Peter Have Rasmussen

Annette Petersen

Faranak Ghorbani

Annual variation of deoxynivalenol in Danish wheat flour 1998 -2003 and daily intake by the Danish population

Keywords: Deoxynivalenol (DON), wheat flour, GC-ECD, LC-MS, annual variation, EU maximum limit, intake calculations

The occurrence of deoxynivalenol (DON) in Danish wheat flour was studied during the period from 1998 to 2003 by respectively capillary gas chromatography with electron capture detection and liquid chromatography coupled to an ion trap mass spectrophotometer. A total of 151 samples were collected from mills and the retail market in Denmark. The contamination level varied considerably from year-to-year with the highest concentrations occurring in samples from the 2002 harvest with mean and median concentrations of 255 µg/kg and 300 µg/kg, respectively. Compared to the other harvest years 2002 had the highest amount of precipitation around flowering time in the end of June and until the beginning of July covering the weeks 25-27. The lowest average levels were found in samples from the harvest of 2001, where the weeks 25, 26 and 27 were dry compared with the other harvest years. The highest value (705 µg/kg) was obtained in a flour sample from the 2002 harvest, but none of the tested samples exceeded the maximum limit of 750 µg/kg, which has been recently introduced by the European Commission for DON in flour used as raw materials in food products.

Calculation of the chronic or usual intake by a deterministic approach showed that the intake did not exceed the TDI of 1 µg/kg bw/day either for the whole population or for children. A probabilistic approach also showed that the intake in general was below the TDI, but the intake for children in the 99 % percentile amounted to more than 75% of the TDI. The highest intake is calculated to be 2.5 µg/kg bw/day.

Fusarium spp varies between cereals. In North America [START_REF] Miller | Epidemiology of Fusarium ear diseases of cereals[END_REF] and to some degree in Europe [START_REF] Gareis | Contamination of cereals and feed with Fusarium mycotoxins in European countries[END_REF] wheat and maize have been regarded as the grain species most susceptible to Fusarium, whereas a survey from Norway revealed oats to be more contaminated with DON than barley and wheat [START_REF] Langseth | The occurrence of deoxynivalenol in Norwegian cerealsdifferences between years and districts, 1988-1996[END_REF]. In a recent Danish study [START_REF] Rasmussen | Deoxynivalenol and other Fusarium toxins in wheat and rye flours on the Danish market[END_REF] both a higher incidence and concentration levels of DON were found in wheat than in rye flour, indicating that wheat grown in Denmark is more susceptible than rye towards attack from DON producing Fusarium spp. Increased levels of DON in cereal grains are often observed in harvest years with frequent rainfall and high humidity during the flowering period. Variation from year-toyear in contamination level has been described in several studies [START_REF] Scott | Multi year monitoring of Canadian grains and grain-based foods for trichothecenes and zearalenone[END_REF][START_REF] Langseth | The occurrence of deoxynivalenol in Norwegian cerealsdifferences between years and districts, 1988-1996[END_REF], indicating that the observation of climate data can be used as a preliminary prediction of contamination levels for a certain field area.

The SCOOP report from the European Commission (2003) shows that the major sources of dietary intake of DON are through products made from cereals, in particular wheat and maize, and especially in infants and young children the dietary intake of DON is often close to the tolerable daily intake (TDI) of 1 µg/kg body weight/day (SCF Scientific Committee on Food, 1999). Similarly, studies from the Netherlands showed that the TDI is easily exceeded in harvest years with a high infection level of Fusarium Thus, in 1998 and[START_REF] Scf | Opinion on Fusarium toxins. Part 1: Deoxynivalenol (DON)[END_REF] the intake of DON could have exceeded the TDI for eighty percent of the 1-year-old children (Pieters et al., 2004).

Based on the results of the SCOOP report and other surveys, in June 2005 the European Union introduced maximum levels of DON in various cereals and cereal products for human consumption. The maximum permitted level of DON in cereal flour including maize flour and maize grits is 750 µg/kg (Commission Regulation (EC) No. 1068/2005 of 6 July 2005), and 200 µg/kg in baby food and processed cereal based food for infants and young children. The regulation is valid from 1 st July 2006. The present study can be regarded as a continuation of our earlier studies [START_REF] Rasmussen | Deoxynivalenol and other Fusarium toxins in wheat and rye flours on the Danish market[END_REF], where not only DON but also other trichothecenes were analysed in flour of wheat and rye. These results showed that the incidence and contamination level of nivalenol, HT-2 toxin and T-2 toxin in Danish wheat flour were low, and for this reason only DON is used to study the variation in incidence and content level during the six year survey period between 1998 and 2003. The data is also used to measure the daily intake of the Danish population with two approaches, the deterministic method and the probabilistic method based on the Monte-Carlo Risk Assessment [START_REF] Boon | Calculations of dietary intake of acrylamide[END_REF].

Materials and methods

Samples

Flour samples of common wheat were collected each harvest year between 1998 and 2003 in two parts, one in the period from October to December, the other part from April to June the following year. Samples from 1998 and 1999 were collected both at the mills as retail packaged samples and from the Danish retail market with Flour samples were collected in retail packaging supplied with production date and batch number. Samples collected at the mill consisted of 5 to 10 bags of approximately two kg and samples obtained at the retail stage consisted of 1 to 4 bags from the same mill each containing two kg of flour. In the laboratory the samples were pooled and fully mixed in the a blending tank to produce one aggregate sample of 1 kg. From the aggregate sample 25 gram was taken for analysis. The number of samples colleted from each harvest year is shown in Table 1.

The mean temperature and precipitation values of June, July and August recorded each year (1998 -2003) at the meteorological stations in Denmark are shown in Table 2 (Danish Meteorological Institute). Precipitation and temperature data recorded on a weekly basis from the end of June to the beginning of July are also shown for each year. This period corresponding to the weeks 25-27 is in the present study assumed to represent the approximate flowering time for wheat grown in Denmark, although it is A general description of the weather conditions during the grain-growing season for each year, published by the Danish Agricultural Advisory Service (Landsudvalget for Planteavl, 1998Planteavl, -2003) ) was also used to evaluate the effect of weather conditions on DON content in flour.

Extraction and clean-up

Standards of DON were purchased from Sigma St. Louis, MO, USA. Stock solutions of 200-500 µg /ml were made in acetonitrile and stored at -20°C in portions of 1 ml for 1 year. Working standards were prepared by dilution of stock solutions with acetonitrile and stored at a concentration of 100 µg/ml at -20°C. The exact concentration was measured by U.V. spectrophotometry at 218 nm using ε 218 = 6400 cm -1 M -1 [START_REF] Widestrand | Effect of time, temperature and solvent on the stability of T-2 toxin, HT-2 toxin, deoxynivalenol and nivalenol calibrants[END_REF]. Working standards that had been stored up to 9 months showed no decrease in concentration. Extraction and clean-up were performed in accordance with the method described by [START_REF] Rasmussen | Deoxynivalenol and other Fusarium toxins in wheat and rye flours on the Danish market[END_REF] Twenty-five grams of ground and homogenised sample were extracted with 100 ml acetonitrile/water (84/16, v/v) in a 250-ml flask on a shaker for two hours at room temperature. The extract was filtered through a cellulose filter, and 5 ml of the extract was slowly pressed through a MycoSep 225 column from Romer Lab. (Washington, USA). An additional 5 ml of acetonitrile/water (84/16) was filled in the culture tube of the MycoSep 225 cartridge and pressed through the column. The eluates were combined and evaporated to dryness at 45°C using a gentle flow of nitrogen.

