Acrylamide in relation to asparagine in baked and toasted wheat and rye bread. Kit Granby, Nikoline Juul Nielsen, Rikke V Hedegaard, Tue Christensen, Mette Kann, Leif H. Skibsted
To cite this version:
Kit Granby, Nikoline Juul Nielsen, Rikke V Hedegaard, Tue Christensen, Mette Kann, et al.. Acry- lamide in relation to asparagine in baked and toasted wheat and rye bread.. Food Additives and Contaminants, 2009, 25 (08), pp.921-929. 10.1080/02652030801958905. hal-00577380
HAL Id: hal-00577380 https://hal.archives-ouvertes.fr/hal-00577380 Submitted on 17 Mar 2011
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Acrylamide in relation to asparagine in baked and toasted wheat and rye bread.
Journal: Food Additives and Contaminants
Manuscript ID: TFAC-2007-450.R1
Manuscript Type: Original Research Paper
Date Submitted by the 23-Jan-2008 Author:
Complete List of Authors: Granby, Kit; Technical University of Denmark, National food Institute Nielsen, Nikoline; University of Copenhagen, Faculty of Life Sciences Hedegaard, Rikke; Technical University of Denmark, National food Institute Christensen, Tue; Tecnical University of Denmark, National Food Institute Kann, Mette; Läntmannen Schulstad A/S Skibsted, Leif; University of Copenhagen, Faculty of Life Sciences
Methods/Techniques: LC/MS
Additives/Contaminants: Acrylamide
Food Types: Cereals
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1 2 3 1 4 2 Acrylamide in relation to asparagine in baked and toasted wheat and rye bread. 5 6 3 7 4 Abstract 8 Deleted: s 9 5 Acrylamide in baked and toasted wheat and rye bread was studied in relation to the contents of 10 11 6 asparagine in flour, dough, bread and toasts. The asparagine was consumed during bread 12 Deleted: 88 13 7 preparation resulting in a reduced acrylamide contents in the products. In wheat bread 12 % of the Deleted: ost 14 -1 15 8 asparagine initially present in the flour (0.14g kg ) was l eft after yeast fermentation and baking, Deleted: during 16 For Peer Review Only Deleted: 1 9 while for rye bread 82% of the asparagine was l eft after sourdough fermentation and baking. The 17 Deleted: ost during 18 10 asparagine present in the untoasted wheat bread had all reacted after hard toasting. Toasted wheat 19 20 11 and rye bread slices contained 11-161 µg kg -1 and 27-205 µg kg -1 acrylamide compared to untoasted 21 22 12 wheat and rye bread containing <5 µg kg -1 and 7-23µg kg -1 acrylamide. The dietary intake of 23 24 13 acrylamide from bread (untoasted) of 2 µg/day is relatively low, however the acrylamide exposure 25 26 14 from bread increases several times for people eating toasted bread. 27 28 15 Keywords: Acrylamide, asparagine, bread, rye, wheat, toast, dietary, intake 29 30 16 31 32 17 Introduction 33 34 35 18 The presence of acrylamide in heat treated food was recognized in 2002 (Tareke et al. 2002) and the 36 37 19 same year it was clear that acrylamide is formed mainly from asparagine and a carbonyl compound 38 39 20 through Maillard browning reactions during food processing (Mottram et al. 2002; Stadler et al. 40 21 2002). Several pathways and intermediates for acrylamide formation have been suggested including 41 42 22 the intermediates: Schiffs bases, decarboxylated Amadori products (Yaylayan 2003), Strecker 43 44 23 aldehydes (Mottram et al. 2002) and the deamination product 3-aminopropionamide which also 45 46 24 generate acrylamide under aqueous conditions (Granvogl and Schieberle, 2006). Besides the main 47 48 25 reaction routes through the Maillard reactions, acrylamide may be formed in bread after protein 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 26 pyrolysis of added gluten (Claus et al. 2006). Also addition of cheese to bread has been found to 4 5 27 increase the acrylamide formation, probably formed with 3-aminopropionamide as an intermediate 6 7 28 in the cheese, which has been found to generate acrylamide (Granvogl and Schieberle 2006, 8 Deleted: 7a 9 29 Hedegaard et al. 200 8). 10 11 12 30 Acrylamide is known to be carcinogenic in rodents and is classified by the International Agency for 13 31 Research on Cancer as probably carcinogenic to humans (IARC 1994). The dietary acrylamide 14 15 32 intake have been estimated to e.g. 0.3-0.8µg (kg bw) -1 day -1 (WHO 2002); appr. 0.5µg (kg bw) -1 16 For Peer Review Only 17 33 day -1(Svensson et al 2003). The dietary intake of acrylamide from bread is about 11% of the total 18 19 34 dietary acrylamide intake of 31µg day -1 as estimated for the Swedish population. A similar 20 21 35 contribution from bread related foods such as crisp bread and biscuits are estimated (Svensson et al. 22 23 36 2003). The acrylamide level in baked bread is relatively low, but due to high consumption of bread 24 25 37 the contribution of acrylamide from bread is still important. 26 27 28 38 Toasting the bread, which is frequently used e.g. for breakfast meals, increases acrylamide 29 -1 30 39 significantly. While untoasted bread contains <50 µg kg the acrylamide content may increase to a 31 32 40 few to several hundreds micrograms per kilo bread after toasting (Konings et al. 2003; Becalski et 33 34 41 al. 2003; Ahn et al. 2002). The acrylamide levels have been found to increase with increasing 35 36 42 toasting time and whole meal rye bread was found to form more acrylamide than white wheat bread 37 43 (Ahn et al. 2002). 38 39 44 The occurrence of acrylamide in bread may depend on several factors. Surdyk et al. (2004) showed 40 41 45 that acrylamide in baked wheat bread correlated with the amount of asparagine added to dough, 42 43 46 while the added reducing sugar fructose did not show a similar correlation, hence the asparagine 44 45 47 was a limiting substrate for the acrylamide formation. The clear relationship between asparagine 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 48 content in the dough and acrylamide formation has also been demonstrated in other wheat and rye 4 5 49 bread model systems (Elmore et al. 2005; Bråthen et al. 2005). 6 7 50 One of the strategies to reduce acrylamide in cereal products may be to choose raw materials low in 8 9 51 asparagine by selecting optimum cultivars and growth conditions. However, variations in free 10 11 52 asparagine in cereals are modestly researched. Claus et al. 2006 investigated agronomic factors 12 13 53 related to the acrylamide formation in cereal products. They showed that nitrogen fertilization 14 Comment [k1]: Left out to shorten 54 resulted in elevated amino acid levels resulting in increased acrylamide levels. The asparagine the introduction 15 Deleted: Favorable light and 16 For Peer Review Only temperature conditions one year gave -1 55 contents of selected European wheat varieties ranged from 0.07-0.66 g kg , that is a factor of five higher acrylamide in bread compared to 17 another year probably by enhancing 18 amino acid and protein contents. Flour 56 between varieties and a factor of two within varieties (Taeymans et al. 2004). derived from sulphur deprived wheat was 19 also found to contain high levels of 20 asparagine (Muttucumaru et al. 2006, 57 Milling will differentiate the asparagine levels in the different milling fractions. Fredriksson et al. Granvogl & Schieberle 2007), so it is 21 important that the cereals are grown at a sufficient sulfur level 22 58 (2004) found that the contents of free asparagine are lower in sifted wheat flour i.e. 0.14- 0.17 g kg- 23 Deleted: . 24 59 1(dw) compared to whole grain wheat flour i.e. 0.5 g kg -1(dw) or wheat bran. Rye generally contains 25 26 60 more asparagine: sifted rye flour 0.6 g kg -1(dw) and whole grain rye flour: 1.1 g kg -1(dw). Not only 27 28 61 milling has been found to influence the levels of the limiting substrate asparagine. It was found that 29 30 62 yeast fermentation of wheat dough depleted a major part of the asparagine (>80%), while 31 32 63 sourdough fermentation of rye dough reduced the asparagine content to a smaller extent 33 34 64 (Fredriksson et al. 2004). During baking, parameters such as water activity, heating time and 35 36 65 temperature, surface to volume ratio, the presence of additives and pH also influence acrylamide 37 38 66 levels (Surdyk et al. 2004; Rydberg et al. 2003). Acrylamide formation may increase when 39 40 67 increasing pH e.g. by using baking soda or ammonium carbonate for bread leavening (Amrein et al. 41 42 68 2005) as it will decrease when lowering pH (Jung et al. 2003; Pedreschi et al. 2004, 2005). 43 69 Fermentation will cause pH in dough to drop due to release of carbon dioxide (Raccach et al. 2004) 44 45 70 and it will reach pH~5 using yeast and pH~4.4-4.8 using sourdough (Suhr et al. 2004). 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 71 The aim of the present study was to find the relationship between asparagine levels and acrylamide 4 5 72 formation in wheat bread and rye bread during baking and toasting in order to be able to reduce the 6 7 73 acrylamide levels. Furthermore to assess the acrylamide levels in bread on the Danish market and to 8 9 74 estimate the dietary intake af acrylamide from wheat and rye bread including toasted bread. 10 11 75 12 13 76 Materials and methods 14 15 77 16 For Peer Review Only 78 Baking wheat bread and rye bread with asparagine additions 17 18 79 An industrial bakery (Lantmännen Schulstad A/S, Hvidovre, Denmark) made the wheat and rye 19 20 80 breads in their laboratory bakery with different amounts of aparagine added (in August 2004). The 21 22 81 experimental design appear s from Figure 1. Four batches of wheat dough were prepared and two 23 24 82 breads selected from each batch: One batch of dough was made without adding asparagine 25 26 83 representing the natural background asparagine level and three batches were added asparagine at 27 28 84 concentration levels of 0.05, 0.13 and 0.27 g kg -1 dough. As the dough contains 60% wheat flour, 29 30 85 the added amounts of asparagine based on flour were 0.09, 0.22 and 0.44 g kg -1 corresponding to 31 32 86 addition at approximately 0.6 - 3 times the actual background level of asparagine. The recipe for a 33 34 87 wheat bread (appr. 800 g dough) included sifted wheat flour, yeast, salt, sugar vegetable oil and 35 36 88 water. Kneading time was 7 min., holding time 20 min. and resting time at 32 °C was 45 min. 37 38 89 Baking was performed initially at 230 °C for 2 min. and subsequently at 200 °C for 30 min. 39 40 90 Rye breads were made using the same experimental design. The asparagine was added to the dough 41 -1 42 91 at concentrations based on rye flour of 0.17, 0.43 and 1.75 g kg respectively corresponding to 43 Deleted: 3 44 92 addition at 0. 2 - 2 times the natural background level. The rye bread (based on appr. 900 g dough) 45 46 93 was made from half and half mixing of sourdough and rye flour and addition of yeast, salt and 47 48 94 water. Kneading time was 10 min, holding time 20 min, and resting time at 37 °C was 77 min. 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 95 Baking was performed initially at 280 °C for 5 min., followed by 210 °C for 35 min. and 190 °C for 4 5 96 12 min. 6 7 97 8 9 98 Toasting wheat bread and rye bread with asparagine additions 10 11 99 Toasting of the bread slices was done in a pushdown toaster (Tefal Delfini Compact Toaster 5396, 12 13100 800 W) with room for two slices. The four batches of bread (background level and the three 14 15101 addition levels) were analyzed either untoasted or after medium or hard toasting, for each level 16 For Peer Review Only 17102 using three slices from each of two breads (n=18). Medium toasting of wheat bread slices of 13 mm 18 Deleted: at level 4 19103 was performed 1.9 min. and hard toasting 2.8 min.. Medium toasting of the rye bread slices of 8 Deleted: at level 6 20 Deleted: at level 6 104 mm was done 2.8 min. and the hard toasting 3.1 min.. All the toasted bread slices were medium to 21 Deleted: at level 7 22 105 dark brown and were considered eatable. The thicknesses of the bread slices were chosen from what 23 24106 is common practice among bakers and consumers. 25
