a clear difference between DON and fumonisins, with DON values reduced by 85% but fumonisins by more than 95%. The effect of milling appears much more variable for residual levels of DON. While concentration of fumonisins up to and just above the EC limits were obtained, those for DON never exceeded 220 µg/kg. World Mycotoxin Journal 1 (4) 445

K.A. Scudamore and S. Patel Historical data (unpublished) relating to Argentine flint maize for the years 1997-2003 imported to the UK show that the maximum levels of DON in raw maize and flaking grits were 410 µg/kg and 113 µg/kg respectively, but were more than 5,000 µg/kg and 400 µg/kg for fumonisins in raw maize and flaking grits respectively. These values are of the same order as reached in the trials here, and can therefore be regarded as representative of levels in normal practice. In total, 20 consignments of flaking grits were examined for cornflake manufacture over a 2-year period (Table 4). In calculating the mean concentrations of FB 1, FB 2 and DON for the 20 consignments of maize grits or cornflakes those samples containing values <10 µg/kg were assigned a nominal value of 5 µg/kg (but 1.7 µg/kg for FB 2 on the basis the FB 1 and FB 2 occur in a ratio of approximately 3:1). During this time the maximum level of FB 1 was 276 µg/kg and was 43 µg/kg for DON, while ZEA was not detected and concentrations of other mycotoxins were very low. The maximum value found for FB 1 + FB 2 was 345 µg/kg. In calculating the reduction achieved for individual runs, only pairs of samples in which maize grits contained values at or above the quantitative limit of 10 µg/kg are included. This therefore limited the number of experimental runs that could be included to 8 for DON and 19 for FB 1 (and FB 1 +FB 2 ) and 18 for FB 2. The results from the 8 runs show both reduction in some runs and an apparent increase in others for DON, but any increases were only of 10-20 µg/kg in DON concentration just above the qualitative limit of determination and this is insignificant in relation to the legislative limit of 500 µg/kg. These concentration changes are very small and might be affected by factors such as the presence of other ingredients or sampling variability (they are however similar to those found in historical data as discussed earlier and suggest that it would be very difficult to source commercial flaking grits containing the concentrations needed to substantiate this). However, DON has been reported elsewhere as being generally stable during aqueous cooking under neutral conditions with losses never greater than 50% (e.g. Lauren and Smith, 2001; Hazel and Patel, 2004) and during dry heating. It must therefore be assumed here that little reduction could be expected during the manufacturing steps. The two samples containing the highest concentrations of DON in flaking grits do however both show a reduction in the manufactured cornflakes within the constraints discussed. The loss for fumonisins is much greater, so that only an estimated overall mean value for the cornflakes can be obtained by using nominal values of 5 for FB 1 and 1.7 for FB 2 in cornflakes for samples less than the quantitative limit (that is, for all but one consignment). It can then be stated that the loss is at least a certain amount, which in this case is 93%. The concentrations of DON in flaking grits during this study were low and cannot define with any certainty the extent or nature of the change in levels of DON during cornflake manufacture. The evidence suggests that there is little change in DON levels between grits and cornflakes. The reduction of mycotoxins from raw maize to flaking grits and then to cornflakes is based on relatively few runs from one large commercial enterprise over a 2-year period. The precise value may change depending on the operation of the specific mill preparing the flaking grits and cornflake manufacture and would probably be specific to those plants, although it is highly likely to be representative of what would occur elsewhere. Moreover, there appears to be little change in DON concentrations when producing cornflakes from grits and these results are very variable. Previous experience and literature referring to the stability of DON again suggest that little change would be expected here. Thus, the reduction of DON in manufactured cornflakes relies purely on that occurring when producing the maize flaking grits from raw maize (85%). Thus, for intake maize containing 1750 µg/kg of DON, cornflakes would be expected with approximately 288 µg/kg DON, on an as is basis. The percentage of fumonisins remaining in the grits is considerably less than that for DON. The mean reduction from intake maize to maize grits for FB 1 +B 2 was 94% (reducing 4,000 µg/kg to 240 µg/kg) and a further reduction from grits to cornflakes was greater than 93% giving a further reduction to less than 17 µg/kg in cornflakes. This reduction is still likely to underestimate the reduction of fumonisins during manufacture because most of the results for cornflakes are