LC-MS Analysis

Samples from the harvest years 1998-2001 were analysed by capillary gas chromatography with electron capture detection (GC-ECD) by the method previously described [START_REF] Rasmussen | Deoxynivalenol and other Fusarium toxins in wheat and rye flours on the Danish market[END_REF], while samples collected from the harvest years 2002 and 2003 were analysed by LC-MS. Liquid chromatography was performed on a Hewlett Packard Series 1100 HPLC system equipped with the corresponding HP 1100 series pump and autosampler. The column was an X-terra MS C18 (5 µm, 3.0 mm x 250 mm, Waters, Milford, MA, USA) protected by a 3.0 x 20 mm guard column of the same material. Isocratic elution with a flow rate of 300 µl/min was applied over 15 minutes by using the same mobile phase as Razzazi-Fazelli et al. (1999) consisting of acetonitrile:-methanol:water (9 : 9 : 82, v/v/v) . Injection volume was 10 -20 µl. The HPLC system was coupled to an ion trap mass spectrometer (LCQ, ThermoFinnigan, San Jose, CA) used in the negative mode employing atmospheric pressure chemical ionisation (APCI (-)). The MS parameters were optimised separately by direct infusion of 10 µg/ml DON in the mobile phase via a T-piece between the column and the ionisation chamber at a flow rate of 5 µl/min. Mass spectra were registered in the fullscan mode between 200 and 350 Da. The capillary and vaporising temperature were 150 ºC and 400 ºC, respectively, and the capillary voltage was set at -47 V. Nitrogen sheath gas flow was approximately 50 ml/min while the auxillary gas was set at 0. Ionisation current of corona discharge was 5.5 µA. Calibration curves performed in the LC-MS buffer alone or in the presence of matrix showed no significant differences between the measured concentrations of DON in different wheat flour sample. Therefore, calibration curves were made in the LC-MS buffer covering a concentration range between 0.05 µg/ml and 3 µg/ml corresponding to 16 µg and 960 µg per kg flour. The detection limit (signal/noise: 3/1) for DON was the same for both the GC-ECD and the LC-MS method corresponding to 20 µg/kg. When calculating exposure, samples found to contain less than the detection limit were defined as having a concentration of half the detection limit, as suggested by [START_REF] Eriksen | Fusarium toxins in cereals -a risk assessment[END_REF]. In each analytical series, a spiking experiment was performed at levels between 50 -400 µg/kg using flour samples with DON contents below the detection limit. The mean recoveries and standard deviation for DON analysed by LC-MS was 101% ± 12% (range 80-114%, n = 27). The results were not corrected for recovery. A relative standard deviation of 13% was obtained for 14 analyses of an in-house reference material of durum wheat with a mean content of 1470 µg/kg measured over a period of two years. -0.5 (n = 62), 1.4 (n = 64), 0.2 (n = 64), -0.9 (n = 55) and-0.7 (n=73), respectively, where n is the number of participating laboratories. Scores are considered satisfactory if z ≤ 2 . The GC-ECD method has earlier been tested by this procedure and with satisfactory results [START_REF] Rasmussen | Deoxynivalenol and other Fusarium toxins in wheat and rye flours on the Danish market[END_REF]. In addition 10 different samples with DON contents between 20 µg/kg and 1800 µg/kg have been analysed by LC-MS and GC-ECD. All the samples with contents above 550 µg/kg consisted of durum wheat including our in-house reference material mentioned above.

Consumption data and methods for intake calculations

The consumption data used for the intake calculations are based on the nationwide dietary survey conducted in 2000 -2002 by the Department of Nutrition, Danish Institute for Food and Veterinary Research [START_REF] Andersen | The Danes´ dietary habits 2000-2002. Main results[END_REF]. The survey used a seven-day prospective food record with a pre-coded (semi-closed) questionnaire that included answering categories for the most commonly eaten foods and dishes in the Danish diet. The questionnaire was organised according to a normal daily meal pattern.

The body weight and age of the participant was also noted and used in the calculations.

In the present study only data on wheat flour are presented, but through the years of 1998-2001 rye flour was also analysed for DON. It was therefore decided to use data for both rye and wheat in the intake calculations. In the food survey the main consumption of rye and wheat are through bread and therefore a conversion of the contents of DON The content of DON in flour was then converted to contents in bread by using a factor of 0.77 for wheat and 0.62 for rye. These figures were estimated from the content of dry matter, protein and carbohydrates in bread, flour, and grain respectively [START_REF] Poulsen | Pesticides, Food monitoring[END_REF]. Baking or heating generally seems to have little effect on the stability of DON [START_REF] Pestka | Deoxynivalenol: toxicology and potential effects on humans[END_REF], however some studies resulted in a 0 to 40 % reduction of the content [START_REF] Eriksen | Fusarium toxins in cereals -a risk assessment[END_REF]. Other data have shown both increase and decrease in DON levels at dough fermentation and baking [START_REF] Hazel | Influence of processing on trichotecene levels[END_REF] Due to these uncertainties, loss of DON through processing has not been taken into consideration. Levels below LOD have been set to ½ LOD or10 µg/kg.

The calculations of the usual intake were performed for both the total population of age 4 -75 years (4068 persons) and for children, age 4-14 years (807 persons). The usual intake is used as a synonymy for the chronic or the long-time intake. Two approaches have been used in the calculations for both consumer groups. The first method is the deterministic approach where the intake is calculated by a point estimate, e.g. only one value is calculated. The usual intake is calculated for the relevant consumer group by multiplying the mean consumption for the consumer group with the mean level of DON for each commodity and the total usual intakes calculated by summing up the individual intakes. In Denmark the mean concentration of mycotoxins is most often used in intake calculations as the best estimate a person will be exposed to during a whole life span [START_REF] Fromberg | Chemical contaminants, Food monitoring[END_REF]. In the SCOOP report from 2003 the mean concentration of DON is also used for the determination of the intake. However, the median concentration of DON can also be used for this purpose, but to what extent the median will represent a better estimate for the intake calculations is out of the scope of this paper to discuss. The other method is the probabilistic approach, where the total distribution of both the content and consumption are used to derive a distribution of intakes as well as percentiles and uncertainties of the intakes. The calculations have been performed using the "Monte Carlo Risk Assessment" program version 4.0 (http://mcra.rikilt.wur.nl/mcra/) and is described by [START_REF] Boon | Calculations of dietary intake of acrylamide[END_REF]. In the program the calculation is performed by multiplying each participant's intake of respectively wheat bread and rye bread with the obtained average content of DON and then summing up the two intakes to give the intake for each consumer from bread. For the total population this gives about 28.000 intakes including non-eating days which are analysed in the programme with statistical methods for usual intakes [START_REF] Nusser | A semi-parametric transformation approach to estimating usual intake daily intake distributions[END_REF][START_REF] Guenther | Development of an approach for estimating usual nutrient intake distributions at the population level[END_REF])

Results and discussion

Analysis of deoxynivalenol by LC-MS

DON was determined by LC-APCI -MS in the negative ion mode as the observed signal/noise ratio was 10 -15 times higher than in the positive ion mode under the given conditions. The mass spectrum consisted of an intensive M-H -ion at m/z 295 corresponding to the deprotonated form of DON (Figure 1). In accordance with the study of [START_REF] Razzazi-Fazeli | Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography-mass spectrometry with negative ion atmospheric pressure chemical ionisation[END_REF], the fragment ions at m/z 265 and at 247 appeared in all chromatograms. In this study DON has also been detected in the negative mode by LC-MS-MS through collision induced dissociation in the ion trap (collision energy All samples from the 1998 harvest were found to contain DON with relatively high mean and median values of 191 µg/kg and 140 µg/kg, respectively (Table I). The amount of precipitation in June and July for the whole country were 76 mm and 92 mm, and thus considerably higher than the normal precipitation values registered in Denmark between 1961 and 1990 (Table II). In 1998 there were large amounts of precipitation around the flowering stage in the weeks 25, 26 and 27, conditions known to favour Fusarium invasion of the grains. [START_REF] Sutton | Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum[END_REF][START_REF] Teich | Epidemiology of wheat (Triticum aestivul L.) scab caused by Fusarium spp, Fusarium, Mycotoxins, Taxonomy and Pathogenicity[END_REF]. The weeks 25 and 26

were relatively cold compared to the other years and below the normal temperature of 15.6 °C for July. Moreover, due to low temperatures during the harvest season in August with a mean of 14.6 ºC for the entire country which were accompanied by heavy showers in many parts of the country, lodging was especially widespread in winter wheat that year (Landsudvalget for Planteavl, 1998). Such factors may also contribute to increased levels of DON in grains [START_REF] Langseth | The effect of lodging and time of harvest on deoxynivalenol contamination in barley and oats[END_REF].

Insert Table I , 2002).

This is in accordance with the findings that the highest mean and median values were observed for the samples collected from the 2002 harvest although the relative incidence of positive samples was lower than for other years except 2001. This is due to the fact that many of the samples from this year contained relatively high DON contents above 250 µg/kg (Table 1). The highest concentration observed was 706 µg/kg. In 2002 there was large regional differences in precipitation (Landsudvalget for Planteavl, 2002) and it could well be that samples with high DON contents come mainly from areas with high precipitation around flowering. A similar hypothesis could also be valid for the harvest years 1998 and 1999. However, as already mentioned, it is difficult to go into detail on this topic as more information on sample origin is essential. [START_REF] Razzazi-Fazeli | Simultaneous quantification of Atrichothecene mycotoxins in grains using liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry[END_REF]. However, all tested samples had contents below the detection limit corresponding to 20 µg/kg (results not shown).

Several studies have indicated that the contamination level of DON in various cereal grains frequently is correlated with the amount of precipitation during the growing season with the lowest contents occurring during dry weather conditions [START_REF] Muller | Fusarium toxins in wheat harvested during six years in an area of Southwest Germany[END_REF][START_REF] Langseth | The occurrence of deoxynivalenol in Norwegian cerealsdifferences between years and districts, 1988-1996[END_REF][START_REF] Scott | Multi year monitoring of Canadian grains and grain-based foods for trichothecenes and zearalenone[END_REF]. The present results are in accordance with these findings. Although the number of investigated harvest years is too small to allow a firm statistical analysis on this point, there seems to be some relationship between the amount of precipitation in the end of June and beginning of July and the concentration of DON found in the samples. Thus, the highest mean as well as median concentrations As well as being affected by weather conditions other factors may also have contributed to both within and between year variations such as soil treatment, crop rotation and cultivar differences where some are more sensitive toward Fusarium infection than others [START_REF] Jørgensen | Aksfusarium og indhold af deoxynivalenol (DON) i forskellige hvedesorter under svampebehandling[END_REF]. The contamination level can alsobe affected by the degree of Fusarium infection in the previous year [START_REF] Langseth | The occurrence of deoxynivalenol in Norwegian cerealsdifferences between years and districts, 1988-1996[END_REF]. However, it is not possible to evaluate the importance of these factors from the present data.