26107 Deleted: ¶ 27108 Figure 1 28109 Deleted: ¶ 29110 30 Deleted: Baking small wheat breads (baguettes): ¶ 31111 The industrial bakery produced small 32 wheat breads (baguettes) in their laboratory bakery. The recipe for the 33112 Chemicals wheat breads (appr. 3000 g dough ~ 60 breads) included sifted wheat flour, yeast, 34 salt, sucrose, margarine and water. 35113 Acrylamide (>99.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA), asparagine Kneading time was 3+5 min., holding 36 time 20+10 min., and resting time at 114 monohydrate (>99%) from Acros Organics (Geel, Belgium), D -acrylamide (>98%) from Polymer 37 °C was 50 min. Baking was performed 37 3 at 220 °C for 10 min. or 15 min. in a 38 ventilated oven and with water spray 115 Source (Dorval, Quebec, Canada), 15 N -asparagine monohydrate (>98%) from Cambridge Isotope added for 30 s..¶ 39 2 40 116 Labs. (MA, USA), methanol (>98%), formic acid (>98%) and acetonitrile (HPLC-grade) from 41 42117 Rathburn Chemicals (Walkerburn, UK). 43 44118 45 46119 Chemical analysis 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 120 The chemical analyses were performed using a recently developed LC-MS/MS method for 4 5 121 simultaneous analysis of acrylamide, asparagine and reducing sugars (Nielsen et al. 2005) and in 6 7 122 addition some of the acrylamide analyses were made with another LC-MS/MS method as described 8 9 123 in Pedreschi et al. (2005). 10 11124 Sliced toasted or untoasted wheat or rye bread was weighed, dried in an oven at 50 °C over night 12 13125 and homogenized using a Braun handheld mixer (type 4169) fitted with a blender like sample 14 15126 compartment (type 4297) (Braun AG, Frankfurt/m, Germany). The drying temperature was set 16 For Peer Review Only 17127 relatively low in order to avoid acrylamide formation during the drying process. The wet (semidry) 18 19128 and dry weight was determined from weighing before and after drying of the baked and toasted 20 129 bread slices. Aliquots of 4 g of homogenate was extracted with MilliQ water after adding 21 Deleted: 22 -1 -1 15 130 internalstandards: 200 µL 10 µg mL D -acrylamide, 100 µL 880 µg mL N -asparagine. The Deleted: ppm 23 3 2 Formatted 24131 sample was extracted by an Ultra Turrax mixer. After centrifuging, SPE cleanup was performed by 25 Deleted: ppm Deleted: 26132 an automated sampler using LiChroLut RP-C18 SPE-cartridges LC-MS/MS detection was 27 28133 performed on a triple quadrupole instrument (Micromass Quattro Ultima) after separation on a 29 30134 hypercarb LC-column (dimensions 2.1 mm x 100 mm, particle size 5 µm) eluted with 0.1% 31 32135 aqueous formic acid at 0.2 mL min -1. The mass spectrometer was operated in ESI + for acrylamide 33 34136 and saccharides and in ESI - for asparagine. Capillary voltages of 3 kV (ESI +) and 2 kV (ESI -) were 35 36137 applied but for simultaneous detection in positive and negative mode a lower capillary voltage, 37 38138 e.g.1 kV is recommended to prevent sparkling from the capillary. 39 40139 Some of the acrylamide analysis was performed after the methods were optimized to detect low 41 42140 concentrations (e.g. by increasing the HPLC injection volume and by optimizing the MS 43 44141 performance before analysing the bread samples), so the method was able to quantify down to 5 µg Formatted 45 -1 -1 46142 kg . The repeatability and reproducibility of the acrylamide analyses at 50 and 250 µg kg (n=24) 47 48143 were 6-17% and 9-29% respectively. The laboratory has participated in proficiency tests for 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 144 acrylamide in food and all results obtained have been with satisfactory test scores, including the 4 Deleted: recently held 5 145 results obtained in an EU validation study on acrylamide analyses in food with 22 samples (11 6 7 146 matrices including bread and toasted bread: assigned value 38µg kg -1, obtained results 41 and 44µg 8 -1 -1 9 147 kg ) (Wenzl et al. 2006). The detection limit of the asparagine analyses was 0.002 g kg , the 10 -1 11148 repeatability and reproducibility measured at 0.02, 0.1 and 0.4 g kg 0-5% and 10-18% repectively. 12 13149 14 15150 Estimation of dietary acrylamide intakes 16 For Peer Review Only 151 Exposure estimates were accomplished using data from the Danish Nationwide Dietary Survey 17 18 152 made from 2000-2002 by the National Food Institute (Lyhne et al. 2005). Information on food 19 20153 consumption was collected from a representative sample of 4120 individuals aged 4-75 years. The 21 22154 participants in the survey kept a food record for seven consecutive days. The monitoring results of 23 24155 acrylamide in Danish foods (appr. 330 samples) were made 2002-2005 by the National Food 25 Formatted 26156 Institute (data not published elsewhere). 27 28157 29 30158 Results and discussion 31 32159 33 34160 The levels of acrylamide and asparagine in wheat and rye bread slices after baking and medium or 35 36161 hard toasting appear from Table I. The results presented are all means of six bread slices, each 37 38162 wheat bread slice weighing 34 ±2 g and each rye bread slice weighing 32 ±1 g. 39 40163 41 42164 Asparagine in flour and bread slices after asparagine additions to dough and subsequent 43
44165 fermentation, baking and toasting
45 -1 46166 The background content of asparagine in sifted wheat flour was 0.14 ± 0.004 g kg (based on three 47 48167 determinations and with the precision measured as 95% confidence levels) and in whole grain rye 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 168 flour 0.76 ± 0.02 g kg -1, figures comparable to those reported by Frederiksson et al. (2004) of 0.14- 4 5 169 0.17 g kg -1 (dw) in sifted wheat flour and 1.1 g kg -1 (dw) in whole grain rye flour . 6 7 170 The levels of asparagine added to the wheat dough were 0.6, 1.6 and 3.1 times the natural 8 Deleted: 3 9 171 background level and to the rye dough 0. 2, 0.6 and 2 .3 times the natural background level. The Deleted: 1 10 11172 highest concentration after addition correspond to 0.6 g kg -1 asparagine in wheat flour and 2.5 g kg -1 12 13173 asparagine in rye flour; hence levels likely to be present in cereal products used for bread 14 15174 production. 16 For Peer Review Only 17175 In Table II the reacted amounts of asparagine in the batches with different asparagine levels upon 18 19176 fermentation, baking and toasting are presented. Some asparagine had reacted during wheat bread 20 177 production as can be seen from data determined on a dry weight basis (Table I), where differences 21 22 178 from evaporation of water during toasting does not have to be considered. In the wheat batch with a 23 Deleted: 88 24 179 natural background level of asparagine 12 % was left after fermentation and baking . Upon hard Deleted: had reacted during 25 Deleted: , and additional 1% had 26180 toasting all asparagine in the wheat bread slices with the natural background content and the lowest reacted during medium toasting while 27 additional 12% (until depletion) had reacted during hard toasting. At the 28181 addition level of asparagine had reacted. highest asparagine level added 51% had 29 reacted during fermentation and baking, additional 10% had reacted during 30182 medium toasting and additional 16% 31 during hard toasting. 32 Deleted: were depleted in asparagine. 183 Table I The findings provide evidence for 33 asparagine as the limiting factor in acrylamide formation in wheat bread, 34184 Table II especially in toasted wheat bread. 35 Deleted: ¶ 36185 Deleted: ¶ 37186 In rye bread the relative reduction in asparagine during sourdough fermentation and baking was less 38 39187 than for the yeast fermented wheat bread, e.g. 18% at the natural background level and 9% for the 40 41188 highest addition level of asparagine. As the results are based on subtracting two large percentages, 42 189 they may be more uncertain. The results are in accordance with those of Frederiksson et al. (2004), 43 44 190 who also found sourdough fermentation to reduce asparagine less than yeast fermentation and at 45 Deleted: ings 46191 about the same rat e i.e. >80% for yeast fermentation of wheat bread and 17% for sourdough 47 Deleted: o 48 192 ferm enta tion of rye . Deleted: a 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 193 A general conclusion may be that rye flour contains more asparagine than wheat flour and 4 5 194 sourdough fermentation and baking of rye dough reduces asparagine to a smaller extent than yeast 6 7 195 fermentation and baking of wheat dough. Prolonging or optimizing the fermentation process, 8 9 196 especially the yeast fermentation of wheat bread may be an efficient tool to reduce the acrylamide 10 11197 formation in bread products. 12 13198 Deleted: ¶ 199 ¶ 14200 Acrylamide in baked and toasted bread in relation to asparagine 15 16201 Baked wheat bread slicesFor contained <5Peer µg kg -1 acrylamide Review ( http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 10 of 42 1 2 Deleted: sourdough 2 3 218 content of asparagine in the rye flour and acrylamide content in bread after medium (r = 0. 86 ) or Deleted: 91 4 Deleted: 5 5 219 hard (r 2 = 0.8 8) toasting appear (Figure 2b). 