below the quantitative limit. The EC limit of 800 µg/kg for FB 1 +B 2 in breakfast cereals thus allows a safety margin of at least x 50, so that the chance of the 800 µg/kg limit ever being exceeded following the cooking process would be extremely small; raw maize with >20,000 mg/kg would be required! For DON the situation is rather different as the only significant reduction occurs at the milling stage, although there is quite a safety margin built in. 5. Conclusions Natural concentrations of Fusarium mycotoxins in commercial wheat used in both processes were low. An exhaustive cleaning regime of the wheat from farms produced a reduction in DON concentration of more than 50% although this varied considerably with consignments. During processing to manufacture two commercial breakfast cereals, the loss of DON is significantly greater in the product from which excess cooker effluent is drained off, which suggests that this loss was due to extraction of DON into the aqueous cooking liquor and not to hydrolysis. Maize flaking grits are inherently low in mycotoxin concentrations compared to the raw maize so that the cereal ingredient for cornflakes used by this 446 World Mycotoxin Journal 1 (4)

Fusarium mycotoxins in breakfast cereals manufacturing process is rarely likely to approach EC regulatory levels. The fumonisins are almost completely lost although DON was not reduced. However, cornflakes manufactured from maize flour by extrusion processes seem likely to result in higher residual mycotoxin levels due to the initial concentrations in maize flour being much higher than for grits. In addition, less destruction of fumonisins occurs during extrusion (Scudamore et al., in press). Natural concentrations of Fusarium mycotoxins in commercial maize flaking grits are much lower than in raw maize. During processing to manufacture cornflakes, concentrations of fumonisins are reduced further by approximately 95% although there is no apparent loss of DON during this stage. The loss of fumonisins appears to be greater than that suggested by surveys and further analytical quality assurance data are required to remove any doubt about the effectiveness of the methodology. However, the data from this study suggest that fumonisins in excess of the EC limit of 800 µg/kg would be extremely unlikely in cornflakes manufactured by the cooking process. The situation for DON is that EC limits should be applied to ensure that levels are kept within the statutory limits for mycotoxins in most breakfast cereals with some margin for variability and/or error. Although most fumonisins appear to be destroyed, further studies are needed to understand the nature of the reaction products produced. Acknowledgements These studies were funded by the UK DEFRA, Food Quality and Safety LINK Programme and the UK Food Standards Agency and would not have been possible without the cooperation and help of UK breakfast cereal manufacturers. References Abbas, H.K., Mirocha, C.J., Pawlosky R.J. and Pusch, D.J., 1985. Effect of cleaning, milling and baking on deoxynivalenol in wheat. Applied and Environmental Microbiology 50: 482-486. Broggi, L.E., Resnik, S.L., Pacin, A.M., Gonzalez, H.H.L., Cano, G. and Taglieri, D., 2002. Distribution of fumonisins in dry-milled corn fractions in Argentina. Food Additives and Contaminants 19: 465-469. Castelo, M.M., Sumner, S.S. and Bullerman, L.B., 1998. Stability of fumonisins in thermally processed corn products. Journal of Food Protection 61: 1030-1033. Chelkowski, J. and Perkowski, J., 1992. Mycotoxins in cereal grain (part 15). Distribution of deoxynivalenol in naturally contaminated wheat kernels. Mycotoxin Research 8: 27-30. De Girolamo, A., Solfrizzo, M. and Visconti, A., 2001. Effect of processing on fumonisin concentration in corn flakes. Journal of Food Protection 64: 701-705. European Commission (EC), 2006a. Commission Regulation (EC) No 1881/2006, of 10 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L364: 5-24. European Commission (EC), 2006b. 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Paepens, C., De Saeger, S., Van Poucke, C., Dumoulin, F., Van Calenbergh, S. and Van Peteghem, C., 2005. Development of a liquid chromatography/tandem mass spectrometry method for the quantification of fumonisin B 1, B 2 and B 3 in cornflakes. Rapid Communications in Mass Spectrometry 19: 2021-2029. Patel, S., Hazel, C.M., Winterton, A.G. and Mortby, E., 1996. Survey of ethnic foods for mycotoxins. Food Additives and Contaminants 13: 833-841. Patey, A.L. and Gilbert, J., 1989. Fate of Fusarium mycotoxins in cereals during Food Processing and methods for their detoxification. In: Chelkowski, J. (ed.) Fusarium. mycotoxins, taxonomy and pathogenicity. Elsevier Science Publishers B.V., Amsterdam, the Netherlands, pp. 399-420. Scott, P.M., Kanhere, S.R., Dexter, J.E., Brennan, P.W. and Trenholm, H.L., 1984. Distribution of the trichothecene mycotoxin deoxynivalenol (vomitoxin) in hard red spring wheat. Food Additives and Contaminants 1: 313-323. World Mycotoxin Journal 1 (4) 447