The European Commission recently introduced a maximum limit of 750 µg/kg for DON in flour used as raw material in food products (Commission regulation (EC) No.

Insert Table III about here

Usual intake of DON by the Danish population

The background information for the intake calculations performed by the deterministic approach is shown in Table III. The intake of DON for both the total population and the children are higher for wheat bread than for rye bread, which is due to a lower content of DON in rye than in wheat flour combined with a higher consumption of wheat bread.

The intake of DON from bread is calculated to 0.17 µg/kg bw/day for the total population and 0.32 µg/kg bw/day for children. The higher intake for children is due to their lower body weight and their consumption of wheat bread, which is almost the same as for the total population. The TDI for DON is 1 µg/kg bw/day (SCF, 2002), and thus the intake calculated by the deterministic approach will not exceed this value.

Using the median values of DON the intake is calculated to be 0.10 µg/kg bw/day for the total population and 0.18 µg/kg bw/day for children. These values are thus lower than the intakes determined from the mean. Whether the mean or median will represent the best estimate for the level a consumer is exposed to for a whole life span can be discussed. As already mentioned, in Denmark the mean level of mycotoxins is usually used in intake calculations as the best estimate [START_REF] Fromberg | Chemical contaminants, Food monitoring[END_REF]. In the SCOOP project from 2003 the mean was also used for the determination of the intake. The mean intake and percentiles for the usual intake calculated by the probabilistic approach are shown in Figure 3. The intake for the total population and for children is calculated to 0.20 µg/kg bw/day and 0.35 µg/kg bw/day, respectively, which are very similar to the values obtained by the deterministic approach. This reflects that bread is consumed nearly every day and that the majority of the samples have DON contents above the detection limit of the method. The calculations are therefore not very sensitive to extreme intakes. The highest intake is calculated to be 2.5 µg/kg bw/day. At the 99.90 % percentile the intake for children is 0.88 µg/kg bw/day, and children with an intake higher than 1 µg/kg bw/day belongs thus to the upper 0.1% percent. When only considering the data from the 2002 harvest with the highest determined content of DON in wheat bread (Table 1), the corresponding intake is calculated to be 0.31µg/kg bw/day for the total population, which is about twofold higher than the value obtained for the whole period between 1998 and 2003. This stresses the importance of not only using data from a single year when calculating the intake of DON and as well as other substances with wide annual variation. Using a value of zero instead of ½LOD for levels below LOD in the probabilistic approach shows only a minor influence on the intakes, as the mean intake changes to 0.34 µg/kg bw/day for children and is the same for the total population. This is due to the fact that most of the samples had contents above the detection limit of the method and that samples with high DON contents will be of overall importance for the calculated intakes.

Insert Figure 3 The highest intake of cereal products by the Danish population is from bread of wheat and rye, which contribute more than 80 % of the total intakes [START_REF] Jørgensen | Chemical Contaminants, Food monotoring[END_REF].

Other sources from corn based foods of sweet corn, cornflakes, breakfast cereals of oats, rice and pasta, cakes, biscuits and popcorn [START_REF] Scoop | TASK 3.2.10: Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU Member States[END_REF], will also contribute to the intake of DON, and especially children can have a high intake of these products. In the high percentile range above 99 % the intake of wheat and rye bread for children will be about 0.8 µg/ kg bw/day (Figure 3), and by adding these other sources the TDI value can be exceeded for this group of children.

Conclusion

Wheat flour on the Danish market showed a wide annual variation in DON concentration during the period from 1998 to 2003, but none of the tested samples exceeded the maximum EU limit of 750 µg/kg. The highest contamination levels were obtained in harvest years with a relatively high amount of precipitation around flowering in the end of June and beginning of July corresponding to the weeks 25, 26 and 27. The wide annual variation in DON contents emphasises the importance of not using a single year for intake calculations. Intake calculations of DON with reference to the consumption of wheat and rye bread show that the mean usual intake determined both by a deterministic and a probabilistic approach are similar corresponding to about 0.2 and 0.3 µg/kg bw/day for the total population and children, respectively.

  ) is produced by different Fusarium spp. and predominantly found in grains of wheat, barley, oats, rye and maize grown in the temperate regions of Europe, America and Asia. The susceptibility towards infection with DON producing

  distribution between the two places. The samples from 2000 to 2003 were collected at the retail market all over Denmark. Authorized personnel from local food control units performed the sampling. Only flour produced at Danish mills were used in the present study. The mills were located all over Denmark, three in Jutland, one on Funen and two on Zealand. In Denmark most of the wheat flour for human consumption are produced from domestic grains, and information from Danish Industry and the millers show that typically over seventy percent of the grains are from Denmark. The rest of the grain is imported mainly from the southern part of Sweden and Germany.

  may have been variations from region to region and from year to year.

  2004, the LC-MS method has been evaluated five times in proficiency testing by the FAPAS (Food Analysis Performance Assessment Scheme, Central Science Laboratory, York, UK). The z-scores for contaminated wheat and maize flour were

  contents in bread has been performed. Bread is a wide group and many different kinds of bread are eaten in Denmark. To simplify the calculations the different groups of bread were merged into two groups, namely "wheat bread" and "rye bread".

  of the parent ion at m/z 295. The most predominating daughter ions were the same as in the MS mode at m/z 265 and m/z 247, respectively. Although the noise level was lower in the MS-MS mode than in the MS mode, DON at m/z 295 displayed a stronger signal (signal/noise) in the full scan mode than could be obtained for the two daughter ions in the MS-MS mode. Quantification was therefore carried out in the MS mode at m/z 295 and by using the fragment ions detected at m/z 265 and m/z 247 as verification. The content of DON determined in parallel by GC-ECD and LC-MS in 10 different flour samples of common wheat and durum wheat is shown in Figure2. Linear regression analyses showed a significant correlation (y = 1.08x -28, r 2 = 0.983, p< 0.005, x: GC-ECD; y: LC-MS) between the concentration measured by the two methods, confirming that similar results are obtained by GC-ECD and the LC-MS method. This assumption is furthermore confirmed by the results obtained in proficiency testing by FAPAS where the z-scores for both methods showed satisfactory results.''(Insert Figure1and Figure2about here)''Annual variationDuring the period from 1998 to 2003 the content of DON in Danish produced wheat flour varied considerably (TableI). The mean values for the precipitation and temperature recorded each year in June, July and August are shown in TableII togetherwith the precipitation and temperature data approximately covering the last two weeks of June and the first week of July. This period is suggested to represent the time around flowering of wheat grown in Denmark, although there may be variations from year to year and from region to region. Despite Denmark is a small country, some years show differences in precipitation. In the present study it has unfortunately not been possible to evaluate the importance of such factors because it could not be exactly confirmed from where in the country the different mill samples originate.Moreover, a minor part of the samples produced at Danish mills may contain flour of imported wheat grains from mainly southern Sweden and Germany. In spite of these uncertainties it has been possible with proviso to derive some general indications in relation to the importance of summer weather conditions to the overall DON level in Danish produced wheat flour.

  were obtained in years with the highest amount of precipitation in the weeks 25, 26 and 27, and the lowest concentrations were found in the years 2000 and 2001 with low precipitation values around flowering. . The amount of precipitation in August on the other hand showed no apparent relationship to the contamination level of DON. Thus, of precipitation (91 mm) was observed in 2001, where the contamination level of DON was low, and in 2003 August was very dry with a mean precipitation of 41 mm but with a higher DON contamination level than in 2000 and 2001.

  856/2005 of 6. June 2005). As shown in TableI, the mean and the median values for the DON content in Danish wheat flour are far below the maximum EU limit. The highest value of 705 µg/kg was obtained from the harvest 2002, and thus none of the 151 tested samples analysed between 1998 and 2003 exceeded the maximum limit of 750 µg/kg. Furthermore, only five samples had DON contents above 500 µg/kg, one sample from the harvest 1999 and four samples from the harvest year 2002 TableI. Almost 50 percent of the tested samples had DON contents below 100 µg/kg, 34 samples between 101 µg/kg and 250 µg/kg and 25 samples between 251 µg/kg and 500 µg/kg. When comparing the present results with the EU maximum limits, it must be emphasized that the sampling procedure used in this study did not fulfil the guidelines for official testing

  mean. Values below the detection limit (20 µg/kg) are set to have a concentration of half the detection limit.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Total ion chromatogram obtained after clean-up with Mycosep 225 column of a natural contaminated wheat extract containing 250 µg/kg DON. The mass spectrum in the negative ionisation mode (APCI/MS) is also shown.