6 Deleted: In the initial setup of the experiment, the natural asparagine level 7 220 in rye was estimated too low, why the 8 two lowest addition levels are close to the background level i.e. at only 1.4 and 1.6 9 221 times the actual asparagine background 10 level in flour. For untoasted rye bread the asparagine additions all showed 11222 significantly higher acrylamide content compared to the background (p < 0.05) 12 but the correlation between asparagine 13223 In the present study, a major part of the asparagine added to the wheat dough had reacted during addition and acrylamide in baked bread is 2 14 weaker (r = 0.45). The medium and hard 224 preparation of the bread. It is known, that especially yeast fermentation promote elimination of toasted rye bread were significantly 15 higher in acrylamide for all asparagine 16 For Peer Review Only additions (p < 0.005). 225 asparagine, and that the reduction depends on e.g. time of fermentation, the amount of yeast added 17 18 226 and pH (Frederiksson et al. 2004). When comparing the asparagine levels left in baked bread before Deleted: formed 19 Deleted: Acrylamide content in baked 20 bread was found to be proportional to the 227 toasting with the acrylamide content in toasted bread slices good linear relationships are present for asparagine content in dough and the 21 acrylamide content in toasted bread was 22228 both medium toasted (r 2 = 0.87) and hard toasted (r 2 = 0.94) wheat bread and for asparagine levels likewise proportional to the asparagine 23 content in bread before toasting. However Deleted: is different 24229 of 0.017-0.29 g kg -1 (dw) bread. The asparagine levels in baked rye bread also showed good linear Deleted: In wheat and rye bread toasted 25 for 3.1 min., representing the hard toasted 26230 correlations with acrylamide formed after medium toasting (r 2 = 0.90) and hard toasting (r 2 = 0.76). wheat bread and the medium toasted rye bread, the ratio of acrylamide units in the 27 toasted bread per unit asparagine in the 28231 untoasted bread was found to be higher in wheat bread (6.7 * 10 -4) compared to rye 29 bread (0.89 * 10 -4) that is the ratio of 30232 Figure 2 acrylamide formation in wheat bread was seven times higher than in rye bread. 31 Deleted: Lindenmeier & Hofmann 32233 (2004) showed that due to their lower pH 33 value (~4.0) sourdough fermented breads contained higher amounts of the key 34234 Although it is not possible directly to compare between acrylamide formation in wheat and rye antioxidant in bread crust pronyl-L-lysine 35 than breads that have been yeast- fermented only. Hence besides the lower 36235 bread having different slice thicknesses, water contents and asparagine levels it seems as the rate of acrylamide formation rate at lower pH, the increased antioxidant content in the 37 crust of rye bread may further reduce 38236 acrylamide formation is somewhat higher in wheat bread compared to rye bread. This may be acrylamide formation, as some antioxidants have an inhibitory effect on 39 the acrylamide formation. Addition of the 40237 explained by lower pH conditions in sourdough fermented rye bread compared to yeast fermented lysine-rich casein (lysine reduces the acrylamide yield) to the bread recipe also 41 increased the amounts of antioxidant 42238 wheat bread (pH appr. 0.5 units lower in sourdough). A better retention of water in the bread when (Lindenmeier & Hofmann 2004, Claeys et al 2005). Hence the effect of lysine 43 239 toasting rye bread compared to wheat bread may also reduce the acrylamide formation. The average may be from increasing the amounts of 44 antioxidants with inhibitory effects. ¶ 45 Deleted: contribute to the lower 240 water content in hard , medium and untoasted rye bread slices were 24 ±2%, 26 ±2% and 41 ±1% reaction rate of acrylamide from 46 asparagine when toasting rye bread in 47 comparison to wheat bread, as higher 241 while the average water content in hard, medium and untoasted wheat bread slices were somewhat water content will reduce the acrylamide 48 formation. 49 Deleted: and 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 11 of 42 Food Additives and Contaminants 1 2 3 242 lower: 17 ±2%, 19 ±1% and 32 ±1%, respectively. However, the actual water contents at the toasted 4 Deleted: a 5 243 surfaces were probably somewhat lower. Hedegaard et al. (2007) found that at high water activity Deleted: suggested 6 7 244 the rate determining step of acrylamide formation from the Schiffs base is the elimination of water 8 9 245 from an intermediate, while at lower water activity, water elimination becomes easier and 10 11246 decarboxylation of a reaction intermediate leading to acrylamide becomes the rate determining step. 12 13247 Hence the acrylamide formation rate increases when the water activity decreases. Hence if different 14 15248 reactions are rate determining, the overall reaction rate may differ for different water activities, 16 For Peer Review Only 17249 which may explain the exponential increase in acrylamide contents (as the water content are 18 Deleted: Schieberle et al. (2005) found 250 reduced) generally found during thermally processing of food at a fixed temperature. It was also in a model system that the amount of 19 water had a significant effect on the 20 yields of acrylamide, however the 251 found in other model system s that acrylamide formation increased when lowering the water content formation of acrylamide needs a certain 21 amount of water to form acrylamide, and that 25% water content gave higher 22 252 (Schieberle et al. 2005, Blank et al. 2005) . acrylamide yields than 10% water 23 content. However the optimum could be between the two water percentages. 24253 The proportions of asparagine in wheat bread reacting to give acrylamide were also estimated Blank et al. (2005) also found in a 25 Deleted: although 26254 (Table III). Generally the ratio is higher in medium toasted bread suggesting that at higher Deleted: the formation rate drops again 27 below a certain water content 28255 temperatures and/or longer duration of toasting precursors are depleted faster, reacting faster in 29 30256 competing reactions and/or acrylamide is eliminated to a larger extent by subsequent reaction. For 31 32257 the hard toasted bread is seen, that as depletion of the asparagine pool approaches, the formation of 33 Deleted: s 34258 acrylamide diminishes, resulting in lower ratios at lower asparagine addition levels. At the low Deleted: Because much smaller 35 proportions of the asparagine had reacted upon toasting of the rye bread the 36259 asparagine levels in wheat bread asparagine was depleted after hard toasting while at higher fractions reacted were more uncertain and 37 are therefore not measured. ¶ 38260 addition levels some asparagine was left. This may be interpreted as if asparagine was the limiting 39 40261 precursor at low asparagine levels while at higher levels other factors also influenced the 41 42262 acrylamide formation. However the other precursor the reducing sugars was not depleted (data not 43 44263 shown). 45 46264 47 265 Figure 3 48 266 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 12 of 42 1 2 3 267 4 268 Table III. 5 269 6 270 Deleted: ¶ 7 ¶ 8 271 Acrylamide contents in wheat breads and rye breads produced in Denmark and comparison with ¶ ¶ 9 ¶ 10272 other surveys. ¶ 11 Deleted: ¶ 273 In order to estimate the level of acrylamide in rye bread, in 2004 13 sifted and whole meal rye ¶ 12 ¶ 13 274 breads produced for or sold on the Danish market were sampled and analysed. They contained 12 ± 14 15 275 5 µg kg -1acrylamide (range <5-19 µg kg -1, n=13). 16 For Peer Review Only 17 276 In 2005 ten samples of wheat breads, produced for or sold on the Danish market were analysed, 18 19 277 containing 10 ± 6 µg kg -1acrylamide (range <5-22 µg kg -1). 20 Deleted: In order to study the 21 278 The contents of acrylamide in bread found in the present study are comparable with the results acrylamide formation in industrial 22 produced breads, the industrial bread producer made small wheat breads 23279 found in other studies. Becalski et al. (2003) found 290 µg kg -1of acrylamide in toasted wheat bread (baguettes) of 50 g dough in their 24 laboratory bakery. The baguettes were baked in a vented oven at 220 °C for 10 25 -1 - min. (to yellow colour of crust) and 15 280 compared to 19 µg kg in untoasted wheat bread (n=2). Light toasted rye bread contained 28 µg kg min. (to light brown colour of crust). As 26 the baguettes are produced and sold 27 1 -1 frozen for final preparation by the 281 acrylamide and untoasted rye bread 17 µg kg . Eerola et al. (2007) found that toasted wheat bread consumer, the baguettes were also baked 28 at 220 °C for 10 min. and reheated from 29282 contained 111±10 µg kg -1 acrylamide (n=5) and untoasted wheat bread <68 µg kg -1 acrylamide, frozen for 5 min. (to light yellow-brow colour). The crumb of all breads (15 30 analyses, 5 x 15 breads) only contained a 31283 which was the limit of detection (n=5). Ahn et al. (2002) found that acrylamide increased with few µg kg -1 acrylamide (<5 µg kg -1) while the crust contained 7-22 µg kg - 32 1acrylamide. The whole baguettes 33284 longer toasting time. After 3.1 min. of toasting when the toasts were still considered eatable wheat contained on average 10 µg kg -1 acrylamide.¶ 34 35285 bread contained appr. 60 µg kg -1 and rye bread contained appr. 80 µg kg -1 acrylamide . After 6 36 37286 minutes wheat bread contained appr. 206 µg kg -1 and rye bread contained appr. 220 µg kg -1 38 39287 acrylamide. Konings et al. (2003) reported acrylamide contents in toasted bread of 183 ± 336 µg kg - 40 41288 1 (range < 30-1430 µg kg -1, n=17). The contents in dark rye bread were 44 ± 13 µg kg -1 (range 30- 42 43289 60 µg kg -1, n=5) and in wheat bread < 30 µg kg -1 (n=6). 44 45290 46 47291 Dietary acrylamide intake from bread 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 13 of 42 Food Additives and Contaminants 1 2 Comment [k2]: the first paragraph 3 292 The average dietary intake of wheat bread in Denmark is 125 g day -1 and in addition Danes has a moved to materials and methods 4 Deleted: Exposure estimates were accomplished using consumption data 5 293 tradition for eating whole meal sourdough fermented rye bread as part of nearly every lunch meal from the Danish¶ 6 Nationwide dietary survey made from -1 2000-2002 by the National Food Institute 7 294 comprising an average daily intake of 59 g rye bread day (Lyhne et al. 2005) In case all bread is (Lyhne et al. 2005). Information on food 8 consumption was collected from a -1 representative sample of 4120 individuals 9 295 consumed without toasting and the mean figures for acrylamide in bread is used: 10 µg kg in aged 4-75 years. The participants in the survey kept a food record for seven 10 consecutive days 296 wheat bread and 12 µg kg -1 in rye bread, the daily intake comprises 1.3 µg from wheat bread and 11 Deleted: . 12 Deleted: . 