  However, problems with the presence of Fusarium in wheat have not been observed that year(Landsudvalget for Planteavl, 2001).Insert TableIIabout here During the grain growing season 2002 there was a large amount of precipitation from the middle of June and throughout the summer (Landudvalget for Planteavl, 2002), and as shown in Table 2, the weeks 25-27 around flowering had the highest observed mean precipitation values. This year the infection level with different Fusarium spp. was more pronounced than in normal harvest years (Landsudvalget for Planteavl

	harvested at all.
	In 1999, the amount of precipitation in June was high with a mean value of 120 mm for
	the whole country and as shown in Table II, the end of June (week 25) and beginning of
	July (week 26) were also relatively wet. Lower amounts of rain were observed in week
	27 than in the previous year. The end of July and the beginning of August were warm
	F o r and dry but accompanied by many showers later in August, and in 1999 harvest conditions were especially difficult in many parts of the country. (Landsudvalget for Planteavl, 1999). These conditions may further explain that the incidence and DON F o r
	P content in the samples were relatively high and showed similar levels compared to P
	1998.	e e r e e r
	R e During 2000 and 2001, the contamination level of DON continued to decrease with the R lowest values occurring in samples from the 2001 harvest, where the average levels e
	v i e decreased about 6 times compared to 1998. The incidence of DON was also low in 2001, where DON was found in about 60% of samples tested (Table 1). Weeks 26 -27 v i e
	w were dry in both 2000 and 2001 compared to the other harvest years, and also week 25 w
	O n l in 2001. Especially July of 2001 was relatively warm with a mean temperature of 17.5 ºC, and this combined with low precipitation resulted in a widespread need for watering crops on many locations in Denmark (Landsudvalget for Planteavl, 2000 and 2001). O n l
	y The low contamination level of DON found in samples from these two harvest years y
	may thus be expected due to the dry weather during the growing season. August of 2001
	was very rainy with a mean precipitation of 91 mm and 17 days with rain. Harvest
	conditions were difficult and quite exceptionally for Denmark, much grain could not be
	about here
		14 15

  samples and all samples had contents below 224 µg/kg. In June the mean precipitation was well above the normal value of 55 mm and July was warm with a day and night mean temperature of 18.1°C, which is 2.5 °C above the normal temperature. The mean precipitation for the whole country was low in week 25 whereas week 27 had the highest observed mean precipitation value of 42 mm. August was dry and relatively warm.It should be mentioned that HT-2 toxin and T-2 toxin have been measured in 13 of the flour wheat samples from the harvest 2003 by a LC-MS method described by Razzazi-

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	Compared to 2002, the contamination level of DON decreased in samples from the
	harvest in 2003 with average and median values of 148µg/kg and 151 µg/kg,
	respectively. A total of 27 samples were analysed and DON was detected in 26 of the

Table I .

 I Occurrence of deoxynivalenol (DON) in flour samples of wheat collected during the survey period 1998 -2003.

	Page 25 of 31	Food Additives and Contaminants
	exceeded by adding the contribution from cereal products other than wheat and rye
	bread.
	Harvest year 1998 1999 2000 2001 2002 2003	F o r P e e r R e F o r P e e r R e F o r P e e r R e F o r P e e r R e F o r P e e r R e DON (µ µ µ µg/kg) Number of positives/total Mean value a 14/14 16/16 26/28 19/30 29/36 F o r P e e r R e v i e w Number of samples in each range (µ µ µ µg/kg) Median value Max. value < 100 101-250 251-500 >500 O n l y 26/27
		v i e v i e v i e v i e v i e
		w w w w w
		O n l O n l O n l O n l l n O 1
		y y y y y	These
	products contribute more than 80 % of the total intake of cereals indicating that DON
	does not represent a general food safety problem in Denmark. However, for children in
	the high percentile range above 99 % the TDI value of 1 µg/kg bw/day can easily be
		21

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Table II

 II Mean values of precipitation and temperature for the whole country recorded each year between 1998 and 2003 for June, July and August. Precipitation and temperature data for the weeks 25, 26 and 27 from each year are also shown. The mean values of the precipitation and temperature recorded each year between 1962 and 1995 are shown in parenthesis. Data were obtained from the Danish Meteorological Institute (DMI).

			Precipitation	Temperature
			(mm)		(ºC)
	Year and week	June	July	August	June	July	August
	(55) week 26, June 30 -July 6 120 1998 week 25, June 22-29 F o r	(66) 92 20 25 56	(67) 60 88	(14.3) 13.9 15.4 13.5	(15.6) 14.4 13.9 13.4 17.2	(15.7) 14.6 16.5
		22 53 21 8 P e e r 18 12 42	48	12.7 13.7 17.3 12.6	15.7 17.5 14.9	15.2
		62 9 12 100 39 24 80 11	10 47 5 113 36 72 R e 91 71 41 v i e w 12.8 13.6 16.6 15.6 16.8 13.9 15.8 14.7	14.4 17.5 19.9 17.1 14.3 18.1	17.0 19.6 17.7
		23	42		16.3 O n 15.8 l
						y
			3

Table III .

 III Background information for the deterministic calculation of the usual mean intake of DON from wheat and rye flour

		Total population	Children
		Wheat	Rye	Wheat	Rye
	Number of participants	4068	4068	807	807
	Consumption days/days	28476/24269 28476/21513 5649/4749	5649/4151
	bread is consumed
	Mean consumption of	92	60	89	48
	bread (g)
	Estimated mean level of	109	26	109	26
	DON (µg/kg) in bread
	Mean body weight	66.2 kg	66.2 kg	35.1 kg	35.1 kg
	Mean intake in µg/kg	0.15	0.02	0.28	0.04
	bw/day
	Sum of intakes from	0.17 µg/kg bw/day	0.32 µg/kg bw/day
	wheat and rye bread

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Acknowledgements

We wish to thank Henrik Hartkopp and Anders Möller, Department of Nutrition, Danish Institute for Food and Veterinary Research for delivering the consumption data and compiling the flour data into rye and wheat bread.


Erik J M Konings

Peter Ashby

Colin Gregory Hamlet

Geoff A K Thompson

Erik ; Konings

E J M Konings

Erik Konings

Acrylamide in cereal and cereal products

Keywords: Acrylamide, asparagine, asparaginase, cereals, cereal products, mitigation, 23 processing, recipe, yeast

published or not. The documents may come

Introduction 1

In a status report of December 2004, the Confederation of the food and drink industries of 2 the EU (CIAA) reported the progress made until that date in lowering acrylamide levels in the 3 major food categories of concern (CIAA 2004). In March 2006 a joint workshop was 4 organized by the European Commission and the CIAA for government, industry and 5 academia to discuss the key knowledge and achievements to date, results of the current 6 studies and projects, opportunities, gaps and constraints. One aim of the workshop was to 18

The text below focuses on issues since that date. It is now widely accepted that acrylamide is primarily formed in cereal products from 23 asparagine reacting with an intermediate, which is typically a carbonyl source formed from a 24 reaction between amino acids and reducing sugars. The simplest possible reaction scheme 25 for the formation of acrylamide is summarised in figure 1.

26

[insert Figure 1 about here]

27

The value of these simplified reaction schemes or kinetic models lies in their predictive

24

Breakfast cereal manufacturers using wheat sourced from the neighbourhood of their The implication is that the asparagine content of wheat is significantly influenced by 1 seasonal factors.

2

Earlier studies showed that the ash content of flour influenced acrylamide content in 3 bakery products. Flours high in ash have significantly elevated amino acid contents, resulting 4 in higher acrylamide contents. Furthermore, dusting of the breads with flour prior to baking

5

bicarbonate in a recipe, which did not contain added reducing sugar failed to give an 6 increase in acrylamide relative to the basic wheat/water recipe. This is important since the 7 added sugar used in the majority of biscuit recipes is sucrose, a non-reducing sugar.

8

Cysteine, sodium bisulfite, and ascorbate also enhanced elimination. Asparagine, but not 9 reducing sugar, caused a large increase in acrylamide formation.

4

The addition of ingredients inhibiting acrylamide formation by reaction with precursors 5 of acrylamide may be more suitable, eg. flavenoids in apples, tea leaves and onions might grain wheat flour contained 0.5 g kg -1 and whole grain rye flour 1.1 g kg -1 . The lowest content 10 was found in sifted wheat flour (0.2 g kg -1 ). Wheat germ had the highest content (4.9 g kg -1 ).

11

Fermentation (baker's yeast or baker's yeast and sourdough) of doughs made with the 12 different milling fractions was performed to investigate whether the content of free 13 asparagine was reduced by this process. In general, most of the asparagine was utilized 14 after 2 hr of fermentation with yeast. Sourdough fermentation, on the other hand, did not 15 reduce the content of free asparagine as efficiently but had a strong negative impact on 16 asparagine utilization by yeast. This indicated that this type of fermentation may result in 17 breads with higher acrylamide content than in breads fermented with yeast only. The effect 18 of fermentation time on acrylamide formation in yeast-leavened bread was studied in a 19 model system. Compared with short fermentation time, longer fermentation reduced 20 acrylamide content in bread made with whole grain wheat 87%. For breads made with rye 21 bran, the corresponding reduction was 77%.

22

In laboratory and small scale trials several groups have reported that added amino 23 acids can reduce acrylamide [START_REF] Amrein | Acrylamide in 2 gingerbread: critical factors for formation and possible ways for reduction[END_REF]Bråthen et al. 2005). Cysteine competes 24 with asparagine for reducing sugars in the Maillard reaction; and the thiol group may react 25 with the double bond of acrylamide. On the other hand its application in dough is limited 26 because cysteine also reacts with gluten proteins, which has a significant impact on the 27 rheological properties of the wheat gluten network and hence the quality of the product.

6

The pH value of the dough is another factor affecting acrylamide formation. Lowering

8

This can be accomplished by adding consumable acids such as citric acid or by lactic 9 fermentation, as applied during sourdough preparation. It is well known that pH is an 10 important determinant of the rate and formation of key Maillard reaction intermediates, e.g.

11

Schiff base formation and the acidic medium can also lead to deamination of asparagine.

12

The practical pH range required to produce viable bread dough is however very limited and 13 hence this application may be more suitable as a surface treatment.

2

Acrylamide formation not only depends on temperature as such, but also on heat transfer to 3 the product. Tunnel or travelling ovens are used for large scale commercial bread production 4 (e.g. in the UK) and these can use both radiant and convective heat transfer. For smaller 5 scale bread production, two types of oven are usually used. In the traditional deck oven heat 6 is transferred mainly by radiation, while air circulation is used in the convection oven.

22

and Davies and Wedzicha 1994); it may be that this is so volatile (bp -19.3°C) that it is lost 23 in processing. However, further work is required to assure that this is the case when it 24 pervades a food rather than being a surface treatment. Figure 3

2

We should not lose sight of the effect of the choice of grain. Wheat is the dominant 3 grain and dominates in bread and biscuit. The experience of sectors, which use other grains, 4 is enlightening. Figure 4 shows the acrylamide content of a breakfast cereal made to the 5 same process but from four different grains.

6

[insert figure 4 about here]

7

One cannot substitute rice or oats for wheat and hope to have the same food. There are many processes in use and even within a process e.g. extrusion puffing 1 there are many variations in how it is affected. Thus some manufacturers extrude at or near 2 final product moisture, others extrude at higher moisture and toast the product to the target 3 moisture.

4

Many processes involve two cooking steps, a pressure-cooking of dough or grain

15

A detailed study of oven baking was made because this platform lends itself better to 16 instrumental measurement in process than other types of toaster. Figure 5 shows the 17 influence of oven temperature on formation of acrylamide in a baked cereal product.

18

[insert figure 5 about here]

19

The results in figure 5 clearly demonstrate that lower temperatures are associated 20 with less acrylamide formation. The most interesting feature was that the system was 21 already at the lowest temperature at which the product successfully bakes, it was set to this 22 point some years ago as part of a project to minimise energy consumption. Some other 23 manufacturers have reported similar findings (personal communication). Simply reducing the 24 temperature of an oven can be very costly for a manufacturer because the throughput is 25 reduced and one may not be able to meet demand. Thus some points in figure 5

4

have been beneficial in reducing acrylamide in some bakery products. For example, figure 7 5 shows the effect of divalent metal ion additions on acrylamide generation in wheat bread 6 doughs. However, these metals are also important micronutrients and the impact on nutrition 7 from increasing e.g. calcium levels to reduce acrylamide formation will need careful 8 consideration.

9

[insert figure 7

19

Future outlook. Our objective should be to reduce acrylamide without compromising the 20 safety (toxicological), quality (organoleptic properties) and nutritional aspects of cereal 21 products and to achieve this, it will be necessary to engage experts from all these areas. although regulatory approvals may be prolonged. When and if it is accepted there is a good 14 reason to believe that the enzyme could allow "standard" recipes to be used so that the 15 sensory quality of the food is unchanged. There are a limited number of successes for particular products. They are achieved 13 in the areas of recipe and processing. There are some promising leads for future, for 14 example asparaginase. We need more involvement of agriculture not only in hope of 15 developing crops, but also to avoid steps backward from the crops (asparagine is going up 16 and down). Involvement of experts in nutrition could help assure mitigation steps would not 17 cause an inverse negative effect in diet. Finally, we need a mechanism to assure that 18 mitigation of acrylamide does not enhance other process contaminants.

19

Cereal foods represent a very large and diverse range of raw materials and processes and it 20 is likely that each product / technology will require individual optimisation.

21

While we await the conclusions from further toxicological studies it is important that the 22 cereals sector continues to take the issue of acrylamide seriously and proactively 23 investigates how to reduce acrylamide in affected products. Regardless of the acrylamide 24 issue, cereals play an important role in the diet. In the meantime, the general advice to 25 consumers should be to continue to eat a varied diet composed of foods from many sources 26 and with moderate consumption of fried and fatty foods.

7

  update the toolbox approach on acrylamide (CIAA 2005) and this was published in October 8 2006 (CIAA 2006a).

9 10

 9 Following the workshop, a number of detailed actions were agreed at the latest Expert 11 Committee on Environmental and Industrial Contaminants meeting of the EU Commission so 12 as to ensure that voluntary measures such as the toolbox approach are effectively applied 13 by food operators and their effects monitored. These actions include the development of a 14 European monitoring programme, contribution to the development and future distribution of 15 sector specific brochures giving guidance to small and medium sized food operators on how 16 to take steps to minimise acrylamide formation, and the development of a European 17 framework on consumer advice for home cooking. These actions will be carried out in close 18 co-operation with the relevant stakeholder organisations (SANCO 2006). Since 2003 both 19 the European Commission and the World Health Organization posted calls for data and 20 placed their spreadsheets for the submission of data on the web. The goal of the databases 21 is to collect data for a reliable estimation of the exposure of consumers to acrylamide via the 22 food chain (Lineback et al., 2005) 23 24 This paper focuses on issues, achievements and results of current studies and projects on 25 cereal and cereal products since the publication of the CIAA toolbox in 2005. Furthermore, 26 aspects of future issues and gaps in knowledge are discussed.

1 29

 1 Cereal foods encompass a vast range of different products with many ingredients, 3 processes, recipes and scales of operation. Research has confirmed that there is no simple 4 route to reduce acrylamide. To date a limited number of successes, for example through 5 work on raising agents, have been achieved. Asparaginase also looks very promising for the 6 near future and longer-term work on generating cereal varieties naturally low in asparagine 7 may prove successful 8 Within the CIAA toolbox, four key compartments encompassing 13 different parameters 10 have been listed with the potential for reduction of acrylamide: (2004) and Stadler (2005) summarized the most important findings with 16 regard to formation and possible mitigation of acrylamide in cereal products and bakery 17 products. Raw materials, ingredients and processing conditions were taken into account.

28(

  applications for "what-if" experiments to determine conditions that can lead to reduced 29 and Hamlet (2005) and Wedzicha et al. (2005) validated traditional kinetic 1 models for the formation and loss of acrylamide in wheat and rye products and 2 demonstrated that levels of acrylamide in cereal products are limited by amino acids running 3 out as well as direct decay or loss of the acrylamide produced. Corradini and Peleg 2006 4 used mathematical simulations based on Fermian decay to demonstrate how temperature 5 can be controlled to minimise acrylamide without losing the desirable effects of a heat 6 process. Perhaps the most interesting finding from these model studies, however, was that 7 the levels of Maillard products (browning) do not necessarily align with acrylamide levels 8 Sadd and Hamlet 2005; Hamlet et al. 2005b); low moisture content is a more important 9 promoter of acrylamide than temperature, so crust moisture is a key factor controlling 10 acrylamide levels, and this accounts for much of the variation seen between different types 11 of cereal products. 12 13 Agronomical parameters 14 The determinant factor for acrylamide in cereal products is free asparagine. The free 15 asparagine content varies widely. There is not enough information available to determine a 16 clear trend: wheat has been reported at 69 to 443 mg kg -1 , while in rye the concentration ranged from 319-791 mg kg -1 (Amrein et al., 2004; Springer et al. 2003; Taeymans et al., 18 2004; Hamlet et al. 2005). . Investigations of Claus et al. (2006) (unpublished data) revealed 19 a significant impact of cultivar and fertilisation on acrylamide levels in bakery products due to 20 variations in the asparagine and crude protein contents of the raw material. A recent 21 publication (Muttucumaru et al. 2006) suggests that sulphate deficiency in soils causes 22 wheat to accumulate asparagine. The effects of sulphate were greater than varietal 23 differences.

57Figure 2 1 (

 21 Figure2shows the effect of recipe changes on the acrylamide content of wheat crackers

6

  react with asparagine (Claus et al. 2006).