13297 0.7 µg from rye bread, respectively. The daily intake of acrylamide from wheat and rye bread Deleted: intake 14 -1 Deleted: together 15298 comprising 2.0 µg is about 8 % of the average daily acrylamide intake of appr. 25 µg day Deleted: 16 For Peer Review Only of 299 estimated for Danes (Figure 4). The acrylamide intake from bread is relatively low compared to Deleted: only 17 18 300 other surveys, estimating e.g. 3.4 µg (Svensson et al. 2003) and 3.6 µg (Matthys et al. 2005). An 19 20301 explanation for the low Danish dietary acrylamide intake from bread may be that most Danish bread 21 22302 are produced using either yeast fermentation or sourdough fermentation, hence the contents of the 23 24303 precursor asparagine are reduced during preparation of the breads. Another explanation for the low 25 26304 acrylamide intake from bread may be that in previous studies on acrylamide in bread the limit of 27 28305 detection (LOD) was higher and the levels in bread often below the LOD. Hence in many cases 29 30306 LOD was used as the acrylamide level in bread. Finally in the present estimate, as the Danish 31 32307 Nationwide dietary survey do not have information on how frequently people eat toasted bread, the 33 34308 acrylamide intake from toasted bread is not included in the dietary acrylamide intake from bread. 35 36309 However, in case the average daily bread intake includes two hard toasted wheat bread slices 37 Deleted: 34 -1 38310 (acrylamide 161 µg kg (ww) ) each of 28 g and with the high pre-processing asparagine level of 0.6 39 -1 -1 40311 g kg (dw) which is about the concentration found in wholegrain wheat flour i.e. 0.5 g kg 41 Deleted: 12 42312 (Frederiksson et al. 2004 ), the daily acrylamide intake is estimated to 9 µg (3 6 % of the average Deleted: 9 43 Deleted: 32 313 Danish acrylamide intake ). If a consumer eats an average amount of bread of which two slices of 25 44 Deleted: r 45 -1 314 g rye bread are hard toasted with asparagine and acrylamide concentrations of 1.2 g kg and 123 µg Deleted: rye bread 46 Deleted: 8 47315 kg -1 respectively, the daily acrylamide intake from bread comprises 7 µg (2 8 % of the average 48 Deleted: 8 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 14 of 42 1 2 3 316 Danish acrylamide intake ). Hence dietary habits with consumption of toasted bread, which are 4 5 317 common throughout at least Europe, may increase the acrylamide intake from bread considerable. 6 Deleted: s 7 318 Especially intake of toasted wholemeal wheat bread and bread produced without leavening by Deleted: s 8 9 319 fermentation may further increase the acrylamide exposure. 10 11320 12 13321 Figure 4. 14 15322 16 For Peer Review Only 17323 Conclusions 18 324 19 20325 The contents of acrylamide in baked rye bread and in toasted wheat and rye bread are correlated to 21 22326 the contents of asparagine in the cereal product used for the bread production and at asparagine 23 24327 concentration ranges that may occur in grain products for bread production. The content of 25 26328 asparagine in the dough for wheat bread preparation is reduced considerably during bread 27 28329 production. After 65 min. raising time and subsequent baking, the asparagine content in the baked 29 30330 wheat bread was reduced to 12% compared to the content in the flour before yeast fermentation. 31 32331 The sourdough fermentation of rye bread showed a modest asparagine reduction to 82% of the 33 34332 content in the flour. This is probably due to the more acidic conditions in sourdough fermented rye 35 36333 bread compared to yeast fermented wheat dough. The contents of acrylamide formed in toasted 37 Deleted: also 38334 wheat bread slices and toasted rye bread slices are proportional to the asparagine concentration of Deleted: The formation of acrylamide 39 -1 in toasted bread per unit of asparagine in 40335 the untoasted bread. The dietary intake of acrylamide from untoasted bread of 2.0 µg day is the untoasted bread was appr. seven times 41 higher in wheat bread than in rye bread. 42336 relatively low but the dietary acrylamide intake increases somewhat for people eating toasted bread. 43 337 44 45 338 Ackowledgement 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 15 of 42 Food Additives and Contaminants 1 2 3 339 This research was supported by the Danish Ministry of Food, Agriculture and Fisheries as a part of 4 5 340 the project “Reduction of the formation and the occurrence of acrylamide in food” and The Nordic 6 7 341 Innovation Centre Project NORDACRYL (Acrylamide precursors; Limiting substrates and in vivo 8 9 342 effects) . Lone Hertz is acknowledged for performing parts of the chemical analyses. 10 11343 12 13344 References 14 345 Ahn JS, Castle L, Clarke DB, Lloyd AS, Philo MR, Speck DR. 2002. Verification of the findings of acrylamide in 15346 heated foods. Food Additives and Contaminants 19:1116-1124. 16347 Amrein TM, Schönbachler B,For Escher F, Amado Peer R. 2005. Acrylamide Review in gingerbread: critical factors Only for formation and 17348 possible ways for reduction. Journal of Agricultural and Food Chemistry 52:4282-4288. 18349 Becalski A, Lau BPY, Lewis D, Seaman SW 2003. Acrylamide in foods: occurrence, sources, and modeling. Journal of 350 Agricultural and Food Chemistry 51:802-08. 19351 Blank I, Robert F, Goldmann T, Pollien P, Varga N, Devaud F, Hyunh-Ba T, Stadler RH. 2005. Mechanisms of 20352 acrylamide formation: Maillard-induced transformations of asparagine. Advanced experimental medicine and Deleted: s 21353 Biology 561: 171-189 22354 Bråthen E, Knutsen S. 2005. Effect of temperature and time on the formation of acrylamide in a starch based and cereal 355 model system. Food Chemistry 92:693-700. 23 356 Claus A, Weisz GM, Schiber A, Carle R. 2006. Pyrolytic acrylamide formation from purified wheat gluten and Deleted: Claeys WL, De Vleeschouwer 24357 gluten-suplemented wheat bread rolls. Molecular Nutrition and Food Research 50: 87-93. K, Hendrickx ME., 2005. Effect of amino 358 Eerola S, Hollebekkers K, Hallikainen A, Peltonen K. 2007. Acrylamide levels in Finnish foodstuffs analysed with acids on acrylamide formation and 25 elimination kinetics. Biotechnology 26359 liquid chromatography tandem mass spectrometry. Molecular Nutrition and Food Research 51: 239-247 progress 21:1525-1530.¶ 360 Elmore JS, Koutsidis G, Dodson AT, Mottram DS, Wedzicha BL. 2005. Measurements of acrylamide and its precursors 27361 in potato, wheat and rye model systems. Journal of Agricultural and Food Chemistry 53:1286-93. 28362 Frederiksson H, Tallving J, Rosén J, Åman P. 2004. Fermentation reduces free asparagine in dough and acrylamide 29363 content in bread. Cereal Chemistry 81:650-653. 30364 Hedegaard RV, Frandsen H, Granby K, Apostolopoulou A, Skibsted LH. 2007. Model studies on acrylamide Deleted: Granvogl M, Wieser H, Koehler P, Tucher SV, Scieberle P. 2007. 31365 generation from glucose/asparagine in aqueous glycerol. Journal of Agricultural and Food Chemistry 55:486-492. 366 Hedegaard RV, Sobrintio LGA, Granby K, Skibsted LH. 2008. Formation of acrylamide in cheese bread. Influence of sulfur fertilization on the 32 amounts of free amino acids in Wheat. 367 Milshwissenschaft (In Press). Correlation with baking properties as well 33368 International Agency of Research on Cancer Monographs (IARC), 1994, 60. as with 3-aminopropionamide and 34369 Jung MY, Choi DS, Ju JW. 2003. A novel technique for limitation of acrylamide formation in fried and baked corn acrylamide generation during baking. 370 chips and in French fries. Journal of Food Science 68:1287-1290. Journal of Agricultural and Food 35 Chemistry 55:4271-4277.¶ 371 Konings EJM, Baars AJ, van Klaveren JD, Spanjer MC, Rensen PM, Hiemstra M, van Kooij JA, Peters PWJ. 2003. Hedegaard RV, Sobrintio LGA, Granby 36372 Acrylamide exposure from foods of the Dutch population and an assessment of the consequent risks. Food and K, Skibsted LH. 2007a. Formation of 37373 Chemical Toxicology 41:1569-1579. acrylamide in cheese bread. 38374 Lyhne N, Christensen T, Groth MV, Fagt S, Biltoft-Jensen A, Hartkopp H, Hinsch HJ, Matthiessen J, Møller A, Saxholt Milshwissenschaft (accepted).¶ 39375 E, Trolle E. 2005 Danskernes Kostvaner 2000-2002, Hovedresultater, The Danish Institute for Food and Veterinary Deleted: b 376 Research, April 2005. Report. No. 11 (In Danish, Summary in English) Available at http://www.Food.DTU.dk. Deleted: Lindenmeier M, Hofmann T. 40377 Matthys C, Bilau M, Govaert Y, Moons E, De Henauw S, Willems JL. 2005. Risk assessment of dietary acrylamide 2004. Influence of baking conditions and 41378 intake in Flemish adolescents. Food and Chemical Toxicology 43:271-278. precursor supplementation on the 42379 Mottram DS, Wedzicha BL, Dodson AT. 2002. Acrylamide is formed in the Maillard reaction. Nature 419: 448-449. amounts of the oxidant pronyl-L-lysine 380 Nielsen NJ, Granby K, Hedegaard RV, Skibsted LH. 2005. A LC-MS/MS method for simultaneous analysis of in bakery products. Journal of 43 Agricultural and Food Chemistry 52:350- 381 acrylamide and the precursors, asparagine and reducing sugars in bread. Analytica Chimica Acta 557:211-220. 354.¶ 44382 Pedreschi F, Kaack K, Granby K. 2004. Reduction of acrylamide formation in potato slices during frying. Food Science Deleted: Muttucumaru N., Halford 45383 and Technology 37,679-685. N.G., Elmore J.S., Dodson A.T., Parry 46384 Pedreschi F, Kaack K, Granby K. 2005. Colour change and acrylamide formation in fried potato slices. Food Research M., Shewry P.R. and Mottram D.S., 47385 International 38:1-9. 2006.Formation of high Levels of 386 Pedreschi F, Kaack K, Granby K. 2006. Acrylamide content and color development in fried potato strip. Food Research Acrylamide during the Processing of 48 Flour Derived from Sulfat-Deprived 387 International 39:40-46. Wheat. J. Agric. Food Chem., 54, 8951- 49 8955.¶ 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 16 of 42 1 2 3 388 Raccach M, Bamiro T, Clinch J, Combs G, Gierczynski A, Karam R. 2004. Natural fermentation of wheat flours. Food 4 389 Control 15:191-195. 390 Rydberg P, Eriksson S, Tareke E, Karlsson P, Ehlenberg L, Törnqvist M. 2003. Investigations of factors that influence 5 391 the acrylamide content of heated foodstuffs. Journal of Agricultural and Food Chemistry 51:7012-7018. 6 392 Schieberle P, Köhler P, Ganvogl M. 2005. New aspects on the formation and analysis of acrylamide. Advanced 7 393 experimental medicine and Biology 561: 191-203. 8 394 Stadler, RH, Blank I, Varga N, Robert F, Hau J, Guy PA, Robert MC, Riediker S. 2002. Acrylamide from Maillard 395 reaction products. Nature 419:449. 9 396 Suhr KI, Nielsen PV. 2004. Effect of weak acid preservatives on growth of bakery product spoilage fungi at different 10397 water activities and pH values. International Journal of Food Microbiology 95:67-78. 