7A7

  wide range of different processing techniques are used in the manufacture of 8 biscuits in order to arrive at the different shapes, textures and flavours typical of such 9 disparate products as shortbread, sandwich creams, crackers, soft cookies, digestives, 10 crackers, gingerbreads crispbreads etc. The influence of the different mixing and forming 11 operations on acrylamide levels in the finished product is far from understood. Some 12 multifactorial experiments carried out by Danone looking at the role of mixing time, pH and 13 fructose content at the dough mixing stage of a model short sweet biscuit recipe have shown 14 that these factors interact strongly to influence final acrylamide levels, but without a clear explanation for the magnitude or the direction of the effects. The work does highlight 16 however the impact which the condition of the dough during processing can have on 17 acrylamide production and perhaps suggests that the state of ingredients entering the mixer 18 -syrup, powder, solution -and the impact of physical factors such as work and temperature 19 on the dough are areas needing further study. That they do influence acrylamide formation is 20 probably one of the reasons why measured acrylamide in different batches of the same 21 finished product are found to vary so widely.22, Although changes in the oven baking profile resulted in significant reduction of 23 acrylamide in non-fermented crispbread, less positive results have been achieved with other 24 biscuit products. Most manufacturers who have optimized their oven process so as to 25 increase heat flux in zone 1 and/or decrease it in later zones to balance the overall heat flux, 26 have found that reductions in acrylamide are marginal (10-20%) and that they must also 27 accept an increase in product moisture and/or a lighter colour in order to achieve this 28 also must often be accompanied by adjustments to raising agents and dosing 1 points, dough forming modifications and extended baking times. Given the substantial 2 acrylamide variability between and within batches of biscuits, often +/-20%, these mitigation 3 measures based on changes to oven baking profile look weak.Fermented crispbread generally contains substantially less acrylamide than non-8 fermented crispbread, but each has it's own distinctive characteristics (CIAA, 2006b).

95

  Changes to the oven baking profile have been suggested as one possible route towards 10 reduction of acrylamide (CIAA Toolbox), and a recent survey carried out by the EU 11 Association of Chocolate, Biscuit and Confectionery Industries (CAOBISCO) assessed the 12 extent to which manufacturers have found success with this, as with other, possible 13 mitigation measures. In the case of non-fermented crispbread changes to the oven profile 14 have led to significant (25 -75%) reduction in acrylamide. 15 Mustafa et al. (2005) described that added asparagine had a significant effect on the 16 formation of acrylamide in rye crispbread, although surprisingly the addition of fructose did 17 not have an effect on the formation of acrylamide. Added oat-bran concentrate was added 18 for its water-binding capacity, and the enzyme lichenase was used to selectively degrade ß-19 glucan into fragments that were expected to influence viscosity and water-binding capacity, 20 but these did not influence acrylamide content. most commercial bread is produced by a rapid mechanical dough 26 development procedure, i.e. the Chorleywood baking process (Chamberlain et al. 1962) and 27 the levels of acrylamide formed in these breads are typically very low (Food Standards 28 . Hamlet et al. (2003; 2005b; 2006) investigated the factors contributing to the 1 low levels of acrylamide generated in UK commercial and domestic breads. In addition to a 2 dilution of acrylamide (formed exclusively in the crust) by the acrylamide free crumb, it was 3 shown that the Statutory addition of calcium (carbonate) to flour (see figure 7), yeast 4 fermentation (see figure 8) and oven profiling were the main controlling parameters. Extensive fermentation with yeast may be one possible way to reduce acrylamide 6 content in bread by reducing free asparagine (Fredriksson et al. 2004; Hamlet et al. 2005a, 7 b), an important precursor for acrylamide in cereal products. The content of free asparagine 8 was determined by Fredriksson (2004) in 11 milling fractions from wheat and rye. Whole

  9

  al. (2005) described that the addition of glycine during dough making significantly 1 reduced acrylamide in both flat breads and bread crusts. In bread crusts the reduction of 2 acrylamide ranged from 50 to >90%, depending on the baking condition. In flat bread the 3 reduction varied between 60 and >95%. However these reductions require relatively high 4 dosages of amino acids, e.g. 80-400 mmol kg -1 (Hamlet 2005b), and unpleasant odours can 5 occur at these levels of addition (Claus et al. 2006).

7

  the pH values in dough can decrease acrylamide (Claus et al. 2006; Hamlet et al 2005b).

14

  Surdyk et al. (2004) studied the effects of asparagine, fructose and baking conditions 15 on acrylamide content in yeast-leavened wheat bread. More than 99% of the acrylamide was 16 found in the crust. Added asparagine dramatically increased the content of acrylamide in 17 crusts dry matter, while added fructose did not influence the content. The effects of 18 temperature and time of baking were studied in another experiment. Mainly temperature 19 (above 200ºC) but also time increased the acrylamide content in crust dry matter, and a 20 significant interaction was seen between these two factors. When baked at different 21 conditions with the same ingredients, a highly significant relationship between colour and 22 acrylamide content in crust was found. Added asparagine, however, did not increase colour. 23 Time and temperature parameters are strongly affecting acrylamide formation. In 24 order to obtain products that are low in acrylamide, while still being acceptable to the 25 consumer, reduced temperatures and prolonged baking times should be given priority (Claus 180ºC, probably due to polymerisation (Claus et al. 2006) and 1 volatilisation.

7

  Compared to the situation in the deck oven, heat transfer is accelerated in the convection 8 oven, which results in faster drying of the product surface and thus in facilitating acrylamide 9 formation. Therefore, acrylamide levels in bread baked in a convection oven are usually 10 higher than those in bread originating from conventional baking processes (Claus et al.112006). In model systems with cakes of wholemeal wheat, and wholemeal rye, it was 12 concluded that acrylamide production was maximized between 25 and 30 minutes, followed 13 by a slow linear reduction. Acrylamide did not occur to a large degree until the moisture 14 contents of the cakes fell below 5% (Elmore et al.2005).

15 • Breakfast Cereals 16 17

 16 Glycine can reduce the acrylamide content of a breakfast cereal although at higher 18 concentrations a very bitter taste develops (Lagrange J. and Vickers C., 2006 personal 19 communication). Acceptable tasting flakes could be made with a reduction of up to half or 20 three quarters of acrylamide. However the toaster operation had to be radically altered. It is 21 known that glycine gives rise to the carcinogen formaldehyde on heating (Low et al. 2006

Food

  shows the effect of glycine 25 addition on acrylamide formation. 26 [Insert figure 3 about here] 27 Page 12 of 43 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk made on a pilot scale, those with more than 3g glycine per kilo dry 1 weight of ingredients were unacceptably bitter.

8

  However, figure4makes the point that wheat stands out from the other common grains. It 9 prompts the suggestion that if the agronomists could develop wheat that contained no more 10 asparagine than rice then more would be achieved than by any other measure presently 11 available. It is certainly desirable that plant breeders monitor new varieties in development to 12 assure that they do not inadvertently increase their asparagine content. 13 Fredriksson et al. (2004) have reported that the germ and the bran of wheat contain 14 more asparagine than the endosperm. Thus the use of refined flour would be expected to 15 yield less acrylamide than wholemeal flour. Industrial experience in this area indicates the 16 difficulty of writing general rules about recipe and process. One breakfast cereal 17 manufacturer recently adapted its extrusion-puff recipes to contain up to 25% wholegrain 18 wheat. The change was assessed for potential impact on acrylamide formation. The results19in table3show that wholemeal does not in all circumstances give rise to more acrylamide.20Results were also provided by a manufacturer who had experimented with wholegrain 21 biscuits; in this case wholemeal does give rise to more acrylamide. The difference may arise 22 from the fact that the mode of extrusion used creates gelatinised starch but with relatively 23 low thermal input and there is little maillard reaction. Such products are very pale unless 24 cocoa or one of the permitted yellow or brown colours is used. In contrast the biscuit was 25 made by a high temperature short time process that generates a significant brown maillard

5

  then toasting after the dough is formed into a flake or other form. Several fundamentally 6 different toasting processes are in use e.g baking ovens, rotary toasters and fluidised bed 7 systems. Observations made by Cereal Partners Worldwide indicate that although most 8 acrylamide forms in toasting, there can be measurable amounts (50-100 µg kg -1 ) formed in 9 at moisture content from 10% to the low 30s%. It should not be assumed that acrylamide 10 only forms at very low moisture content. That said, there is no doubt that most acrylamide 11 forms in the toaster. Manufacturers normally control the process to deliver a uniform and 12 tightly defined target colour. Too light a colour is associated with a higher moisture content, 13 weak or raw taste and shorter storage life. Too dark a colour is associated with complaints of 14 over-baked taste and, of course with too low a moisture content and reduced yield.