11398 Surdyk N, Rosén J, Andersson R, Åman P. 2004. Effects of asparagine, fructose, and baking conditions on acrylamide 12399 contents in yeast-leavened wheat bread. Journal of Agricultural and Food Chemistry 52:2047-2051. 13400 Svensson K, Abramsson L, Becker W, Glynn A, Hellenäs KE, Lind Y, Rosén J. 2003. Dietary intake of acrylamide in 401 Sweden. Food and Chemical Toxicology 41:1581-1586. 14402 Taeymans D, Wood J, Ashby P, Blank I, Studer A, Stadler RH, Gondé P, Van Eijck P, Lalljie S, Lingnert H, Lindblom 15403 M, Matissek R, Müller D, Tallmadge D, O’brian J, Thompson S, Silvani D, Whitmore T. 2004. A review of 16404 acrylamide: An industry perspectiveFor on research, Peer analysis, formation Review and control. Critical Review Only in Food Sciences 17405 and Nutrition 44:323-347. 406 Tareke E, Rydberg P, Karlsson P, Eriksson S, Törnqvist M. 2002. Analysis of acrylamide, a carcinogen formed in 18407 heated foodstuffs. Journal of Agricultural and Food Chemistry 50:4998-5006. 19408 Wenzl T, Karasek L, Rosen J, Hellenaes K-E, Castle L, Anklam E. 2006. Collaborative trial validation study of two 20409 methods, one based on high performance liquid chromatography-tandem mass spectrometry and on gas 21410 chromatography- mass spectrometry for the determination of acrylamide in bakery and potato products. Journal of 411 Chromatography A 1132: 211-218. 22412 World Health Organization 2002. Health Implications of Acrylamide in Food. 23413 http://www.who.int/foodsafety/publications/chem/en/acrylamide_full.pdf . 24414 25415 26416 Table I. Levels of asparagine added to a) wheat dough and b) rye dough, asparagine content in 27417 wheat and rye bread slices and the acrylamide contents after medium or hard toasting of the bread 28418 slices (mean ±2sd). 29419 30420 Table II. Asparagine left after fermentation and baking, and subsequent medium or hard toasting of 31421 wheat and rye bread (in percentage of asparagine in the flour). 422 32 423 Table III. Amount of acrylamide formed and present [µg] from asparagine reacted [mg] when 33 424 toasting wheat bread (ratios calculated by subtracting analyte concentrations determined in medium 34 425 or hard toasted bread from analyte concentrations determined in untoasted bread). 35426 36427 Figure 1. Experimental design for baking wheat and rye bread with addition of asparagine and 37428 subsequent medium or hard toasting of bread slices. All bread slices were analysed for acrylamide 38429 and asparagine. 39430 40431 Figure 2. Correlation between naturally occurring and added asparagine in flour and acrylamide in 41432 baked and toasted bread slices of a) wheat or b) rye. 42433 43434 Figure 3. Correlation between asparagine in untoasted bread and acrylamide in toasted bread slices 44435 of a) wheat or b) rye. 45436 46437 Figure 4. The proportions from different foods to the daily mean acrylamide intake for Danish 47438 adults of appr. 25 µg. 48439 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 17 of 42 Food Additives and Contaminants 1 2 3 4 Table I 5 a) 6 7 Asparagine in 8 flour Asparagine in Asparagine in Acrylamide in Wheat (background + bread bread bread 9 added level) [g kg -1 (dw)] [g kg -1(ww)] [µg kg -1 (ww)] 10 [g kg -1 (dw)] 11 12 0.14 (no addition) 0.017 ± 0.01 0.012 ± 0.01 <5* 13 0.22 ( +0.09) 0.037 ± 0.01 0.025 ± 0.01 <5* Untoasted 14 0.37 ( +0.22) 0.11 ± 0.01 0.074 ± 0.01 <5* 15 0.60 ( +0.44) 0.29 ± 0.02 0.20 ± 0.01 <5* 16 For Peer Review Only 17 0.14 (no addition) 0.015 ± 0.02 0.012 ± 0.01 11 ± 4 18 0.22 ( +0.09) 0.032 ± 0.01 0.026 ± 0.01 26 ± 9 Medium toasted 19 0.37 ( +0.22) 0.092 ± 0.01 0.075 ± 0.01 46 ± 9 20 0.60 ( +0.44) 0.24 ± 0.01 0.19 ± 0.01 130 ± 23 21 22 0.14 (no addition) <0.002* <0.002* 15 ± 6 23 Hard toasted 0.22 ( +0.09) <0.002* <0.002* 34 ± 9 24 0.37 ( +0.22) 0.014 ± 0.03 0.011 ± 0.02 65 ± 6 25 0.60 ( +0.44) 0.20 ± 0.01 0.17 ± 0.01 161 ± 15 26 27 28 29 b) 30 31 Asparagine in 32 Rye flour (total of Asparagine in Asparagine in Acrylamide in 33 background and bread bread bread added level) [g kg -1 (dw)] [g kg -1(ww)] [µg kg -1 (ww)] 34 -1 35 [g kg (dw)] 36 0.76 (no add.) 0.62 ± 0.02 0.36 ± 0.02 12 ± 3 37 0.93 ( +0.17) 0.78 ± 0.03 0.45 ± 0.01 7 ± 4 Untoasted 38 1.19 ( +0.43) 0.99 ± 0.04 0.58 ± 0.03 17 ± 2 39 2.49 ( +1.7) 2.3 ± 0.1 1. 3 ± 0.1 23 ± 8 40 41 0.76 (no add.) 0.72 ± 0.03 0.53 ± 0.02 27 ± 3 42 Medium 0.93 ( +0.17) 0.82 ± 0.03 0.62 ± 0.02 54 ± 9 43 toasted 1.19 ( +0.43) 1.08 ± 0.03 0.80 ± 0.03 65 ± 17 44 2.49 ( +1.7) 2.2 ± 0.2 1.6 ± 0.1 155 ± 14 45 46 0.76 (no add.) 0.64 ± 0.04 0.48 ± 0.02 68 ± 4 Hard 47 0.93 ( +0.17) 0.75 ± 0.03 0.57 ± 0.02 100 ± 8 toasted 48 + 1.19 ( 0.43) 1.01 ± 0.03 0.79 ± 0.04 123 ± 12 49 2.49 ( +1.7) 2.2 ± 0.1 1.6 ± 0.04 205 ± 41 50 51 52 * Limit of detection (LOD) 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 18 of 42 1 2 3 4 Table II 5 Asparagine 6 Asparagine Asparagine after after 7 Asparagine in after fermentation, fermentation, 8 flour fermentation baking and baking, and 9 [g kg -1] and baking medium toasting hard toasting 10 % % % 11 WHEAT 12 BREAD: 13 0.14 12% 11% 0% 14 0.22 17% 14% 0% 15 0.37 30% 25% 4% 16 0.60 For 49% Peer 39% Review 33% Only 17 RYE 18 BREAD: 19 0.76 82% 95% 84% 20 0.93 83% 88% 80% 21 1.19 83% 91% 85% 22 2.49 91% 89% 86% 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 19 of 42 Food Additives and Contaminants 1 2 3 4 Table III. 5 Medium toasted Hard toasted 6 Asparagine [µg acrylamide [µg acrylamide 7 level in dough formed per mg formed per mg 8 g kg -1 (dw) asparagine reacted asparagine reacted 9 upon toasting] upon toasting] 10 0.14 ± 0.01 5 ± 2 1 ± 0.4 11 12 0.22 ± 0.01 8 ± 4 1 ± 0.3 13 0.37 ± 0.01 4 ± 1 1 ± 1.0 14 0.60 ± 0.01 3 ± 1 2 ± 0.3 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 20 of 42 1 2 3 4 5 TYPE OF Rye bread 6 BREAD: Wheat bread 7 8 9 10 LEVEL OF 0. Natural asparagine level 11 ASPARAGINE 1. Low asparagine addition 12 ADDITION: 2. Medium asparagine addition 13 3. High asparagine addition 14 15 16 Untoasted slices (n=6) For PeerLEVEL OF Review Only Medium toasted slices (n=6) 17 TOASTING: Hard toasted slices (n=6) 18 19 20 21 Fig. 1 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 21 of 42 Food Additives and Contaminants 1 2 3 4 5 a) 6 7 8 9 200 10 y = 8.4e 0.0051x 11 R2 = 0.87 12 13 150 (ww)] (ww)] 14 -1 15 16 For Peer Review Only 17 100 18 19 y = 6.0e 0.0052x 20 R2 = 0.86 21 50 22 Acrylamide[µg in bread kg 23 24 25 0 26 0 100 200 300 400 500 600 700 27 Asparagine in flour [mg kg -1 (dw)] 28 29 Not toasted Medium toasted Hard toasted 30 b) 31 32 33 34 35 36 300 37 38 0.9153 250 y = 0.18x 39 R2 = 0.88 40 (ww)] 41 -1 200 42 43 44 150 45 y = 0.0044x 1.3442 46 R2 = 0.86 47 100 48 49 Acrylamide in bread [µg kg 50 50 51 52 0 53 54 0 500 1000 1500 2000 2500 3000 55 Asparagine in flour [mg kg -1 (dw)] 56 Not toasted Medium toasted Hard toasted 57 58 59 60 Fig 2 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 22 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 23 of 42 Food Additives and Contaminants 1 2 3 4 a) 5 6 7 250 8 y = 0.67x 9 2 dw) R = 0.94 10 -1 200 11 y = 0.53x 12 R2 = 0.87 13 150 14 15 16 100 For Peer Review Only 17 18 19 50 20 21 Acrylamide (µgin toasted bread kg 0 22 23 0 50 100 150 200 250 300 350 24 Asparagine in un-toasted bread (mg kg -1 dw) 25 Medium toasted Hard toasted Lineær (Hard toasted) 26 27 b) 28 29 30 31 400 y = 0.13x 32 2 dw) 350 R = 0.76 33 -1 34 300 35 36 250 y = 0.089x 37 R2 = 0.90 38 200 39 150 40 41 100 42 43 50 44 Acrylamide (µgin toasted bread kg 0 45 0 500 1000 1500 2000 2500 3000 46 -1 47 Asparagine in un-toasted bread (mg kg dw) 48 Medium toasted Hard toasted 49 50 Fig. 3. 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 24 of 42 1 2 3 4 5 6 rye bread 7 crispbread wheat bread 3% 1% 8 4% 9 popcorn/snack 1% 10 coffee chocolate/cocoa 24% 11 4% 12 13 cakes/biscuits 14 5% 15 16 For Peer Review Onlycrisp 17 6% 18 breakfast cereals 19 2% 20 convenience food 21 2% 22 23 24 25 26 27 28 29 fried potatoes 30 48% 31 32 33 34 Fig. 4 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 25 of 42 Food Additives and Contaminants 1 2 3 1 4 2 Acrylamide in relation to asparagine in baked and toasted wheat and rye bread. 5 6 3 7 4 Abstract 8 9 5 Acrylamide in baked and toasted wheat and rye bread was studied in relation to the content of 10 11 6 asparagine in flour, dough, bread and toasted bread. Asparagine was consumed during bread 12 13 7 preparation resulting in a reduced acrylamide contents in the products. In wheat bread 12% of the 14 -1 15 8 asparagine initially present in the flour (0.14 g kg ) was left after yeast fermentation and baking, 16 For Peer Review Only 9 while for rye bread 82% of the asparagine was left after sourdough fermentation and baking. The 17 18 10 asparagine present in the untoasted wheat bread had all reacted after hard toasting. Toasted wheat 19 20 11 and rye bread slices contained 11-161 µg kg -1 and 27-205 µg kg -1 acrylamide compared to untoasted 21 22 12 wheat and rye bread containing <5 µg kg -1 and 7-23 µg kg -1 acrylamide. The dietary intake of 23 24 13 acrylamide from bread (untoasted) of 2 µg/day is relatively low, however the acrylamide exposure 25 26 14 from bread increases several times for people eating toasted bread. 27 28 15 29 30 16 Keywords: Acrylamide, asparagine, bread, rye, wheat, toast, dietary, intake 31 32 17 33 34 18 Introduction 35 36 37 19 The presence of acrylamide in heat-treated food was recognized in 2002 (Tareke et al. 2002) and 38 39 20 the same year it was clear that acrylamide is formed mainly from asparagine and a carbonyl 40 21 compound through Maillard browning reactions during food processing (Mottram et al. 2002; 41 42 22 Stadler et al. 2002). Several pathways and intermediates for acrylamide formation have been 43 44 23 suggested including the intermediates: Schiffs bases, decarboxylated Amadori products (Yaylayan 45 46 24 2003), Strecker aldehydes (Mottram et al. 2002) and the deamination product 3-aminopropionamide 47 48 25 which also generate acrylamide under aqueous conditions (Granvogl and Schieberle, 2006). Besides 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 26 of 42 1 2 3 26 the main reaction routes through the Maillard reactions, acrylamide may be formed in bread after 4 5 27 protein pyrolysis of added gluten (Claus et al. 2006). Also addition of cheese to bread has been 6 7 28 found to increase the acrylamide formation, probably formed with 3-aminopropionamide as an 8 9 29 intermediate in the cheese, which has been found to generate acrylamide (Granvogl and Schieberle 10 11 30 2006, Hedegaard et al. 2008). 