1 2 9 ••••

 19 Modifications of conventional technologies and the use of novel ingredients may alter the 3 sensory properties of products. Consumers are quick to pick up changes in a well-liked 4 foodstuff and generally reject and complain about it. Furthermore, changes to production 5 lines may be associated with higher costs or with loss of capacity such that manufacturers 6 run out-of-stock. Simple reduction of toasting/baking temperature generally reduces 7 capacity. Therefore the ideal changes to a recipe from a manufacturing perspective would 8 be: Availability of grains with little or no asparagine content 10 The ability to destroy asparagine through a simple addition 11 The ability to destroy acrylamide or one of its precursors by a simple ingredient now recognise the need to include the so called "risk / risk" 17 and "risk / benefit" analyses in potential mitigation strategies for acrylamide. These strategies 18 need to consider whether inventions to reduce the risk of acrylamide might increase the risk 19 of other process contaminants, e.g. furan or 3-monochloropropane-1,2-diol (3-MCPD), as 20 well as the impact on health benefits associated with nutrition. The Process-Related21Compounds Task Force of The International Life Science Institute (ILSI) started a new Impact of mitigation on the possible formation of other of other process contaminants.

1 2(

 1 While strategies to reduce all process-derived contaminants based on temperature 3 moderation are well defined (Arrhenius temperature dependence), i.e. reducing temperature 4 will reduce acrylamide, the impact of recipe interventions on all known contaminants is not 5 clear. For example, investigations have shown that extended yeast fermentation 6 Frederickson 2004; Hamlet et al. 2005a), pH reduction (Hamlet et al. 2005b) and the 7 addition of sodium chloride or glycerol (FEI / BLL 2005) can individually reduce acrylamide 8 formation in baked cereal products. However, all of these interventions can also lead to 9 significant increases in the generation of the process contaminant 3-MCPD in cereal 10 products (Hamlet et al. 2004; Hamlet et al. 2005c). An example of the effects of changing 11 dough pH on both acrylamide and 3-MCPD can be seen in figure 6.

  that asparagine is naturally concentrated in the bran and the germ of 17 wheat(Taeymans et al. 2004). One might be concerned that this could give rise to increased 18 levels of acrylamide in wholegrain or bran enriched products. However, there is much science that testifies to the health benefits associated with these products e.g. from cereal 20 antioxidants and fibre. Many epidemiological studies demonstrate that diets richer in 21 wholegrain or fibre are associated with benefits such as reduced risk of cardiovascular 22 disease, type 2 diabetes and overweight (Slavin 2004, Pereira et al. 2004). Intervention 23 studies are in progress to identify the causality of the effects and work such as the European 24 Healtgrain projects (www.healthgrain.org) seeks to identify the specific chemical entities in 25 bran and germ that drive the benefit. Careful consideration of the evidence for a significant 26 overall benefit of fibre and wholegrain is needed should we consider moving to more refined flour does not invariably lead to a higher acrylamide 1 concentration. Statutory (Statutory Instrument (SI 1998)) or additional fortification of flours 2 with sodium chloride (FEI / BLL 2005), divalent metal ions (Hamlet et al. 2005b) and the 3 replacement of ammonium raising agents by sodium raising agents[START_REF] Amrein | Acrylamide in 2 gingerbread: critical factors for formation and possible ways for reduction[END_REF]

about here] 10 •

 10 Impact on Maillard reaction products (MRP).

11 12

 11 In addition to the colours, tastes, and odours desired by producers and consumers of 13 cereal products alike, other positive effects such as antioxidant activity (AOA) have also 14 been attributed to MRPs. Some of these MRPs have beneficial effects on human health 15 (Lindenmeier et al. 2002, Nicoli et al. 1997, Yen et al. 2005) as well as contributing to the 16 shelf life of cooked products (Manzocco et al. 2000). Consequently strategies to reduce 17 acrylamide formation by temperature and recipe modifications e.g. limiting reducing sugars, 18 can impact significantly on AOA (Suma et al. 2006).

5

  to reduce acrylamide in cereals products is to reduce the 26 amount of asparagine that can react with reducing sugars. While investigations into the use is known about the factors that affect the natural variations in asparagine 1 levels, both within and between cereal cultivars(Hamlet et al. 2005a). As we approach the 2 fifth crop year since the acrylamide issue, asparagine data for varieties of wheat, mill 3 fractions and other grains is still sparse.4Future outlook. More analytical effort is required to understand the apparent seasonal 6 variation in asparagine, the effect of milling interactions and the impact of environmental 7 factors on asparagine levels. There is a need to develop low asparagine cereal varieties that 8 retain the desired yield, milling and baking characteristics. Consequently, breeding programs 9 are needed, including the application of marker assisted breeding, which, in some markets, 10 will raise an interesting debate concerning acrylamide and genetically modified organisms.11An early dialogue with all crop breeders in conjunction with DG Agriculture should also be 12 established to ensure that accidental increases in asparagine levels do not result from 13 current breeding programmes.

18 19 1

 181 Bakers yeast (Saccharomyces cerevisciae) can selectively consume asparagine and trials 20 with model breads have shown reductions of up to 80% for a proof time of 60 min (Hamlet et 21 al. 2005b). Investigations with yeast variants have shown that: a) asparagine consumption is 22 proportional to yeast addition level; and b) the rate of consumption of asparagine is 23 equivalent for both yeast low and high gassing yeasts (see figure 8). Consequently it should 24 be possible to optimise for both yeast gassing and asparagine reduction and this behaviour 25 should be explored further with yeast manufacturers. 26 [insert figure 8 about here] Removal of asparagine by the degradation of the amide moiety can be accomplished by the 2 use of the enzyme asparaginase, which catalyses the hydrolysis of Asn to asparatic acid . In 3 fact, L-asparaginase is widely used for the treatment of leukaemia (Friedman et al. 2003) 4 and its therapeutic basis exploits the greater dependence of the malignant cells on an 5 exogenous source of Asn compared to normal cells. Zyzac et al. (2003) demonstrated that 6 asparaginase pre-treatment of a potato product could produce an Asn reduction of 88%, and 7 a corresponding acrylamide reduction of greater that 99% in the cooked material. Although 8 laboratory trials have been encouraging for bakery products, commercial scale trials are 9 required to demonstrate the effectiveness of asparaginases, particularly in low moisture 10 products such as biscuits types. Since one of the known minor side effects of clinical 11 asparaginase treatment is allergenicity, an evaluation of the safety of treated foods will be 12 required. Commercial food grade asparaginases are expected to be available shortly 13

16 17

 16 Process factors: From laboratory trials to commercial production.18Ways to reduce acrylamide are often demonstrated on a laboratory scale, but they also need 19 to be feasible in a commercial environment.

20

  Sadd and Hamlet (2005) demonstrated that acrylamide formation is very sensitive to 21 small changes in moisture, particularly at the lower range of moisture contents. Furthermore, 22 changes to mitigate for acrylamide e.g. increasing moisture and decreasing temperature 23 were also favourable for reducing other processing contaminants such as a 3-MCPD (figure249). Although optimisation of moisture control can achieve a 15-20 % reduction in acrylamide 25 in laboratory trials, this does not necessarily carry over into an industrial production 26 environment where commercial ovens may not have the necessary degree of control.

  the potential to reduce asparagine in fermented dough, trials have 1 shown that this does not necessarily equate to less acrylamide in the cooked product. When 2 the yeast-fermented doughs were baked to generate acrylamide the corresponding reduction 3 in acrylamide was less than expected due to the release of fructose by yeast early in the 4 fermentation cycle (Hamlet et al. 2005a). Furthermore the addition of bakery fat also reduced 5 the effectiveness of asparagine removal by yeast (Hamlet et al. 2005b) indicating that this 6application may not be effective in high fat products such as biscuits. Dough is complex and 7 dynamic system and further work will be required to understand and optimise processes to 8

  16 CIAA, 2005, The CIAA acrylamide toolbox. Brussels: Confederation of the food and drink 17 industries of the EU (CIAA).

  18 CIAA, 2006a, The CIAA acrylamide toolbox. Brussels: Confederation of the food and drink 19 industries of the EU (CIAA).

20 1 and

 1 CIAA 2006b, The CIAA acrylamide toolbox for the reduction of acrylamide in biscuits, 21 crackers & crispbreads. Brussels: Confederation of the food and drink industries of 22 the EU (CIAA). 23 Claus, A., Carle, R., and Schieber, A., 2006, Reducing acrylamide in bakery products. New 24 Food, 2, 10-14. 25 Corradini, M.G., Peleg, M., 2006, Linear and non-linear kinetics in the synthesis and 26 degradation of acrylamide in foods and model systems. Crit. Rev. Food Science. And . G., and Sadd, P. A., 2003, Acrylamide generation in bread and toast. Report No 21 C005 compiled for the Federation of Bakers. High Wycombe, UK: RHM Technology. 22 Hamlet, C. G., Sadd, P. A., and Gray, D. A., 2004, Generation of monochloropropanediols 23 (MCPDs) in model dough systems. 1. Leavened doughs. Journal of Agricultural and 24 Food Chemistry, 52, 2059-2066. 25 Hamlet, C. G., Sadd, P. A., and Liang, L., 2005a, Wheat variation, proving and acrylamide . G., Baxter, D. E., Sadd, P. A., Slaiding, I., Liang, L., Muller, R., Jayaratne, S. M., Booer, C., 2005b, Exploiting process factors to reduce acrylamide in cereal-2 based foods: project codes C03032 and C03026. Report No C014 prepared for the 3 UK Food Standards Agency. High Wycombe, UK: RHM Technology. 4 Hamlet, C. G., and Sadd, P. A., 2005c, Effects of yeast stress and pH on 3-MCPD producing 5 reactions in model dough systems. Food Additives and Contaminants, 22, 616-623. 6 Hamlet, C. G., Jayaratne, S. M., and Morrison, C., 2006, Processing contaminants in bread Lindenmeier, M., Faist, V., and Hofmann, T., 2002, Structural and functional 16 characterization of pronyl-lysine, a novel protein modification in bread crust 17 melanoidins showing in vitro antioxidative and phase I/II enzyme. Journal of 18 Agricultural and Food Chemistry, 50, 6997-7006.