12 13 31 Acrylamide is known to be carcinogenic in rodents and is classified by the International Agency for 14 15 32 Research on Cancer as probably carcinogenic to humans (IARC 1994). The dietary acrylamide 16 For Peer Review Only 17 33 intake have been estimated to e.g. 0.3-0.8 µg (kg bw) -1 day -1 (WHO 2002); appr. 0.5µg (kg bw) -1 18 19 34 day -1(Svensson et al 2003). The dietary intake of acrylamide from bread is about 11% of the total 20 21 35 dietary acrylamide intake of 31 µg day -1 as estimated for the Swedish population. A similar 22 23 36 contribution from bread related foods such as crisp bread and biscuits are estimated (Svensson et al. 24 25 37 2003). The acrylamide level in baked bread is relatively low, but due to high consumption of bread 26 27 38 the contribution of acrylamide from bread is still important. 28 29 30 39 Toasting the bread, which is frequently used e.g. for breakfast meals, increases acrylamide 31 -1 32 40 significantly. While untoasted bread contains <50 µg kg the acrylamide content may increase to a 33 34 41 few to several hundreds micrograms per kilo bread after toasting (Konings et al. 2003; Becalski et 35 36 42 al. 2003; Ahn et al. 2002). The acrylamide levels have been found to increase with increasing 37 43 toasting time and whole meal rye bread was found to form more acrylamide than white wheat bread 38 39 44 (Ahn et al. 2002). 40 41 45 42 43 46 The occurrence of acrylamide in bread may depend on several factors. Surdyk et al. (2004) showed 44 45 47 that acrylamide in baked wheat bread correlated with the amount of asparagine added to dough, 46 47 48 while the added reducing sugar fructose did not show a similar correlation, hence the asparagine 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 27 of 42 Food Additives and Contaminants 1 2 3 49 was a limiting substrate for the acrylamide formation. The clear relationship between asparagine 4 5 50 content in the dough and acrylamide formation has also been demonstrated in other wheat and rye 6 7 51 bread model systems (Elmore et al. 2005; Bråthen et al. 2005). One of the strategies to reduce 8 9 52 acrylamide in cereal products may be to choose raw materials low in asparagine by selecting 10 11 53 optimum cultivars and growth conditions. However, variations in free asparagine in cereals are 12 13 54 modestly researched. Claus et al. 2006 investigated agronomic factors related to the acrylamide 14 15 55 formation in cereal products. They showed that nitrogen fertilization resulted in elevated amino acid 16 For Peer Review Only Comment [k1]: Left out to shorten 56 levels resulting in increased acrylamide levels. The asparagine contents of selected European wheat the introduction 17 18 57 varieties ranged from 0.07-0.66 g kg -1, that is a factor of five between varieties and a factor of two 19 20 58 within varieties (Taeymans et al. 2004). 21 22 59 23 24 60 Milling will differentiate the asparagine levels in the different milling fractions. Fredriksson et al. 25 26 61 (2004) found that the contents of free asparagine are lower in sifted wheat flour i.e. 0.14- 0.17 g kg- 27 28 62 1(dw) compared to whole grain wheat flour i.e. 0.5 g kg -1(dw) or wheat bran. Rye generally contains 29 30 63 more asparagine: sifted rye flour 0.6 g kg -1(dw) and whole grain rye flour: 1.1 g kg -1(dw). Not only 31 32 64 milling has been found to influence the levels of the limiting substrate asparagine. It was found that 33 34 65 yeast fermentation of wheat dough depleted a major part of the asparagine (>80%), while 35 36 66 sourdough fermentation of rye dough reduced the asparagine content to a smaller extent 37 38 67 (Fredriksson et al. 2004). During baking, parameters such as water activity, heating time and 39 40 68 temperature, surface to volume ratio, the presence of additives and pH also influence acrylamide 41 42 69 levels (Surdyk et al. 2004; Rydberg et al. 2003). Acrylamide formation may increase when 43 70 increasing pH e.g. by using baking soda or ammonium carbonate for bread leavening (Amrein et al. 44 45 71 2005) as it will decrease when lowering pH (Jung et al. 2003; Pedreschi et al. 2004, 2005). 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 28 of 42 1 2 3 72 Fermentation will cause pH in dough to drop due to release of carbon dioxide (Raccach et al. 2004) 4 5 73 and it will reach pH~5 using yeast and pH~4.4-4.8 using sourdough (Suhr et al. 2004). 6 7 74 8 9 75 The aim of the present study was to find the relationship between asparagine levels and acrylamide 10 11 76 formation in wheat bread and rye bread during baking and toasting in order to be able to reduce the 12 13 77 acrylamide levels. Furthermore to assess the acrylamide levels in bread on the Danish market and to 14 15 78 estimate the dietary intake of acrylamide from wheat and rye bread including toasted bread. 16 For Peer Review Only 17 79 18 80 Materials and methods 19 20 81 Baking wheat bread and rye bread with asparagine additions 21 22 82 An industrial bakery (Lantmännen Schulstad A/S, Hvidovre, Denmark) made the wheat and rye 23 24 83 breads in their laboratory bakery with different amounts of asparagine added (in August 2004). The 25 26 84 experimental design appears from Figure 1. Four batches of wheat dough were prepared and two 27 28 85 breads selected from each batch: One batch of dough was made without adding asparagine 29 30 86 representing the natural background asparagine level and three batches were added asparagine at 31 32 87 concentration levels of 0.05, 0.13 and 0.27 g kg -1 dough. As the dough contains 60% wheat flour, 33 34 88 the added amounts of asparagine based on flour were 0.09, 0.22 and 0.44 g kg -1 corresponding to 35 36 89 addition at approximately 0.6 - 3 times the actual background level of asparagine. The recipe for a 37 38 90 wheat bread (appr. 800 g dough) included sifted wheat flour, yeast, salt, sugar vegetable oil and 39 40 91 water. Kneading time was 7 min., holding time 20 min. and resting time at 32 °C was 45 min. 41 42 92 Baking was performed initially at 230 °C for 2 min. and subsequently at 200 °C for 30 min. 43 44 93 45 46 94 Rye breads were made using the same experimental design. The asparagine was added to the dough 47 -1 48 95 at concentrations based on rye flour of 0.17, 0.43 and 1.75 g kg respectively corresponding to 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 29 of 42 Food Additives and Contaminants 1 2 3 96 addition at 0.2 - 2 times the natural background level. The rye bread (based on appr. 900 g dough) 4 5 97 was made from half and half mixing of sourdough and rye flour and addition of yeast, salt and 6 7 98 water. Kneading time was 10 min, holding time 20 min, and resting time at 37 °C was 77 min. 8 9 99 Baking was performed initially at 280 °C for 5 min., followed by 210 °C for 35 min. and 190 °C for 10 11100 12 min. 12 13101 14 15102 Toasting wheat bread and rye bread with asparagine additions 16 For Peer Review Only 17103 Toasting of the bread slices was done in a pushdown toaster (Tefal Delfini Compact Toaster 5396, 18 19104 800 W) with room for two slices. The four batches of bread (background level and the three 20 21105 addition levels) were analyzed either untoasted or after medium or hard toasting, for each level 22 23106 using three slices from each of two breads (n=18). Medium toasting of wheat bread slices of 13 mm 24 107 was performed 1.9 min. and hard toasting 2.8 min.. Medium toasting of the rye bread slices of 8 25 26 108 mm was done 2.8 min. and the hard toasting 3.1 min.. All the toasted bread slices were medium to 27 28109 dark brown and were considered eatable. The thicknesses of the bread slices were chosen from what 29 30110 is common practice among bakers and consumers. 31 32111 33112 Figure 1 34113 35114 36 37115 Chemicals 38 39116 Acrylamide (>99.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA), asparagine 40 41117 monohydrate (>99%) from Acros Organics (Geel, Belgium), D 3-acrylamide (>98%) from Polymer 42 118 Source (Dorval, Quebec, Canada), 15 N -asparagine monohydrate (>98%) from Cambridge Isotope 43 2 44 119 Labs. (MA, USA), methanol (>98%), formic acid (>98%) and acetonitrile (HPLC-grade) from 45 46120 Rathburn Chemicals (Walkerburn, UK). 47 48121 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 30 of 42 1 2 3 122 Chemical analysis 4 5 123 The chemical analyses were performed using a recently developed LC-MS/MS method for 6 7 124 simultaneous analysis of acrylamide, asparagine and reducing sugars (Nielsen et al. 2005) and in 8 9 125 addition some of the acrylamide analyses were made with another LC-MS/MS method as described 10 11126 in Pedreschi et al. (2005). 12 13127 Sliced toasted or untoasted wheat or rye bread was weighed, dried in an oven at 50 °C over night 14 15128 and homogenized using a Braun handheld mixer (type 4169) fitted with a blender like sample 16 For Peer Review Only 17129 compartment (type 4297) (Braun AG, Frankfurt/m, Germany). The drying temperature was set 18 19130 relatively low in order to avoid acrylamide formation during the drying process. The wet (semidry) 20 131 and dry weight was determined from weighing before and after drying of the baked and toasted 21 22 132 bread slices. Aliquots of 4 g of homogenate was extracted with MilliQ water after adding 23 24133 internalstandards: 200 µL 10 µg mL -1 D -acrylamide, 100 µL 880 µg mL -1 15 N -asparagine. The 25 3 2 26134 sample was extracted by an Ultra Turrax mixer. After centrifuging, SPE cleanup was performed by 27 28135 an automated sampler using LiChroLut RP-C18 SPE-cartridges LC-MS/MS detection was 29 30136 performed on a triple quadrupole instrument (Micromass Quattro Ultima) after separation on a 31 32137 hypercarb LC-column (dimensions 2.1 mm x 100 mm, particle size 5 µm) eluted with 0.1% 33 34138 aqueous formic acid at 0.2 mL min -1. The mass spectrometer was operated in ESI + for acrylamide 35 36139 and saccharides and in ESI - for asparagine. Capillary voltages of 3 kV (ESI +) and 2 kV (ESI -) were 37 38140 applied but for simultaneous detection in positive and negative mode a lower capillary voltage, 39 40141 e.g.1 kV is recommended to prevent sparkling from the capillary. 41 42142 43 44143 Some of the acrylamide analysis was performed after the methods were optimized to detect low 45 46144 concentrations (e.g. by increasing the HPLC injection volume and by optimizing the MS 47 48145 performance before analysing the bread samples), so the method was able to quantify down to 5 µg 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 31 of 42 Food Additives and Contaminants 1 2 3 146 kg -1. The repeatability and reproducibility of the acrylamide analyses at 50 and 250 µg kg -1 (n=24) 4 5 147 were 6-17% and 9-29% respectively. The laboratory has participated in proficiency tests for 6 7 148 acrylamide in food and all results obtained have been with satisfactory test scores, including the 8 9 149 results obtained in an EU validation study on acrylamide analyses in food with 22 samples (11 10 -1 11150 matrices including bread and toasted bread: assigned value 38µg kg , obtained results 41 and 44µg 12 -1 -1 13151 kg ) (Wenzl et al. 2006). The detection limit of the asparagine analyses was 0.002 g kg , the 14 -1 15152 repeatability and reproducibility measured at 0.02, 0.1 and 0.4 g kg 0-5% and 10-18% repectively. 16 For Peer Review Only 153 17 18 154 Estimation of dietary acrylamide intakes 19 20155 Exposure estimates were accomplished using data from the Danish Nationwide Dietary Survey 21 22156 made from 2000-2002 by the National Food Institute (Lyhne et al. 2005). Information on food 23 24157 consumption was collected from a representative sample of 4120 individuals aged 4-75 years. The 25 26158 participants in the survey kept a food record for seven consecutive days. The monitoring results of 27 28159 acrylamide in Danish foods (appr. 330 samples) were made 2002-2005 by the National Food 29 30160 Institute (data not published elsewhere). 31 32161 33 34162 Results and discussion 35 36163 The levels of acrylamide and asparagine in wheat and rye bread slices after baking and medium or 37 38164 hard toasting appear from Table I. The results presented are all means of six bread slices, each 39 40165 wheat bread slice weighing 34 ±2 g and each rye bread slice weighing 32 ±1 g. 41 42166 43 44167 Asparagine in flour and bread slices after asparagine additions to dough and subsequent 45 46168 fermentation, baking and toasting 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 32 of 42 1 2 3 169 The background content of asparagine in sifted wheat flour was 0.14 ± 0.004 g kg -1 (based on three 4 5 170 determinations and with the precision measured as 95% confidence levels) and in whole grain rye 6 7 171 flour 0.76 ± 0.02 g kg -1, figures comparable to those reported by Frederiksson et al. (2004) of 0.14- 8 9 172 0.17 g kg -1 (dw) in sifted wheat flour and 1.1 g kg -1 (dw) in whole grain rye flour . 10 11173 The levels of asparagine added to the wheat dough were 0.6, 1.6 and 3.1 times the natural 12 13174 background level and to the rye dough 0.2, 0.6 and 2.3 times the natural background level. The 14 15175 highest concentration after addition correspond to 0.6 g kg -1 asparagine in wheat flour and 2.5 g kg -1 16 For Peer Review Only 17176 asparagine in rye flour; hence levels likely to be present in cereal products used for bread 18 19177 production. 20 21178 In Table II the reacted amounts of asparagine in the batches with different asparagine levels upon 22 23179 fermentation, baking and toasting are presented. Some asparagine had reacted during wheat bread 24 180 production as can be seen from data determined on a dry weight basis (Table I), where differences 25 26 181 from evaporation of water during toasting does not have to be considered. In the wheat batch with a 27 28182 natural background level of asparagine 12% was left after fermentation and baking. Upon hard 29 30183 toasting all asparagine in the wheat bread slices with the natural background content and the lowest 31 32184 addition level of asparagine had reacted. 33 34185 35 36186 Table I 37 38187 Table II 39 40188 41189 In rye bread the relative reduction in asparagine during sourdough fermentation and baking was less 42 43190 than for the yeast fermented wheat bread, e.g. 18% at the natural background level and 9% for the 44 45191 highest addition level of asparagine. As the results are based on subtracting two large percentages, 46 192 they may be more uncertain. The results are in accordance with those of Frederiksson et al. (2004), 47 48 193 who also found sourdough fermentation to reduce asparagine less than yeast fermentation and at 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 33 of 42 Food Additives and Contaminants 1 2 3 194 about the same rate i.e. >80% for yeast fermentation of wheat bread and 17% for sourdough 4 5 195 fermentation of rye . 6 7 196 8 9 197 A general conclusion may be that rye flour contains more asparagine than wheat flour and 10 11198 sourdough fermentation and baking of rye dough reduces asparagine to a smaller extent than yeast 12 13199 fermentation and baking of wheat dough. Prolonging or optimizing the fermentation process, 14 15200 especially the yeast fermentation of wheat bread may be an efficient tool to reduce the acrylamide 16 For Peer Review Only 201 formation in bread products. 17 18202 19203 20204 Acrylamide in baked and toasted bread in relation to asparagine 21 22205 Baked wheat bread slices contained <5 µg kg -1 acrylamide ( 29 -1 -1 30209 27-155 µg kg acrylamide; hard toasted 68-205 µg kg acrylamide for asparagine levels in flour of 31 -1 32210 0.76-2.5 g kg . 33 34211 35 212 In Figure 2 acrylamide concentrations in wheat and rye bread, initially baked with different 36 37 213 asparagine addition levels, and after baking toasted to medium or hard level are plotted and marked 38 39214 with correlation coefficients. Due to the low acrylamide contents in the baked wheat bread slices no 40 41215 correlation was found between asparagine content in dough and acrylamide content in baked wheat 42 43216 bread. However, acrylamide contents in wheat bread correlated well with the asparagine levels 44 45217 added to the dough for medium (r 2 = 0.86) and hard toasted bread slices (r 2 = 0.87). As smaller 46 47218 proportions of asparagine were consumed during yeast fermentation and baking for the higher 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 34 of 42 1 2 3 219 asparagine additions the acrylamide contents increased exponentially with increasing asparagine 4 5 220 levels (quadratic equations). 6 7 221 8 -1 9 222 The untoasted rye bread slices contained naturally 0.62 g kg (dw) asparagine , about 36 times 10 11223 more than the wheat bread slices. As for the wheat bread quadratic relationships between the 12 2 13224 content of asparagine in the rye flour and acrylamide content in bread after medium (r = 0.86) or 14 2 15225 hard (r = 0.88) toasting appear (Figure 2b). 16 For Peer Review Only 226 17 18 227 In the present study, a major part of the asparagine added to the wheat dough had reacted during 19 20228 preparation of the bread. It is known, that especially yeast fermentation promote elimination of 21 22229 asparagine, and that the reduction depends on e.g. time of fermentation, the amount of yeast added 23 24230 and pH (Frederiksson et al. 2004). When comparing the asparagine levels left in baked bread before 25 26231 toasting with the acrylamide content in toasted bread slices good linear relationships are present for 27 28232 both medium toasted (r 2 = 0.87) and hard toasted (r 2 = 0.94) wheat bread and for asparagine levels 29 30233 of 0.017-0.29 g kg -1 (dw) bread. The asparagine levels in baked rye bread also showed good linear 31 32234 correlations with acrylamide formed after medium toasting (r 2 = 0.90) and hard toasting (r 2 = 0.76). 33 34235 35 36236 Figure 2 37 38237 39 40238 Although it is not possible directly to compare between acrylamide formation in wheat and rye 41 42239 bread having different slice thicknesses, water contents and asparagine levels it seems as the rate of 43 240 acrylamide formation is somewhat higher in wheat bread compared to rye bread. This may be 44 45 241 explained by lower pH conditions in sourdough fermented rye bread compared to yeast fermented 46 47242 wheat bread (pH appr. 0.5 units lower in sourdough). A better retention of water in the bread when 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 35 of 42 Food Additives and Contaminants 1 2 3 243 toasting rye bread compared to wheat bread may also reduce the acrylamide formation. The average 4 5 244 water content in hard, medium and untoasted rye bread slices were 24 ±2%, 26 ±2% and 41 ±1% 6 7 245 while the average water content in hard, medium and untoasted wheat bread slices were somewhat 8 9 246 lower: 17 ±2%, 19 ±1% and 32 ±1%, respectively. However, the actual water contents at the toasted 10 11247 surfaces were probably somewhat lower. Hedegaard et al. (2007) found that at high water activity 12 13248 the rate determining step of acrylamide formation from the Schiffs base is the elimination of water 14 15249 from an intermediate, while at lower water activity, water elimination becomes easier and 16 For Peer Review Only 17250 decarboxylation of a reaction intermediate leading to acrylamide becomes the rate determining step. 18 19251 Hence the acrylamide formation rate increases when the water activity decreases. Hence if different 20 21252 reactions are rate determining, the overall reaction rate may differ for different water activities, 22 23253 which may explain the exponential increase in acrylamide contents (as the water content are 24 254 reduced) generally found during thermally processing of food at a fixed temperature. It was also 25 26 255 found in other model systems that acrylamide formation increased when lowering the water content 27 28256 (Schieberle et al. 2005, Blank et al. 2005) . 29 30257 31 32258 The proportions of asparagine in wheat bread reacting to give acrylamide were also estimated 33 34259 (Table III). Generally the ratio is higher in medium toasted bread suggesting that at higher 35 36260 temperatures and/or longer duration of toasting precursors are depleted faster, reacting faster in 37 38261 competing reactions and/or acrylamide is eliminated to a larger extent by subsequent reaction. For 39 40262 the hard toasted bread is seen, that as depletion of the asparagine pool approaches, the formation of 41 42263 acrylamide diminishes, resulting in lower ratios at lower asparagine addition levels. At the low 43 44264 asparagine levels in wheat bread asparagine was depleted after hard toasting while at higher 45 46265 addition levels some asparagine was left. This may be interpreted as if asparagine was the limiting 47 48266 precursor at low asparagine levels while at higher levels other factors also influenced the 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 36 of 42 1 2 3 267 acrylamide formation. However the other precursor the reducing sugars was not depleted (data not 4 5 268 shown). 6 7 269 8 9 270 Figure 3 10271 11272 12273 Table III. 13274 14 15275 Acrylamide contents in wheat breads and rye breads produced in Denmark and comparison with 16 For Peer Review Only 276 other surveys. 