Figure 1 .Figure 2 .Figure 4 .

 124 Figure 1. Minimum reaction scheme used to model acrylamide formation a bakery products (adapted from Sadd and Hamlet 2005; Wedzicha et al. 2005) Figure 2. The effect of recipe changes on the acrylamide content of wheat crackers Figure 3. The effect of glycine on acrylamide formation. (Lagrange J. and Vickers C of Cereal Partners Worldwide) Figure 4. The acrylamide content of breakfast cereal made to a common recipe and process but with different grains. (The experiments were made by Besson A. and Byrne S. in trials by Cereal Partners Worldwide).

Figure 5 .

 5 Figure 5. The influence of oven temperature on formation of acrylamide in a baked cereal product. Points in red indicate that the product tasted raw; in blue that the baked taste was acceptable. (The experiments were made by Cereal Partners Worldwide in conjunction with Thistle Consulting and Prof. D. Mottram of the Department of Food Science ar Reading University).

Figure 6 .

 6 Figure 6. Effects of initial dough pH on acrylamide and 3-MCPD generation in wheat bread doughs.

Figure 7 .

 7 Figure 7. Effect of divalent metal ion additions on acrylamide generated in wheat bread doughs.

Figure 8 .

 8 Figure 8. Effects of high (control) and low gassing rate bakers yeasts on asn levels in yeasted wheat bread doughs.

Figure 9 .

 9 Figure 9. Effects of moisture and temperature on acrylamide (solid contours) and 3-MCPD (dashed contours) generated in wheat doughs. Data from: Sadd and Hamlet 2005; Hamlet et al.2004.

  25factories have observed significant changes in acrylamide content from season to season as 26 shown in table 1 and the CIAA status report 2004 (CIAA 2004).

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	6

the use of ammonium bicarbonate. Dutch spiced cake accounts for 22 16% of the total acrylamide exposure from foods for the whole population in the Netherlands 23 (Konings et al. 2003).

	1
	2	acrylamide by 70% in semi-finished biscuits at an industrial scale. The use of sucrose
	3	instead of inverted sugar syrup had a similar effect. Addition of extra tartaric acid reduced
		acrylamide by about one-third.
	10 11	Amrein et al. (2004) reported that ammonium bicarbonate strongly enhanced F o r
	12 13	acrylamide formation in gingerbread and that replacement with sodium hydrogencarbonate as baking agent reduced the acrylamide concentration by >60%. Free asparagine was a P
	14	limiting factor for acrylamide formation, but the acrylamide content could also be lowered by e
	15 16	replacing reducing sugars with sucrose or by adding organic acids. Acrylamide concentration and browning intensity both increased with baking time and correlated with each other. e r
		Results from monitoring activities of the Food and Consumer Product Safety R
		e
	20 21	information on the product label, ammonium bicarbonate is not used anymore in products analysed in 2006, while product labels of the same products in 2003, which were high in v i e
	w In the UK an on-going study (Hamlet et al. 2005) is looking at the relationship acrylamide, showed 24 [insert table 2 about here] 25 26 O n l
	27	between acrylamide in retail products and declared raising agents, and researching ways of y
	28	counteracting the negative effects of added ammonium.

  bake times. However, it requires an oven capable of such regulation and a 1 scientific capability probably beyond the reach of smaller businesses.

	problem of longer 2
	3	A summary of major papers on mitigation of acrylamide in cereals and cereal
	4	products since 2004 is given in table 4.
		[insert table 4 about here]
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
	26	would n O l
		reduce factory output to a third of normal. A more sophisticated approach reducing y
		15

Table 1 :

 1 The effect of the seasons on acrylamide levels of two breakfast cereals made by different processes but using the same wheat supply. Application of the t-test to the pooled data confirms that 2004 is different from 2003 and 2005 (P<0.05).

	19
	20	Lineback, D., Wenzl, T., Ostermann O.P., de la Call, B., Anklam, E., and Taeymans, D.,
	21	2005, Overview of acrylamide monitoring databases. Journal of AOAC International,
	22	88, 1, 246-252.
	23	Low, M.Y., Koutsidis, G., Parker, J.K., Elmore, J.S., Dodson, A.T., and Mottram, D.S., 2006,
	24	Effect of citric acid and glycine addition on acrylamide and flavor in a potato model
		system. Journal of Agricultural and Food Chemistry, 54, 5976-5983.

Table 2 .

 2 Monitoring results of acrylamide in Dutch spiced cake available on the Dutch

	market.
	Year	Number of samples	Acrylamide µg kg-1	Acrylamide µg kg-1
			Mean	Range
	2002	13	1018	260-1410
	2003	10	796	300-1480
	2006	15	353	271-521

Table 3 .

 3 Effect of adding wholegrain to recipes. The number analysed refers to the number of trials with distinct batches of raw materials. (The results for breakfast cereal are from trials made by Cereal Partners Worldwide. Those for biscuit were provided by a biscuit manufacturer).

	Page 29 of 43		Food Additives and Contaminants
		Wholegrain	Acrylamide	Standard deviation	Number analysed
		%	ug kg -1	ug kg -1
	Cereal 1	0	121	24	8
	Cereal 1	25	114	25	7
	Cereal 2	0	69	1	5
	Cereal 2	12	44	7	6
	Biscuit	-	75 -302		4
	Biscuit	+	1144 -1275		4

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Table 4

 4 Summary of major papers on mitigation of acrylamide (AA) in cereals and cereal products since 2004. NaHCO 3 eliminated acrylamide. To lesser extent, NH 4 HCO 3 , cysteine, sodium bisulfite and ascorbate enhanced elimination. Some ingredients: citric acid, ferulic acid, and NaCl were found to decrease AA produced but had no effect on elimination.

	Application	Outcome

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[insert table 1 about here]

Vass et al.(2004) concluded that the application of an asparaginase during dough 18 preparation can reduce the acrylamide content of cracker products by at least 70%. The

Dutch spiced cake available on the Dutch market in 2006 (table 2). According to the

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etal. 2006). However, acrylamide is subject to partial degradation when the surface

(lower bran) flours to reduce acrylamide. Further work such as that reported in table3

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• Asparaginase.

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lowering levels of acrylamide in food. Brussels: Confederation of the food and drink

Nutrition., 46 (6): 489-517.

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Friedman, M., 2003, Chemistry, biochemistry, and safety of acrylamide: a review, Journal of

Graf , M., Amrein, T.M., Graf, S., Szalay, R., Escher, F., and Amadò, R. 2006. Reducing the

acrylamide content of a semi-finished biscuit on industrial scale., LWT Food Science

formation in bread. In: R. E. Poms and S. P. Cauvain, eds., Cereals -the future

challenge, ICC conference, 3-6 July 2005. Vienna: ICC, p. 87.

Model. Journal of Agricultural and Food Chemistry, 53, 4410-4416.

Manzocco, l., Calligaris, S., Mastrocola, D., Nicoli, M. C., and Lerici, C. R., 2000, Review of 26 non-enzymatic browning and antioxidant capacity in processed foods. Trends in

Sadd, P. A., and Hamlet, C. G., 2005, The formation of acrylamide in UK cereal products.

Stadler, R. H., 2005, Acrylamide formation in different foods and potential strategies for

O'brien, J., Thompson, S., Silvani, D., and Whitmore, T., 2004, A Review of

. Medcine and. Biology., 561: 235-53.

Authority (VWA) in the Netherlands showed a decrease (p<0.05) in acrylamide levels of Agricultural and Food Chemistry. 52, 4282-4288.

4

Biedermann, M., and Grob, K., 2003, Model studies on acrylamide formation in potato, 5 wheat flour and corn starch; ways to reduce acrylamide contents in bakery ware.

6

Mitteilungen Gebiete Lebensmittel Hygiene, 94, 406-422.

7

Bråthen, E., Kita, A., Knutsen, S. H., and Wicklund, T., 2005, Addition of glycine reduces the Taeymans, D., Wood, J., Ashby, P., Blank, I., Studer, A., Stadler, R., Gondé, P., Eijck, P., 9 Lalljie, S., Lingnert, H., Lindblom, M., Matissek, R., Müller, D., Tallmadge, D.,