17 18 277 In 2004, in order to estimate the level of acrylamide in rye bread, 13 sifted and whole meal rye 19 20 278 breads produced for or sold on the Danish market were sampled and analysed. They contained 12 ± 21 22 279 5 µg kg -1acrylamide (range <5-19 µg kg -1, n=13). In 2005, ten samples of wheat breads, produced 23 24 280 for or sold on the Danish market were analysed, containing 10 ± 6 µg kg -1acrylamide (range <5-22 25 26 281 µg kg -1). The content of acrylamide in bread found in the present study are comparable with the 27 28282 results found in other studies. Becalski et al. (2003) found 290 µg kg -1of acrylamide in toasted 29 30283 wheat bread compared to 19 µg kg -1 in untoasted wheat bread (n=2). Light toasted rye bread 31 32284 contained 28 µg kg -1 acrylamide and untoasted rye bread 17 µg kg -1. Eerola et al. (2007) found that 33 34285 toasted wheat bread contained 111±10 µg kg -1 acrylamide (n=5) and untoasted wheat bread <68 µg 35 36286 kg -1 acrylamide, which was the limit of detection (n=5). Ahn et al. (2002) found that acrylamide 37 38287 increased with longer toasting time. After 3.1 min. of toasting when the toasts were still considered 39 40288 eatable wheat bread contained appr. 60 µg kg -1 and rye bread contained appr. 80 µg kg -1 acrylamide . 41 -1 -1 42289 After 6 minutes wheat bread contained appr. 206 µg kg and rye bread contained appr. 220 µg kg 43 - 44290 acrylamide. Konings et al. (2003) reported acrylamide contents in toasted bread of 183 ± 336 µg kg 45 46291 1 (range < 30-1430 µg kg -1, n=17) . The contents in dark rye bread were 44 ± 13 µg kg -1 (range 30- 47 48292 60 µg kg -1, n=5) and in wheat bread < 30 µg kg -1 (n=6). 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 37 of 42 Food Additives and Contaminants 1 2 3 293 4 5 294 Dietary acrylamide intake from bread 6 Comment [k2]: the first paragraph 7 295 The average dietary intake of wheat bread in Denmark is 125 g day -1 and in addition Danes has a moved to materials and methods 8 9 296 tradition for eating whole meal sourdough fermented rye bread as part of nearly every lunch meal 10 -1 11297 comprising an average daily intake of 59 g rye bread day (Lyhne et al. 2005) In case all bread is 12 -1 13298 consumed without toasting and the mean figures for acrylamide in bread is used: 10 µg kg in 14 -1 15299 wheat bread and 12 µg kg in rye bread, the daily intake comprises 1.3 µg from wheat bread and 16 For Peer Review Only 300 0.7 µg from rye bread, respectively. The daily intake of acrylamide from wheat and rye bread 17 18 301 comprising 2.0 µg is about 8 % of the average daily acrylamide intake of appr. 25 µg day -1 19 20302 estimated for Danes (Figure 4). The acrylamide intake from bread is relatively low compared to 21 22303 other surveys, estimating e.g. 3.4 µg (Svensson et al. 2003) and 3.6 µg (Matthys et al. 2005). An 23 24304 explanation for the low Danish dietary acrylamide intake from bread may be that most Danish bread 25 26305 are produced using either yeast fermentation or sourdough fermentation, hence the contents of the 27 28306 precursor asparagine are reduced during preparation of the breads. Another explanation for the low 29 30307 acrylamide intake from bread may be that in previous studies on acrylamide in bread the limit of 31 32308 detection (LOD) was higher and the levels in bread often below the LOD. Hence in many cases 33 34309 LOD was used as the acrylamide level in bread. Finally in the present estimate, as the Danish 35 36310 Nationwide dietary survey do not have information on how frequently people eat toasted bread, the 37 38311 acrylamide intake from toasted bread is not included in the dietary acrylamide intake from bread. 39 40312 However, in case the average daily bread intake includes two hard toasted wheat bread slices 41 -1 42313 (acrylamide 161 µg kg (ww)) each of 28 g and with the high pre-processing asparagine level of 0.6 43 314 g kg -1 (dw) which is about the concentration found in wholegrain wheat flour i.e. 0.5 g kg -1 44 45 315 (Frederiksson et al. 2004 ), the daily acrylamide intake is estimated to 9 µg (36 % of the average 46 47316 Danish acrylamide intake). If a consumer eats an average amount of bread of which two slices of 25 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 38 of 42 1 2 3 317 g rye bread are hard toasted with asparagine and acrylamide concentrations of 1.2 g kg -1 and 123 µg 4 5 318 kg -1 respectively, the daily acrylamide intake from bread comprises 7 µg (28 % of the average 6 7 319 Danish acrylamide intake). Hence dietary habits with consumption of toasted bread, which are 8 9 320 common throughout at least Europe, may increase the acrylamide intake from bread considerable. 10 11321 Especially intake of toasted wholemeal wheat bread and bread produced without leavening by 12 13322 fermentation may further increase the acrylamide exposure. 14 15323 16 For Peer Review Only 324 Figure 4. 17 18 325 19 20 326 Conclusions 21 22327 The contents of acrylamide in baked rye bread and in toasted wheat and rye bread are correlated to 23 24328 the contents of asparagine in the cereal product used for the bread production and at asparagine 25 26329 concentration ranges that may occur in grain products for bread production. The content of 27 28330 asparagine in the dough for wheat bread preparation is reduced considerably during bread 29 30331 production. After 65 min. raising time and subsequent baking, the asparagine content in the baked 31 32332 wheat bread was reduced to 12% compared to the content in the flour before yeast fermentation. 33 34333 The sourdough fermentation of rye bread showed a modest asparagine reduction to 82% of the 35 36334 content in the flour. This is probably due to the more acidic conditions in sourdough fermented rye 37 38335 bread compared to yeast fermented wheat dough. The contents of acrylamide formed in toasted 39 40336 wheat bread slices and toasted rye bread slices are proportional to the asparagine concentration of 41 -1 42337 the untoasted bread. The dietary intake of acrylamide from untoasted bread of 2.0 µg day is 43 338 relatively low but the dietary acrylamide intake increases somewhat for people eating toasted bread. 44 45 339 46 47340 Ackowledgement 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 39 of 42 Food Additives and Contaminants 1 2 3 341 This research was supported by the Danish Ministry of Food, Agriculture and Fisheries as a part of 4 5 342 the project “Reduction of the formation and the occurrence of acrylamide in food” and The Nordic 6 7 343 Innovation Centre Project NORDACRYL (Acrylamide precursors; Limiting substrates and in vivo 8 9 344 effects). Lone Hertz is acknowledged for performing parts of the chemical analyses. 10 11345 12 13346 References 14347 Ahn JS, Castle L, Clarke DB, Lloyd AS, Philo MR, Speck DR. 2002. Verification of the 15 16348 findings of acrylamide inFor heated foods. Peer Food Additives Review and Contaminants 19:1116-1124. 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Food Control 15:191-195. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 41 of 42 Food Additives and Contaminants 1 2 3 400 Rydberg P, Eriksson S, Tareke E, Karlsson P, Ehlenberg L, Törnqvist M. 2003. Investigations 4 401 of factors that influence the acrylamide content of heated foodstuffs. Journal of Agricultural and 5 6 402 Food Chemistry 51:7012-7018. 7 403 Schieberle P, Köhler P, Ganvogl M. 2005. New aspects on the formation and analysis of 8 9 404 acrylamide. Advanced experimental medicine and Biology 561: 191-203. 10405 Stadler, RH, Blank I, Varga N, Robert F, Hau J, Guy PA, Robert MC, Riediker S. 2002. 11 12406 Acrylamide from Maillard reaction products. Nature 419:449. 13407 Suhr KI, Nielsen PV. 2004. Effect of weak acid preservatives on growth of bakery product 14 15408 spoilage fungi at different water activities and pH values. International Journal of Food 16409 Microbiology 95:67-78. For Peer Review Only 17 410 Surdyk N, Rosén J, Andersson R, Åman P. 2004. Effects of asparagine, fructose, and baking 18 19411 conditions on acrylamide contents in yeast-leavened wheat bread. Journal of Agricultural and Food 20412 Chemistry 52:2047-2051. 21 22413 Svensson K, Abramsson L, Becker W, Glynn A, Hellenäs KE, Lind Y, Rosén J. 2003. Dietary 23414 intake of acrylamide in Sweden. Food and Chemical Toxicology 41:1581-1586. 24 25415 Taeymans D, Wood J, Ashby P, Blank I, Studer A, Stadler RH, Gondé P, Van Eijck P, Lalljie S, 26416 Lingnert H, Lindblom M, Matissek R, Müller D, Tallmadge D, O’brian J, Thompson S, Silvani D, 27 28417 Whitmore T. 2004. A review of acrylamide: An industry perspective on research, analysis, 29418 formation and control. Critical Review in Food Sciences and Nutrition 44:323-347. 30 419 Tareke E, Rydberg P, Karlsson P, Eriksson S, Törnqvist M. 2002. Analysis of acrylamide, a 31 32420 carcinogen formed in heated foodstuffs. Journal of Agricultural and Food Chemistry 50:4998-5006. 33 421 Wenzl T, Karasek L, Rosen J, Hellenaes K-E, Castle L, Anklam E. 2006. Collaborative trial 34 35422 validation study of two methods, one based on high performance liquid chromatography-tandem 36423 mass spectrometry and on gas chromatography- mass spectrometry for the determination of 37 38424 acrylamide in bakery and potato products. Journal of Chromatography A 1132: 211-218. 39425 World Health Organization 2002. Health Implications of Acrylamide in Food. 40 41426 http://www.who.int/foodsafety/publications/chem/en/acrylamide_full.pdf . 42427 43428 44429 Table I. Levels of asparagine added to a) wheat dough and b) rye dough, asparagine content in 45430 wheat and rye bread slices and the acrylamide contents after medium or hard toasting of the bread 46431 slices (mean ±2sd). 47432 48433 Table II. Asparagine left after fermentation and baking, and subsequent medium or hard toasting of 49434 wheat and rye bread (in percentage of asparagine in the flour). 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 42 of 42 1 2 3 435 4 436 Table III. Amount of acrylamide formed and present [µg] from asparagine reacted [mg] when 5 437 toasting wheat bread (ratios calculated by subtracting analyte concentrations determined in medium 6 438 or hard toasted bread from analyte concentrations determined in untoasted bread). 7 439 8 440 Figure 1. Experimental design for baking wheat and rye bread with addition of asparagine and 9 441 subsequent medium or hard toasting of bread slices. All bread slices were analysed for acrylamide 10442 and asparagine. 11443 12444 Figure 2. Correlation between naturally occurring and added asparagine in flour and acrylamide in 13445 baked and toasted bread slices of a) wheat or b) rye. 14446 15447 Figure 3. Correlation between asparagine in untoasted bread and acrylamide in toasted bread slices 16448 of a) wheat or b) rye. For Peer Review Only 449 17 450 Figure 4. The proportions from different foods to the daily mean acrylamide intake for Danish 18 451 adults of appr. 25 µg. 19452 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected]